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Sample records for replication factor subunit

  1. The eukaryotic translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and facilitates viral replication.

    Science.gov (United States)

    Liu, Xiaofeng; Wang, Xiaoyu; Wang, Qian; Luo, Mingyang; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

    2018-02-01

    Classical swine fever virus (CSFV) NS5A protein is a multifunctional protein, playing critical roles in viral RNA replication, translation and assembly. To further explore its functions in viral replication, interaction of NS5A with host factors was assayed using a his-tag "pull down" assay coupled with shotgun LC-MS/MS. Host protein translation initiation factor 3 subunit E was identified as a binding partner of NS5A, and confirmed by co-immunoprecipitation and co-localization analysis. Overexpression of eIF3E markedly enhanced CSFV genomic replication, viral protein expression and production of progeny virus, and downregulation of eIF3E by siRNA significantly decreased viral proliferation in PK-15 cells. Luciferase reporter assay showed an enhancement of translational activity of the internal ribosome entry site of CSFV by eIF3E and a decrease in cellular translation by NS5A. These data indicate that eIF3E plays an important role in CSFV replication, thereby identifying it as a potential target for inhibition of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. uvsF RFC1, the large subunit of replication factor C in Aspergillus nidulans, is essential for DNA replication, functions in UV repair and is upregulated in response to MMS-induced DNA damage.

    Science.gov (United States)

    Kafer, Etta; Chae, Suhn-Kee

    2008-09-01

    uvsF201 was the first highly UV-sensitive repair-defective mutation isolated in Aspergillus nidulans. It showed epistasis only with postreplication repair mutations, but caused lethal interactions with many other repair-defective strains. Unexpectedly, closest homology of uvsF was found to the large subunit of human DNA replication factor RFC that is essential for DNA replication. Sequencing of the uvsF201 region identified changes at two close base pairs and the corresponding amino acids in the 5'-region of uvsF(RFC1). This viable mutant represents a novel and possibly important type. Additional sequencing of the uvsF region confirmed a mitochondrial ribosomal protein gene, mrpA(L16), closely adjacent, head-to-head with a 0.2kb joint promoter region. MMS-induced transcription of both the genes, but especially uvsF(RFC1), providing evidence for a function in DNA damage response.

  3. The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication.

    Science.gov (United States)

    Morais, Ana Ts; Terzian, Ana Cb; Duarte, Danilo Vb; Bronzoni, Roberta Vm; Madrid, Maria Cfs; Gavioli, Arieli F; Gil, Laura Hvg; Oliveira, Amanda G; Zanelli, Cleslei F; Valentini, Sandro R; Rahal, Paula; Nogueira, Mauricio L

    2013-06-22

    Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role

  4. The gene for replication factor C subunit 2 (RFC2) is within the 7q11.23 Williams syndrome deletion

    Energy Technology Data Exchange (ETDEWEB)

    Peoples, R.; Perez-Jurado, L.; Francke, U.; Yu-Ker Wang [Stanford Univ. Medical Center, CA (United States); Kaplan, P. [Children`s Hospital of Philadelphia, PA (United States)

    1996-06-01

    Williams syndrome (WS) is a developmental disorder with multiple system manifestations, including supraval var aortic stenosis (SVAS), peripheral pulmonic stenosis, connective tissue abnormalities, short stature, characteristic personality profile and cognitive deficits, and variable hypercalcemia in infancy. It is caused by heterozygosity for a chromosomal deletion of part of band 7q11.23 including the elastin locus (ELN). Since disruption of the ELN gene causes autosomal dominant SVAS, it is assumed that ELN haploinsufficiency is responsible for the cardiovascular features of WS. The deletion that extends from the ELN locus in both directions is {ge}200 kb in size, although estimates of {ge}2 Mb are suggested by high-resolution chromosome banding and physical mapping studies. We have searched for additional dosage-sensitive genes within the deletion that may be responsible for the noncardiovascular features. We report here that the gene for replication factor C subunit 2 (RFC2) maps within the WS deletion region and was found to be deleted in all of 18 WS patients studied. The protein product of RFC2 is part of a multimeric complex involved in DNA elongation during replication. 14 refs., 3 figs.

  5. Two subunits of human ORC are dispensable for DNA replication and proliferation.

    Science.gov (United States)

    Shibata, Etsuko; Kiran, Manjari; Shibata, Yoshiyuki; Singh, Samarendra; Kiran, Shashi; Dutta, Anindya

    2016-12-01

    The six-subunit Origin Recognition Complex (ORC) is believed to be an essential eukaryotic ATPase that binds to origins of replication as a ring-shaped heterohexamer to load MCM2-7 and initiate DNA replication. We have discovered that human cell lines in culture proliferate with intact chromosomal origins of replication after disruption of both alleles of ORC2 or of the ATPase subunit, ORC1 . The ORC1 or ORC2 -depleted cells replicate with decreased chromatin loading of MCM2-7 and become critically dependent on another ATPase, CDC6, for survival and DNA replication. Thus, either the ORC ring lacking a subunit, even its ATPase subunit, can load enough MCM2-7 in partnership with CDC6 to initiate DNA replication, or cells have an ORC-independent, CDC6-dependent mechanism to load MCM2-7 on origins of replication.

  6. Chromatin Immunoprecipitation of Replication Factors Moving with the Replication Fork

    OpenAIRE

    Rapp, Jordan B.; Ansbach, Alison B.; Noguchi, Chiaki; Noguchi, Eishi

    2009-01-01

    Replication of chromosomes involves a variety of replication proteins including DNA polymerases, DNA helicases, and other accessory factors. Many of these proteins are known to localize at replication forks and travel with them as components of the replisome complex. Other proteins do not move with replication forks but still play an essential role in DNA replication. Therefore, in order to understand the mechanisms of DNA replication and its controls, it is important to examine localization ...

  7. Inhibition of herpesvirus and influenza virus replication by blocking polymerase subunit interactions.

    Science.gov (United States)

    Palù, Giorgio; Loregian, Arianna

    2013-09-01

    Protein-protein interactions (PPIs) play a key role in many biological processes, including virus replication in the host cell. Since most of the PPIs are functionally essential, a possible strategy to inhibit virus replication is based on the disruption of viral protein complexes by peptides or small molecules that interfere with subunit interactions. In particular, an attractive target for antiviral drugs is the binding between the subunits of essential viral enzymes. This review describes the development of new antiviral compounds that inhibit herpesvirus and influenza virus replication by blocking interactions between subunit proteins of their polymerase complexes. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Rpa4, a homolog of the 34-kilodalton subunit of the replication protein A complex.

    OpenAIRE

    Keshav, K F; Chen, C; Dutta, A

    1995-01-01

    Replication protein A (RPA) is a complex of three polypeptides of 70, 34, and 13 kDa isolated from diverse eukaryotes. The complex is a single-stranded DNA-binding protein essential for simian virus 40-based DNA replication in vitro and for viability in the yeast Saccharomyces cerevisiae. We have identified a new 30-kDa human protein which interacts with the 70- and 13-kDa subunits of RPA, with a yeast two-hybrid/interaction trap method. This protein, Rpa4, has 47% identity with Rpa2, the 34-...

  9. The p150 subunit of CAF-1 causes association of SUMO2/3 with the DNA replication foci

    International Nuclear Information System (INIS)

    Uwada, Junsuke; Tanaka, Niina; Yamaguchi, Yutaro; Uchimura, Yasuhiro; Shibahara, Kei-ichi; Nakao, Mitsuyoshi; Saitoh, Hisato

    2010-01-01

    The small ubiquitin-related modifier 2/3 (SUMO2/3) can be post-translationally conjugated to a wide variety of proteins constituting chromatin, the platform for genetic and epigenetic regulation. Nevertheless, it is unclear how SUMO2/3 and SUMO2/3-modified proteins are delivered to the chromatin fibers. Here we report that the largest subunit of chromatin assembly factor 1 (CAF-1), human p150, interacts directly and preferentially with SUMO2/3. Amino acid residue of 98-105 in p150 is essential and sufficient for SUMO2/3 interaction. p150-SUMO2/3 interaction coincided with regions that replicate chromatin fibers, because accumulation of the proliferating cell nuclear antigen (PCNA), and incorporation of bromodeoxyuridine (BrdU) were detected at foci co-localized with both p150 and SUMO2/3 during the S-phase in a cell line expressing epitope-tagged p150. Although inhibition of SUMO2/3 expression had only a small effect on p150 deposition on the replication sites, depletion of p150 led to delocalization of SUMO2/3 from the replication foci. Furthermore, p150 mutants deficient in SUMO2/3 interaction, caused a major reduction of SUMO2/3 at the replication foci. Thus, our findings suggest an expanded role of p150 as a SUMO2/3-interacting factor, and raise the intriguing possibility that p150 plays a role in promoting delivery of SUMO2/3 or SUMO2/3-modified proteins (or both) on chromatin fibers during replication.

  10. Factors influencing microinjection molding replication quality

    Science.gov (United States)

    Vera, Julie; Brulez, Anne-Catherine; Contraires, Elise; Larochette, Mathieu; Trannoy-Orban, Nathalie; Pignon, Maxime; Mauclair, Cyril; Valette, Stéphane; Benayoun, Stéphane

    2018-01-01

    In recent years, there has been increased interest in producing and providing high-precision plastic parts that can be manufactured by microinjection molding: gears, pumps, optical grating elements, and so on. For all of these applications, the replication quality is essential. This study has two goals: (1) fabrication of high-precision parts using the conventional injection molding machine; (2) identification of robust parameters that ensure production quality. Thus, different technological solutions have been used: cavity vacuuming and the use of a mold coated with DLC or CrN deposits. AFM and SEM analyses were carried out to characterize the replication profile. The replication quality was studied in terms of the process parameters, coated and uncoated molds and crystallinity of the polymer. Specific studies were processed to quantify the replicability of injection molded parts (ABS, PC and PP). Analysis of the Taguchi experimental designs permits prioritization of the impact of each parameter on the replication quality. A discussion taking into account these new parameters and the thermal and spreading properties on the coatings is proposed. It appeared that, in general, increasing the mold temperature improves the molten polymer fill in submicron features except for the steel insert (for which the presence of a vacuum is the most important factor). Moreover, the DLC coating was the best coating to increase the quality of the replication. This result could be explained by the lower thermal diffusivity of this coating. We noted that the viscosity of the polymers is not a primordial factor of the replication quality.

  11. CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

    Science.gov (United States)

    Langston, Lance D; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E; Finkelstein, Jeff; Yao, Nina Y; Indiani, Chiara; O'Donnell, Mike E

    2014-10-28

    DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

  12. The host-dependent interaction of alpha-importins with influenza PB2 polymerase subunit is required for virus RNA replication.

    Directory of Open Access Journals (Sweden)

    Patricia Resa-Infante

    Full Text Available The influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. As PB2 is a relevant host-range determinant we expressed a TAP-tagged PB2 in human cells and isolated intracellular complexes. Alpha-importin was identified as a PB2-associated factor by proteomic analyses. To study the relevance of this interaction for virus replication we mutated the PB2 NLS and analysed the phenotype of mutant subunits, polymerase complexes and RNPs. While mutant PB2 proteins showed reduced nuclear accumulation, they formed polymerase complexes normally when co expressed with PB1 and PA. However, mutant RNPs generated with a viral CAT replicon showed up to hundred-fold reduced CAT accumulation. Rescue of nuclear localisation of mutant PB2 by insertion of an additional SV40 TAg-derived NLS did not revert the mutant phenotype of RNPs. Furthermore, determination of recombinant RNP accumulation in vivo indicated that PB2 NLS mutations drastically reduced virus RNA replication. These results indicate that, above and beyond its role in nuclear accumulation, PB2 interaction with alpha-importins is required for virus RNA replication. To ascertain whether PB2-alpha-importin binding could contribute to the adaptation of H5N1 avian viruses to man, their association in vivo was determined. Human alpha importin isoforms associated efficiently to PB2 protein of an H3N2 human virus but bound to diminished and variable extents to PB2 from H5N1 avian or human strains, suggesting that the function of alpha importin during RNA replication is important for the adaptation of avian viruses to the human host.

  13. Factor Copula Models for Replicated Spatial Data

    KAUST Repository

    Krupskii, Pavel

    2016-12-19

    We propose a new copula model that can be used with replicated spatial data. Unlike the multivariate normal copula, the proposed copula is based on the assumption that a common factor exists and affects the joint dependence of all measurements of the process. Moreover, the proposed copula can model tail dependence and tail asymmetry. The model is parameterized in terms of a covariance function that may be chosen from the many models proposed in the literature, such as the Matérn model. For some choice of common factors, the joint copula density is given in closed form and therefore likelihood estimation is very fast. In the general case, one-dimensional numerical integration is needed to calculate the likelihood, but estimation is still reasonably fast even with large data sets. We use simulation studies to show the wide range of dependence structures that can be generated by the proposed model with different choices of common factors. We apply the proposed model to spatial temperature data and compare its performance with some popular geostatistics models.

  14. Factor Copula Models for Replicated Spatial Data

    KAUST Repository

    Krupskii, Pavel; Huser, Raphaë l; Genton, Marc G.

    2016-01-01

    We propose a new copula model that can be used with replicated spatial data. Unlike the multivariate normal copula, the proposed copula is based on the assumption that a common factor exists and affects the joint dependence of all measurements of the process. Moreover, the proposed copula can model tail dependence and tail asymmetry. The model is parameterized in terms of a covariance function that may be chosen from the many models proposed in the literature, such as the Matérn model. For some choice of common factors, the joint copula density is given in closed form and therefore likelihood estimation is very fast. In the general case, one-dimensional numerical integration is needed to calculate the likelihood, but estimation is still reasonably fast even with large data sets. We use simulation studies to show the wide range of dependence structures that can be generated by the proposed model with different choices of common factors. We apply the proposed model to spatial temperature data and compare its performance with some popular geostatistics models.

  15. Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage.

    Science.gov (United States)

    Wang, Yan; Mao, Lili; Kankanala, Jayakanth; Wang, Zhengqiang; Geraghty, Robert J

    2017-02-01

    The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease. Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus

  16. Ionizing radiation-dependent and independent phosphorylation of the 32-kDa subunit of replication protein A during mitosis.

    LENUS (Irish Health Repository)

    Stephan, Holger

    2009-10-01

    The human single-stranded DNA-binding protein, replication protein A (RPA), is regulated by the N-terminal phosphorylation of its 32-kDa subunit, RPA2. RPA2 is hyperphosphorylated in response to various DNA-damaging agents and also phosphorylated in a cell-cycle-dependent manner during S- and M-phase, primarily at two CDK consensus sites, S23 and S29. Here we generated two monoclonal phospho-specific antibodies directed against these CDK sites. These phospho-specific RPA2-(P)-S23 and RPA2-(P)-S29 antibodies recognized mitotically phosphorylated RPA2 with high specificity. In addition, the RPA2-(P)-S23 antibody recognized the S-phase-specific phosphorylation of RPA2, suggesting that during S-phase only S23 is phosphorylated, whereas during M-phase both CDK sites, S23 and S29, are phosphorylated. Immunofluorescence microscopy revealed that the mitotic phosphorylation of RPA2 starts at the onset of mitosis, and dephosphorylation occurs during late cytokinesis. In mitotic cells treated with ionizing radiation (IR), we observed a rapid hyperphosphorylation of RPA2 in addition to its mitotic phosphorylation at S23 and S29, associated with a significant change in the subcellular localization of RPA. Our data also indicate that the RPA2 hyperphosphorylation in response to IR is facilitated by the activity of both ATM and DNA-PK, and is associated with activation of the Chk2 pathway.

  17. Topography measurements for determining the decay factors in surface replication

    International Nuclear Information System (INIS)

    Song, J; Zheng, A; Vorburger, T V; Rubert, P

    2008-01-01

    The electro-forming technique is used at National Institute of Standards and Technology (NIST) for the production of standard reference material (SRM) 2461 standard casings to support nationwide ballistics measurement traceability and measurement quality control in the US. In order to ensure that the SRM casings are produced with virtually the same surface topography, it is necessary to test the decay factors of the replication process. Twenty-six replica casings are replicated from the same master casing for the decay factor tests. The NIST topography measurement system is used for measurements and correlations of surface topography. The topography decays are quantified by the cross-correlation function maximum CCF max . Based on the test, it is expected that 256 SRM casings can be replicated from the same master with CCF max values higher than 95%

  18. A replication of a factor analysis of motivations for trapping

    Science.gov (United States)

    Schroeder, Susan; Fulton, David C.

    2015-01-01

    Using a 2013 sample of Minnesota trappers, we employed confirmatory factor analysis to replicate an exploratory factor analysis of trapping motivations conducted by Daigle, Muth, Zwick, and Glass (1998).  We employed the same 25 items used by Daigle et al. and tested the same five-factor structure using a recent sample of Minnesota trappers. We also compared motivations in our sample to those reported by Daigle et el.

  19. Novel host restriction factors implicated in HIV-1 replication.

    Science.gov (United States)

    Ghimire, Dibya; Rai, Madhu; Gaur, Ritu

    2018-04-01

    Human immunodeficiency virus-1 (HIV-1) is known to interact with multiple host cellular proteins during its replication in the target cell. While many of these host cellular proteins facilitate viral replication, a number of them are reported to inhibit HIV-1 replication at various stages of its life cycle. These host cellular proteins, which are known as restriction factors, constitute an integral part of the host's first line of defence against the viral pathogen. Since the discovery of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G) as an HIV-1 restriction factor, several human proteins have been identified that exhibit anti-HIV-1 restriction. While each restriction factor employs a distinct mechanism of inhibition, the HIV-1 virus has equally evolved complex counter strategies to neutralize their inhibitory effect. APOBEC3G, tetherin, sterile alpha motif and histidine-aspartate domain 1 (SAMHD1), and trim-5α are some of the best known HIV-1 restriction factors that have been studied in great detail. Recently, six novel restriction factors were discovered that exhibit significant antiviral activity: endoplasmic reticulum α1,2-mannosidase I (ERManI), translocator protein (TSPO), guanylate-binding protein 5 (GBP5), serine incorporator (SERINC3/5) and zinc-finger antiviral protein (ZAP). The focus of this review is to discuss the antiviral mechanism of action of these six restriction factors and provide insights into the probable counter-evasion strategies employed by the HIV-1 virus. The recent discovery of new restriction factors substantiates the complex host-pathogen interactions occurring during HIV-1 pathogenesis and makes it imperative that further investigations are conducted to elucidate the molecular basis of HIV-1 replication.

  20. Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase δ revealed in individual cells by cytometry.

    Science.gov (United States)

    Darzynkiewicz, Zbigniew; Zhao, Hong; Zhang, Sufang; Lee, Marietta Y W T; Lee, Ernest Y C; Zhang, Zhongtao

    2015-05-20

    During our recent studies on mechanism of the regulation of human DNA polymerase δ in preparation for DNA replication or repair, multiparameter imaging cytometry as exemplified by laser scanning cytometry (LSC) has been used to assess changes in expression of the following nuclear proteins associated with initiation of DNA replication: cyclin A, PCNA, Ki-67, p21(WAF1), DNA replication factor Cdt1 and the smallest subunit of DNA polymerase δ, p12. In the present review, rather than focusing on Pol δ, we emphasize the application of LSC in these studies and outline possibilities offered by the concurrent differential analysis of DNA replication in conjunction with expression of the nuclear proteins. A more extensive analysis of the data on a correlation between rates of EdU incorporation, likely reporting DNA replication, and expression of these proteins, is presently provided. New data, specifically on the expression of cyclin D1 and cyclin E with respect to EdU incorporation as well as on a relationship between expression of cyclin A vs. p21(WAF1) and Ki-67 vs. Cdt1, are also reported. Of particular interest is the observation that this approach makes it possible to assess the temporal sequence of degradation of cyclin D1, p21(WAF1), Cdt1 and p12, each with respect to initiation of DNA replication and with respect to each other. Also the sequence or reappearance of these proteins in G2 after termination of DNA replication is assessed. The reviewed data provide a more comprehensive presentation of potential markers, whose presence or absence marks the DNA replicating cells. Discussed is also usefulness of these markers as indicators of proliferative activity in cancer tissues that may bear information on tumor progression and have a prognostic value.

  1. Risk Factors as Major Determinants of Resilience: A Replication Study.

    Science.gov (United States)

    Eshel, Yohanan; Kimhi, Shaul; Lahad, Mooli; Leykin, Dmitry; Goroshit, Marina

    2018-03-16

    The present study was conducted in the context of current concerns about replication in psychological research. It claims that risk factors should be regarded as an integral part of the definition of individual resilience, which should be defined in terms of the balance between individual strength or protective factors, and individual vulnerability or risk factors (IND-SVR). Five independent samples, including 3457 Israeli participants, were employed to determine the effects of resilience promoting and resilience suppressing variables on the IND-SVR index of resilience, and on its two components: recovery from adversity, and distress symptoms. Five path analyses were employed for determining the role of distress symptoms as a measure of psychological resilience, as compared to other indices of this resilience. Results indicated the major role of risk factors (distress symptoms) as an integral component of resilience. This role was generally replicated in the five investigated samples. Risk factors are legitimate, valid, and useful parts of the definition of psychological resilience. Resilience research has shifted away from studying individual risk factors to investigating the process through which individuals overcome the hardships they experience. The present data seem to suggest that this shift should be reexamined.

  2. Identification of a conserved archaeal RNA polymerase subunit contacted by the basal transcription factor TFB.

    Science.gov (United States)

    Magill, C P; Jackson, S P; Bell, S D

    2001-12-14

    Archaea possess two general transcription factors that are required to recruit RNA polymerase (RNAP) to promoters in vitro. These are TBP, the TATA-box-binding protein and TFB, the archaeal homologue of TFIIB. Thus, the archaeal and eucaryal transcription machineries are fundamentally related. In both RNAP II and archaeal transcription systems, direct contacts between TFB/TFIIB and the RNAP have been demonstrated to mediate recruitment of the polymerase to the promoter. However the subunit(s) directly contacted by these factors has not been identified. Using systematic yeast two-hybrid and biochemical analyses we have identified an interaction between the N-terminal domain of TFB and an evolutionarily conserved subunit of the RNA polymerase, RpoK. Intriguingly, homologues of RpoK are found in all three nuclear RNA polymerases (Rpb6) and also in the bacterial RNA polymerase (omega-subunit).

  3. Human cellular restriction factors that target HIV-1 replication

    Directory of Open Access Journals (Sweden)

    Jeang Kuan-Teh

    2009-09-01

    Full Text Available Abstract Recent findings have highlighted roles played by innate cellular factors in restricting intracellular viral replication. In this review, we discuss in brief the activities of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, bone marrow stromal cell antigen 2 (BST-2, cyclophilin A, tripartite motif protein 5 alpha (Trim5α, and cellular microRNAs as examples of host restriction factors that target HIV-1. We point to countermeasures encoded by HIV-1 for moderating the potency of these cellular restriction functions.

  4. The transcription elongation factor Bur1-Bur2 interacts with replication protein A and maintains genome stability during replication stress

    DEFF Research Database (Denmark)

    Clausing, Emanuel; Mayer, Andreas; Chanarat, Sittinan

    2010-01-01

    Multiple DNA-associated processes such as DNA repair, replication, and recombination are crucial for the maintenance of genome integrity. Here, we show a novel interaction between the transcription elongation factor Bur1-Bur2 and replication protein A (RPA), the eukaryotic single-stranded DNA......-binding protein with functions in DNA repair, recombination, and replication. Bur1 interacted via its C-terminal domain with RPA, and bur1-¿C mutants showed a deregulated DNA damage response accompanied by increased sensitivity to DNA damage and replication stress as well as increased levels of persisting Rad52...... foci. Interestingly, the DNA damage sensitivity of an rfa1 mutant was suppressed by bur1 mutation, further underscoring a functional link between these two protein complexes. The transcription elongation factor Bur1-Bur2 interacts with RPA and maintains genome integrity during DNA replication stress....

  5. The beta subunit modulates bypass and termination at UV lesions during in vitro replication with DNA polymerase III holoenzyme of Escherichia coli

    International Nuclear Information System (INIS)

    Shavitt, O.; Livneh, Z.

    1989-01-01

    The cycling time of DNA polymerase III holoenzyme during replication of UV-irradiated single-stranded (ss) DNA was longer than with unirradiated DNA (8 versus 3 min, respectively), most likely due to slow dissociation from lesion-terminated nascent DNA strands. Initiation of elongation on primed ssDNA was not significantly inhibited by the presence of UV lesions as indicated by the identical distribution of replication products synthesized at early and late reaction times and by the identical duration of the initial synthesis bursts on both unirradiated and UV-irradiated DNA templates. When replication was performed with DNA polymerase III* supplemented with increasing quantities of purified beta 2 subunit, the cycling time on UV-irradiated DNA decreased from 14.8 min at 1.7 nM beta 2 down to 6 min at 170 nM beta 2, a concentration in which beta 2 was in large excess over the polymerase. In parallel to the reduction in cycling time, also the bypass frequency of cyclobutane-photodimers decreased with increasing beta 2 concentration, and at 170 nM beta 2, bypass of photodimers was essentially eliminated. It has been shown that polymerase complexes with more than one beta 2 per polymerase molecule were formed at high beta 2 concentrations. It is plausible that polymerase complexes obtained under high beta 2 concentration dissociate from lesion-terminated primers faster than polymerase complexes formed at a low beta 2 concentration. This is expected to favor termination over bypass at pyrimidine photodimers and thus decrease their bypass frequency. These results suggest that the beta 2 subunit might act as a sensor for obstacles to replication caused by DNA damage, and that it terminates elongation at these sites by promoting dissociation. The intracellular concentration of beta 2 was estimated to be 250 nM

  6. Supplementary Material for: Factor Copula Models for Replicated Spatial Data

    KAUST Repository

    Krupskii, Pavel; Huser, Raphaë l; Genton, Marc G.

    2016-01-01

    We propose a new copula model that can be used with replicated spatial data. Unlike the multivariate normal copula, the proposed copula is based on the assumption that a common factor exists and affects the joint dependence of all measurements of the process. Moreover, the proposed copula can model tail dependence and tail asymmetry. The model is parameterized in terms of a covariance function that may be chosen from the many models proposed in the literature, such as the Matérn model. For some choice of common factors, the joint copula density is given in closed form and therefore likelihood estimation is very fast. In the general case, one-dimensional numerical integration is needed to calculate the likelihood, but estimation is still reasonably fast even with large data sets. We use simulation studies to show the wide range of dependence structures that can be generated by the proposed model with different choices of common factors. We apply the proposed model to spatial temperature data and compare its performance with some popular geostatistics models.

  7. Tandem truncated rotavirus VP8* subunit protein with T cell epitope as non-replicating parenteral vaccine is highly immunogenic.

    Science.gov (United States)

    Wen, Xiaobo; Cao, Dianjun; Jones, Ronald W; Hoshino, Yasutaka; Yuan, Lijuan

    2015-01-01

    The two currently available live oral rotavirus vaccines, Rotarix(®) and RotaTeq(®), are highly efficacious in the developed countries. However, the efficacy of such vaccines in resource deprived countries in Africa and Southeast Asia is low. We reported previously that a bacterially-expressed rotavirus P2-P[8] ΔVP8* subunit vaccine candidate administered intramuscularly elicited high-titers of neutralizing antibodies in guinea pigs and mice and significantly shortened the duration of diarrhea in neonatal gnotobiotic pigs upon oral challenge with virulent human rotavirus Wa strain. To further improve its vaccine potential and provide wider coverage against rotavirus strains of global and regional epidemiologic importance, we constructed 2 tandem recombinant VP8* proteins, P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* based on Escherichia coli expression system. The two resulting recombinant tandem proteins were highly soluble and P2-P[8] ΔVP8*-P[8] ΔVP8* was generated with high yield. Moreover, guinea pigs immunized intramuscularly by 3 doses of the P2-P[8] ΔVP8*-P[8] ΔVP8* or P2-P[8] ΔVP8*-P[6] ΔVP8* vaccine with aluminum phosphate adjuvant developed high titers of homotypic and heterotypic neutralizing antibodies against human rotaviruses bearing G1-G4, G8, G9 and G12 with P[8], P[4] or P[6] combination. The results suggest that these 2 subunit vaccines in monovalent or bivalent formulation can provide antigenic coverage to almost all the rotavirus G (VP7) types and major P (VP4) types of global as well as regional epidemiologic importance.

  8. The interaction between endogenous 30S ribosomal subunit protein S11 and Cucumber mosaic virus LS2b protein affects viral replication, infection and gene silencing suppressor activity.

    Directory of Open Access Journals (Sweden)

    Ruilin Wang

    Full Text Available Cucumber mosaic virus (CMV is a model virus for plant-virus protein interaction and mechanism research because of its wide distribution, high-level of replication and simple genome structure. The 2b protein is a multifunctional protein encoded by CMV that suppresses RNA silencing-based antiviral defense and contributes to CMV virulence in host plants. In this report, 12 host proteins were identified as CMV LS2b binding partners using the yeast two-hybrid screen system from the Arabidopsis thaliana cDNA library. Among the host proteins, 30S ribosomal subunit protein S11 (RPS11 was selected for further studies. The interaction between LS2b and full-length RPS11 was confirmed using the yeast two-hybrid system. Bimolecular fluorescence complementation (BIFC assays observed by confocal laser microscopy and Glutathione S-transferase (GST pull-down assays were used to verify the interaction between endogenous NbRPS11 and viral CMVLS2b both in vivo and in vitro. TRV-based gene silencing vector was used to knockdown NbRPS11 transcription, and immunoblot analysis revealed a decline in infectious viral RNA replication and a decrease in CMV infection in RPS11 down-regulated Nicotiana benthamiana plants. Thus, the knockdown of RPS11 likely inhibited CMV replication and accumulation. The gene silencing suppressor activity of CMV2b protein was reduced by the RPS11 knockdown. This study demonstrated that the function of viral LS2b protein was remarkably affected by the interaction with host RPS11 protein.

  9. DNA replication factor C1 mediates genomic stability and transcriptional gene silencing in Arabidopsis

    KAUST Repository

    Liu, Qian; Wang, Junguo; Miki, Daisuke; Xia, Ran; Yu, Wenxiang; He, Junna; Zheng, Zhimin; Zhu, Jian-Kang; Gonga, Zhizhong

    2010-01-01

    Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylationindependent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana. © 2010 American Society of Plant Biologists.

  10. DNA replication factor C1 mediates genomic stability and transcriptional gene silencing in Arabidopsis

    KAUST Repository

    Liu, Qian

    2010-07-01

    Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylationindependent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana. © 2010 American Society of Plant Biologists.

  11. The structure of the TFIIH p34 subunit reveals a von Willebrand factor A like fold.

    Directory of Open Access Journals (Sweden)

    Dominik R Schmitt

    Full Text Available RNA polymerase II dependent transcription and nucleotide excision repair are mediated by a multifaceted interplay of subunits within the general transcription factor II H (TFIIH. A better understanding of the molecular structure of TFIIH is the key to unravel the mechanism of action of this versatile protein complex within these vital cellular processes. The importance of this complex becomes further evident in the context of severe diseases like xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy, that arise from single point mutations in TFIIH subunits. Here we describe the structure of the p34 subunit of the TFIIH complex from the eukaryotic thermophilic fungus Chaetomium thermophilum. The structure revealed that p34 contains a von Willebrand Factor A (vWA like domain, a fold which is generally known to be involved in protein-protein interactions. Within TFIIH p34 strongly interacts with p44, a positive regulator of the helicase XPD. Putative protein-protein interfaces are analyzed and possible binding sites for the p34-p44 interaction suggested.

  12. Regulation of DNA replication in irradiated cells by trans-acting factors

    International Nuclear Information System (INIS)

    Wang, Y.; Huq, M.S.; Cheng, X.; Iliakis, G.

    1995-01-01

    We compared DNA replication activity in cytoplasmic extracts prepared from irradiated and nonirradiated HeLa cells using a simian virus 40 (SV40)-based in vitro replication assay. The assay measures semi-conservative DNA replication in a plasmid carrying the SV40 origin of replication and requires SV40 T antigen as the sole noncellular protein. The plasmid DNA used in the replication reaction is never exposed to radiation. We find that replication of plasmid DNA is significantly reduced when cytoplasmic extracts from irradiated cells are used. Since plasmid replication proceeds to completion in extracts from irradiated cells, the observed reduction in the overall replication activity is probably due to a reduction in the efficiency of initiation events. The degree of inhibition of DNA replication after exposure to 10, 30 and 50 Gy X rays as measured in vitro using this assay is similar to that measured in intact cells immediately before processing for extract preparation. These observations are compatible with the induction or activation by ionizing radiation of a factor(s) that inhibits in trans DNA replication. The results contribute to our understanding of the mechanism(s) developed by the cells to regulate DNA replication when exposed to clastogenic agents. Such processes may be of significance in the restoration of DNA integrity, and may define yet another checkpoint operating during S at the level of clusters of replicons. 26 refs., 4 figs

  13. Chemical shift changes provide evidence for overlapping single-stranded DNA and XPA binding sites on the 70 kDa subunit of human replication protein A

    Energy Technology Data Exchange (ETDEWEB)

    Daughdrill, Gary W.; Buchko, Garry W.; Botuyan, Maria V.; Arrowsmith, Cheryl H.; Wold, Marc S.; Kennedy, Michael A.; Lowry, David F.

    2003-07-15

    Replication protein A (RPA) is a heterotrimeric single-stranded DNA (ssDNA) binding protein that can form a complex with the xeroderma pigmentosum group A protein (XPA). This complex can preferentially recognize UV damaged DNA over undamaged DNA and has been implicated in the stabilization of open complex formation during nucleotide excision repair. In this report, NMR spectroscopy was used to investigate the interaction between a fragment of the 70 kDa subunit of human RPA, residues 1-326 (hRPA701-326), and a fragment of the human XPA protein, residues 98-219 (XPA-MBD). Intensity changes were observed for amide resonances in the 1H-15N correlation spectrum of uniformly 15N-labeled hRPA701-326 after the addition of unlabeled XPA-MBD. The intensity changes observed were restricted to an ssDNA binding domain that is between residues 183 and 296 of the hRPA701-326 fragment. The hRPA701-326 residues with the largest resonance intensity reductions were mapped onto the structure of the ssDNA binding domain to identify the binding surface with XPA-MBD. The XPA-MBD binding surface showed significant overlap with an ssDNA binding surface that was previously identified using NMR spectroscopy and X-ray crystallography.

  14. Virulence factor NSs of rift valley fever virus recruits the F-box protein FBXO3 to degrade subunit p62 of general transcription factor TFIIH.

    Science.gov (United States)

    Kainulainen, Markus; Habjan, Matthias; Hubel, Philipp; Busch, Laura; Lau, Simone; Colinge, Jacques; Superti-Furga, Giulio; Pichlmair, Andreas; Weber, Friedemann

    2014-03-01

    The nonstructural protein NSs is the main virulence factor of Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), a serious pathogen of livestock and humans in Africa. RVFV NSs blocks transcriptional upregulation of antiviral type I interferons (IFN) and destroys the general transcription factor TFIIH subunit p62 via the ubiquitin/proteasome pathway. Here, we identified a subunit of E3 ubiquitin ligases, F-box protein FBXO3, as a host cell interactor of NSs. Small interfering RNA (siRNA)-mediated depletion of FBXO3 rescued p62 protein levels in RVFV-infected cells and elevated IFN transcription by 1 order of magnitude. NSs interacts with the full-length FBXO3 protein as well as with a truncated isoform that lacks the C-terminal acidic and poly(R)-rich domains. These isoforms are present in both the nucleus and the cytoplasm. NSs exclusively removes the nuclear pool of full-length FBXO3, likely due to consumption during the degradation process. F-box proteins form the variable substrate recognition subunit of the so-called SCF ubiquitin ligases, which also contain the constant components Skp1, cullin 1 (or cullin 7), and Rbx1. siRNA knockdown of Skp1 also protected p62 from degradation, suggesting involvement in NSs action. However, knockdown of cullin 1, cullin 7, or Rbx1 could not rescue p62 degradation by NSs. Our data show that the enzymatic removal of p62 via the host cell factor FBXO3 is a major mechanism of IFN suppression by RVFV. Rift Valley fever virus is a serious emerging pathogen of animals and humans. Its main virulence factor, NSs, enables unhindered virus replication by suppressing the antiviral innate immune system. We identified the E3 ubiquitin ligase FBXO3 as a novel host cell interactor of NSs. NSs recruits FBXO3 to destroy the general host cell transcription factor TFIIH-p62, resulting in suppression of the transcriptional upregulation of innate immunity.

  15. Class I ADP-ribosylation factors are involved in enterovirus 71 replication.

    Directory of Open Access Journals (Sweden)

    Jianmin Wang

    Full Text Available Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies.

  16. Class I ADP-ribosylation factors are involved in enterovirus 71 replication.

    Science.gov (United States)

    Wang, Jianmin; Du, Jiang; Jin, Qi

    2014-01-01

    Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies.

  17. Chromosomal Replication Complexity: A Novel DNA Metrics and Genome Instability Factor.

    Directory of Open Access Journals (Sweden)

    Andrei Kuzminov

    2016-10-01

    Full Text Available As the ratio of the copy number of the most replicated to the unreplicated regions in the same chromosome, the definition of chromosomal replication complexity (CRC appears to leave little room for variation, being either two during S-phase or one otherwise. However, bacteria dividing faster than they replicate their chromosome spike CRC to four and even eight. A recent experimental inquiry about the limits of CRC in Escherichia coli revealed two major reasons to avoid elevating it further: (i increased chromosomal fragmentation and (ii complications with subsequent double-strand break repair. Remarkably, examples of stable elevated CRC in eukaryotic chromosomes are well known under various terms like "differential replication," "underreplication," "DNA puffs," "onion-skin replication," or "re-replication" and highlight the phenomenon of static replication fork (sRF. To accurately describe the resulting "amplification by overinitiation," I propose a new term: "replification" (subchromosomal overreplication. In both prokaryotes and eukaryotes, replification, via sRF processing, causes double-strand DNA breaks and, with their repair elevating chromosomal rearrangements, represents a novel genome instability factor. I suggest how static replication bubbles could be stabilized and speculate that some tandem duplications represent such persistent static bubbles. Moreover, I propose how static replication bubbles could be transformed into tandem duplications, double minutes, or inverted triplications. Possible experimental tests of these models are discussed.

  18. Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

    Energy Technology Data Exchange (ETDEWEB)

    Kamada, Katsuhiko; De Angelis, Jacqueline; Roeder, Robert G.; Burley, Stephen K. (Rockefeller)

    2012-12-13

    The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-{angstrom} resolution. The {alpha}/{beta} structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3{gamma} (HNF-3{gamma}), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3{gamma} and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the {beta} subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

  19. Only three personality factors are fully replicable across languages: Reply to Ashton and Lee

    Czech Academy of Sciences Publication Activity Database

    De Raad, B.; Barelds, D.P.H.; Mlacić, B.; Church, A.T.; Katigbak, M. S.; Ostendorf, F.; Hřebíčková, Martina; Di Bias, L.; Szirmak, S.

    2010-01-01

    Roč. 44, č. 4 (2010), s. 442-445 ISSN 0092-6566 R&D Projects: GA ČR GAP407/10/2394 Institutional research plan: CEZ:AV0Z70250504 Keywords : lexical approach * factor replicability * cross - cultural * three factors Subject RIV: AN - Psychology Impact factor: 1.756, year: 2010

  20. miR-370 suppresses HBV gene expression and replication by targeting nuclear factor IA.

    Science.gov (United States)

    Fan, Hongxia; Lv, Ping; Lv, Jing; Zhao, Xiaopei; Liu, Min; Zhang, Guangling; Tang, Hua

    2017-05-01

    Hepatitis B virus (HBV) infection is a major health problem worldwide. The roles of microRNAs in the regulation of HBV expression are being increasingly recognized. In this study, we found that overexpression of miR-370 suppressed HBV gene expression and replication in Huh7 cells, whereas antisense knockdown of endogenous miR-370 enhanced HBV gene expression and replication in Huh7 cells and HepG2.2.15 cells. Further, we identified the transcription factor nuclear factor IA (NFIA) as a new host target of miR-370. Overexpression and knockdown studies showed that NFIA stimulated HBV gene expression and replication. Importantly, overexpression of NFIA counteracted the effect of miR-370 on HBV gene expression and replication. Further mechanistic studies showed that miR-370 suppressed HBV replication and gene expression by repressing HBV Enhancer I activity, and one of the NFIA binding site in the Enhancer I element was responsible for the repressive effect of miR-370 on HBV Enhancer I activity. Altogether, our results demonstrated that miR-370 suppressed HBV gene expression and replication through repressing NFIA expression, which stimulates HBV replication via direct regulation on HBV Enhancer I activities. Our findings may provide a new antiviral strategy for HBV infection. J. Med. Virol. 89:834-844, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

    International Nuclear Information System (INIS)

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

    1979-01-01

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

  2. Characterisation by nuclear magnetic resonance of the β catalytic subunit of the chloroplastic coupling factor

    International Nuclear Information System (INIS)

    Andre, Francois

    1986-09-01

    This academic work addressed the use of nuclear magnetic resonance (NMR) for the structural and dynamic study of the catalytic sub-unit of the extrinsic section of a membrane complex, the chloroplastic H+-ATPase. This work included the development of a protocol of preparation and quantitative purification of β subunits isolated from the CF1 for the elaboration of a concentrated sample for NMR, and then the study of the β subunit by using proton NMR

  3. MUTATION ON WD DIPEPTIDE MOTIFS OF THE p48 SUBUNIT OF CHROMATIN ASSEMBLY FACTOR-1 CAUSING VIABILITY AND GROWTH OF DT40 CHICKEN B CELL LINE

    Directory of Open Access Journals (Sweden)

    Ahyar Ahmad

    2010-07-01

    Full Text Available Chromatin assembly factor-1 (CAF-1, a protein complex consisting of three subunits, p150, p60, and p48, is highly conserved from yeast to humans and facilitated nucleosome assembly of newly replicated DNA. The p48 subunit, CAF-1p48 (p48, with seven WD (Trp-Asp repeat motifs, is a member of the WD protein family. The immunoprecipitation experiment revealed that ß-propeller structure of p48 was less stringent for it's binding to HDAC-1, but more stringent for its binding to both histones H4 and CAF-1p60 but not to ASF-1, indicating that the proper ß-propeller structure of p48 is essential for the binding to these two proteins histone H4 and CAF-1p60. Complementation experiments, involving missense and truncated mutants of FLAG-tagged p48, revealed that mutations of every of seven WD dipeptide motifs, like both the N-terminal and C-terminal truncated mutations, could not rescue for the tet-induced lethality. These results indicate not only that p48 is essential for the viability of vertebrate cells, although the yeast p48 homolog is nonessential, but also that all the seven WD dipeptide motifs are necessary for the maintenance of the proper structure of p48 that is fundamentally important for cell viability.   Keywords: Chromatin assembly factor-1, complementation experiments, viability

  4. Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.

    Science.gov (United States)

    Dembowski, Jill A; DeLuca, Neal A

    2015-05-01

    Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND) was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics during infection and

  5. Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.

    Directory of Open Access Journals (Sweden)

    Jill A Dembowski

    2015-05-01

    Full Text Available Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics

  6. The structure of psychopathology in adolescence : Replication of a general psychopathology factor in the TRAILS study

    NARCIS (Netherlands)

    Laceulle, O.M.; Vollebergh, W.A.M.; Ormel, J.

    2015-01-01

    This study aimed to replicate a study by Caspi and colleagues, which proposed that the structure of psychopathology is characterized by a general psychopathology factor, in addition to smaller internalizing and externalizing factors. Our study expanded the approach of the original by using

  7. Cloning and characterization of p52, the fifth subunit of the core of transcription/repair factor TFIIH.

    NARCIS (Netherlands)

    J.C. Marinoni; R. Roy (Richard); W. Vermeulen (Wim); P. Miniou; Y. Lutz; G. Weeda (Geert); T. Seroz; D.M. Gomez (Denise Molina); J.H.J. Hoeijmakers (Jan); J-M. Egly (Jean-Marc)

    1997-01-01

    textabstractTFIIH is a multiprotein factor involved in transcription and DNA repair and is implicated in DNA repair/transcription deficiency disorders such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Eight out of the nine genes encoding the subunits forming TFIIH have

  8. The origin of the supernumerary subunits and assembly factors of complex I: A treasure trove of pathway evolution

    NARCIS (Netherlands)

    Elurbe, D.M.; Huynen, M.A.

    2016-01-01

    We review and document the evolutionary origin of all complex I assembly factors and nine supernumerary subunits from protein families. Based on experimental data and the conservation of critical residues we identify a spectrum of protein function conservation between the complex I representatives

  9. An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies.

    Science.gov (United States)

    Komatsu, Tetsuro; Nagata, Kyosuke; Wodrich, Harald

    2016-02-01

    Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. The immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and

  10. Downregulation of SWI/SNF chromatin remodeling factor subunits modulates cisplatin cytotoxicity

    International Nuclear Information System (INIS)

    Kothandapani, Anbarasi; Gopalakrishnan, Kathirvel; Kahali, Bhaskar; Reisman, David; Patrick, Steve M.

    2012-01-01

    Chromatin remodeling complex SWI/SNF plays important roles in many cellular processes including transcription, proliferation, differentiation and DNA repair. In this report, we investigated the role of SWI/SNF catalytic subunits Brg1 and Brm in the cellular response to cisplatin in lung cancer and head/neck cancer cells. Stable knockdown of Brg1 and Brm enhanced cellular sensitivity to cisplatin. Repair kinetics of cisplatin DNA adducts revealed that downregulation of Brg1 and Brm impeded the repair of both intrastrand adducts and interstrand crosslinks (ICLs). Cisplatin ICL-induced DNA double strand break repair was also decreased in Brg1 and Brm depleted cells. Altered checkpoint activation with enhanced apoptosis as well as impaired chromatin relaxation was observed in Brg1 and Brm deficient cells. Downregulation of Brg1 and Brm did not affect the recruitment of DNA damage recognition factor XPC to cisplatin DNA lesions, but affected ERCC1 recruitment, which is involved in the later stages of DNA repair. Based on these results, we propose that SWI/SNF chromatin remodeling complex modulates cisplatin cytotoxicity by facilitating efficient repair of the cisplatin DNA lesions. -- Highlights: ► Stable knockdown of Brg1 and Brm enhances cellular sensitivity to cisplatin. ► Downregulation of Brg1 and Brm impedes the repair of cisplatin intrastrand adducts and interstrand crosslinks. ► Brg1 and Brm deficiency results in impaired chromatin relaxation, altered checkpoint activation as well as enhanced apoptosis. ► Downregulation of Brg1 and Brm affects recruitment of ERCC1, but not XPC to cisplatin DNA lesions.

  11. p44 and p34 subunits of the BTF2/TFIIH transcription factor have homologies with SSL1, a yeast protein involved in DNA repair.

    NARCIS (Netherlands)

    S. Humbert; H. van Vuuren; Y. Lutz; J.H.J. Hoeijmakers (Jan); J-M. Egly (Jean-Marc); V. Moncollin

    1994-01-01

    textabstractThe human BTF2 (TFIIH) transcription factor is a multisubunit protein involved in transcription initiation by RNA polymerase II (B) as well as in DNA repair. In addition to the previously characterized p62 and p89/ERCC3 subunits, we have cloned two other subunits of BTF2, p44 and p34.

  12. Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III

    Directory of Open Access Journals (Sweden)

    Matsutani Sachiko

    2004-08-01

    Full Text Available Abstract Background In eukaryotes, RNA polymerase III (RNAP III transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs. The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously. Results Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related. Conclusion Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and α-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants

  13. Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III.

    Science.gov (United States)

    Matsutani, Sachiko

    2004-08-09

    In eukaryotes, RNA polymerase III (RNAP III) transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs). The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously. Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related. Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and alpha-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants than to fungi.

  14. A loss of function analysis of host factors influencing Vaccinia virus replication by RNA interference.

    Directory of Open Access Journals (Sweden)

    Philippa M Beard

    Full Text Available Vaccinia virus (VACV is a large, cytoplasmic, double-stranded DNA virus that requires complex interactions with host proteins in order to replicate. To explore these interactions a functional high throughput small interfering RNA (siRNA screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF influencing the replication and spread of an eGFP-tagged VACV. The experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of VACV. The screen revealed 153 pro- and 149 anti-viral HFs that strongly influenced VACV replication. These HFs were investigated further by comparisons with transcriptional profiling data sets and HFs identified in RNAi screens of other viruses. In addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in VACV replication. This revealed, as anticipated, that many pro-viral HFs are involved in translation of mRNA and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several DNA repair pathways possess anti-viral activity. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. This advancement in our understanding of the VACV life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics.

  15. SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

    Science.gov (United States)

    McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

    1992-01-01

    The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

  16. Evolution of Replication Machines

    Science.gov (United States)

    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

  17. DNA is a co-factor for its own replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Lebofsky, Ronald; van Oijen, Antoine M.; Walter, Johannes C.

    Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below

  18. Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III

    OpenAIRE

    Matsutani Sachiko

    2004-01-01

    Abstract Background In eukaryotes, RNA polymerase III (RNAP III) transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs). The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFII...

  19. The origin of the supernumerary subunits and assembly factors of complex I: A treasure trove of pathway evolution.

    Science.gov (United States)

    Elurbe, Dei M; Huynen, Martijn A

    2016-07-01

    We review and document the evolutionary origin of all complex I assembly factors and nine supernumerary subunits from protein families. Based on experimental data and the conservation of critical residues we identify a spectrum of protein function conservation between the complex I representatives and their non-complex I homologs. This spectrum ranges from proteins that have retained their molecular function but in which the substrate specificity may have changed or have become more specific, like NDUFAF5, to proteins that have lost their original molecular function and critical catalytic residues like NDUFAF6. In between are proteins that have retained their molecular function, which however appears unrelated to complex I, like ACAD9, or proteins in which amino acids of the active site are conserved but for which no enzymatic activity has been reported, like NDUFA10. We interpret complex I evolution against the background of molecular evolution theory. Complex I supernumerary subunits and assembly factors appear to have been recruited from proteins that are mitochondrial and/or that are expressed when complex I is active. Within the evolution of complex I and its assembly there are many cases of neofunctionalization after gene duplication, like ACAD9 and TMEM126B, one case of subfunctionalization: ACPM1 and ACPM2 in Yarrowia lipolytica, and one case in which a complex I protein itself appears to have been the source of a new protein from another complex: NDUFS6 gave rise to cytochrome c oxidase subunit COX4/COX5b. Complex I and its assembly can therewith be regarded as a treasure trove for pathway evolution. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. The acid-labile subunit of the ternary insulin-like growth factor complex in cirrhosis: relation to liver dysfunction

    DEFF Research Database (Denmark)

    Møller, S; Juul, A; Becker, U

    2000-01-01

    BACKGROUND/AIMS: In the circulation, insulin-like growth factor-I (IGF-I) is bound in a trimeric complex of 150 kDa with IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). Whereas circulating IGF-I and IGFBP-3 are reported to be low in patients with chronic liver failure, the leve...... with significant relations to liver dysfunction and other components of the IGF complex. A small hepatic extraction was found in controls, which suggests extrahepatic production of ALS. Future studies should focus on organ-specific removal of ALS.......BACKGROUND/AIMS: In the circulation, insulin-like growth factor-I (IGF-I) is bound in a trimeric complex of 150 kDa with IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). Whereas circulating IGF-I and IGFBP-3 are reported to be low in patients with chronic liver failure, the level...... of ALS has not been described in relation to hepatic dysfunction. The aim of the present study was therefore to measure circulating and hepatic venous concentrations of ALS in relation to hepatic function and the IGF axis. METHODS: Twenty-five patients with cirrhosis (Child class A/B/C:5/10/10) and 30...

  1. RNASEK Is a V-ATPase-Associated Factor Required for Endocytosis and the Replication of Rhinovirus, Influenza A Virus, and Dengue Virus

    Directory of Open Access Journals (Sweden)

    Jill M. Perreira

    2015-08-01

    Full Text Available Human rhinovirus (HRV causes upper respiratory infections and asthma exacerbations. We screened multiple orthologous RNAi reagents and identified host proteins that modulate HRV replication. Here, we show that RNASEK, a transmembrane protein, was needed for the replication of HRV, influenza A virus, and dengue virus. RNASEK localizes to the cell surface and endosomal pathway and closely associates with the vacuolar ATPase (V-ATPase proton pump. RNASEK is required for endocytosis, and its depletion produces enlarged clathrin-coated pits (CCPs at the cell surface. These enlarged CCPs contain endocytic cargo and are bound by the scissioning GTPase, DNM2. Loss of RNASEK alters the localization of multiple V-ATPase subunits and lowers the levels of the ATP6AP1 subunit. Together, our results show that RNASEK closely associates with the V-ATPase and is required for its function; its loss prevents the early events of endocytosis and the replication of multiple pathogenic viruses.

  2. Transcription factor genes essential for cell proliferation and replicative lifespan in budding yeast

    Energy Technology Data Exchange (ETDEWEB)

    Kamei, Yuka; Tai, Akiko; Dakeyama, Shota; Yamamoto, Kaori; Inoue, Yamato; Kishimoto, Yoshifumi; Ohara, Hiroya; Mukai, Yukio, E-mail: y_mukai@nagahama-i-bio.ac.jp

    2015-07-31

    Many of the lifespan-related genes have been identified in eukaryotes ranging from the yeast to human. However, there is limited information available on the longevity genes that are essential for cell proliferation. Here, we investigated whether the essential genes encoding DNA-binding transcription factors modulated the replicative lifespan of Saccharomyces cerevisiae. Heterozygous diploid knockout strains for FHL1, RAP1, REB1, and MCM1 genes showed significantly short lifespan. {sup 1}H-nuclear magnetic resonance analysis indicated a characteristic metabolic profile in the Δfhl1/FHL1 mutant. These results strongly suggest that FHL1 regulates the transcription of lifespan related metabolic genes. Thus, heterozygous knockout strains could be the potential materials for discovering further novel lifespan genes. - Highlights: • Involvement of yeast TF genes essential for cell growth in lifespan was evaluated. • The essential TF genes, FHL1, RAP1, REB1, and MCM1, regulate replicative lifespan. • Heterozygous deletion of FHL1 changes cellular metabolism related to lifespan.

  3. Genome-wide CRISPR/Cas9 Screen Identifies Host Factors Essential for Influenza Virus Replication

    Directory of Open Access Journals (Sweden)

    Julianna Han

    2018-04-01

    Full Text Available Summary: The emergence of influenza A viruses (IAVs from zoonotic reservoirs poses a great threat to human health. As seasonal vaccines are ineffective against zoonotic strains, and newly transmitted viruses can quickly acquire drug resistance, there remains a need for host-directed therapeutics against IAVs. Here, we performed a genome-scale CRISPR/Cas9 knockout screen in human lung epithelial cells with a human isolate of an avian H5N1 strain. Several genes involved in sialic acid biosynthesis and related glycosylation pathways were highly enriched post-H5N1 selection, including SLC35A1, a sialic acid transporter essential for IAV receptor expression and thus viral entry. Importantly, we have identified capicua (CIC as a negative regulator of cell-intrinsic immunity, as loss of CIC resulted in heightened antiviral responses and restricted replication of multiple viruses. Therefore, our study demonstrates that the CRISPR/Cas9 system can be utilized for the discovery of host factors critical for the replication of intracellular pathogens. : Using a genome-wide CRISPR/Cas9 screen, Han et al. demonstrate that the major hit, the sialic acid transporter SLC35A1, is an essential host factor for IAV entry. In addition, they identify the DNA-binding transcriptional repressor CIC as a negative regulator of cell-intrinsic immunity. Keywords: CRISPR/Cas9 screen, GeCKO, influenza virus, host factors, sialic acid pathway, SLC35A1, Capicua, CIC, cell-intrinsic immunity, H5N1

  4. Functional characterization of replication and stability factors of an incompatibility group P-1 plasmid from Xylella fastidiosa.

    Science.gov (United States)

    Lee, Min Woo; Rogers, Elizabeth E; Stenger, Drake C

    2010-12-01

    Xylella fastidiosa strain riv11 harbors a 25-kbp plasmid (pXF-RIV11) belonging to the IncP-1 incompatibility group. Replication and stability factors of pXF-RIV11 were identified and used to construct plasmids able to replicate in X. fastidiosa and Escherichia coli. Replication in X. fastidiosa required a 1.4-kbp region from pXF-RIV11 containing a replication initiation gene (trfA) and the adjacent origin of DNA replication (oriV). Constructs containing trfA and oriV from pVEIS01, a related IncP-1 plasmid of the earthworm symbiont Verminephrobacter eiseniae, also were competent for replication in X. fastidiosa. Constructs derived from pXF-RIV11 but not pVEIS01 replicated in Agrobacterium tumefaciens, Xanthomonas campestris, and Pseudomonas syringae. Although plasmids bearing replication elements from pXF-RIV11 or pVEIS01 could be maintained in X. fastidiosa under antibiotic selection, removal of selection resulted in plasmid extinction after 3 weekly passages. Addition of a toxin-antitoxin addiction system (pemI/pemK) from pXF-RIV11 improved plasmid stability such that >80 to 90% of X. fastidiosa cells retained plasmid after 5 weekly passages in the absence of antibiotic selection. Expression of PemK in E. coli was toxic for cell growth, but toxicity was nullified by coexpression of PemI antitoxin. Deletion of N-terminal sequences of PemK containing the conserved motif RGD abolished toxicity. In vitro assays revealed a direct interaction of PemI with PemK, suggesting that antitoxin activity of PemI is mediated by toxin sequestration. IncP-1 plasmid replication and stability factors were added to an E. coli cloning vector to constitute a stable 6.0-kbp shuttle vector (pXF20-PEMIK) suitable for use in X. fastidiosa.

  5. Role of Chromatin assembly factor 1 in DNA replication of Plasmodium falciparum.

    Science.gov (United States)

    Gupta, Mohit Kumar; Agarawal, Meetu; Banu, Khadija; Reddy, K Sony; Gaur, Deepak; Dhar, Suman Kumar

    2018-01-01

    Nucleosome assembly in P. falciparum could be the key process in maintaining its genomic integrity as DNA replicates more than once per cell cycle during several stages of its life cycle. Here, we report the functional characterization of P. falciparum chromatin assembly factor 1 (CAF1), which interacts with several proteins namely PfCAF2, Histones, PfHP1 and others. Consistent with the above findings, we demonstrate the presence of PfCAF1 at the telomeric repeat regions, central and subtelomeric var genes of multiple var gene family along with PfHP1. Further, we report the upregulation of PfCAF1 after treatment with genotoxic agents like MMS and HU. Together, these findings establish role of PfCAF1 in heterochromatin maintenance and as histone chaperone in nucleosome assembly and DNA damage repair. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Identification of eight novel coagulation factor XIII subunit A mutations: implied consequences for structure and function.

    Science.gov (United States)

    Ivaskevicius, Vytautas; Biswas, Arijit; Bevans, Carville; Schroeder, Verena; Kohler, Hans Peter; Rott, Hannelore; Halimeh, Susan; Petrides, Petro E; Lenk, Harald; Krause, Manuele; Miterski, Bruno; Harbrecht, Ursula; Oldenburg, Johannes

    2010-06-01

    Severe hereditary coagulation factor XIII deficiency is a rare homozygous bleeding disorder affecting one person in every two million individuals. In contrast, heterozygous factor XIII deficiency is more common, but usually not associated with severe hemorrhage such as intracranial bleeding or hemarthrosis. In most cases, the disease is caused by F13A gene mutations. Causative mutations associated with the F13B gene are rarer. We analyzed ten index patients and three relatives for factor XIII activity using a photometric assay and sequenced their F13A and F13B genes. Additionally, structural analysis of the wild-type protein structure from a previously reported X-ray crystallographic model identified potential structural and functional effects of the missense mutations. All individuals except one were heterozygous for factor XIIIA mutations (average factor XIII activity 51%), while the remaining homozygous individual was found to have severe factor XIII deficiency (<5% of normal factor XIII activity). Eight of the 12 heterozygous patients exhibited a bleeding tendency upon provocation. The identified missense (Pro289Arg, Arg611His, Asp668Gly) and nonsense (Gly390X, Trp664X) mutations are causative for factor XIII deficiency. A Gly592Ser variant identified in three unrelated index patients, as well as in 200 healthy controls (minor allele frequency 0.005), and two further Tyr167Cys and Arg540Gln variants, represent possible candidates for rare F13A gene polymorphisms since they apparently do not have a significant influence on the structure of the factor XIIIA protein. Future in vitro expression studies of the factor XIII mutations are required to confirm their pathological mechanisms.

  7. The rice eukaryotic translation initiation factor 3 subunit f (OseIF3f is involved in microgametogenesis

    Directory of Open Access Journals (Sweden)

    Qi eLi

    2016-04-01

    Full Text Available Microgametogenesis is the postmeiotic pollen developmental phase when unicellular microspores develop into mature tricellular pollen. In rice, microgametogenesis can influence grain yields to a great degree because pollen abortion occurs more easily during microgametogenesis than during other stages of pollen development. However, our knowledge of the genes involved in microgametogenesis in rice remains limited. Due to the dependence of pollen development on the regulatory mechanisms of protein expression, we identified the encoding gene of the eukaryotic translation initiation factor 3, subunit f in Oryza sativa (OseIF3f. Immunoprecipitation combined with mass spectrometry confirmed that OseIF3f was a subunit of rice eIF3, which consisted of at least 12 subunits including eIF3a, eIF3b, eIF3c, eIF3d, eIF3e, eIF3f, eIF3g, eIF3h, eIF3i, eIF3k, eIF3l and eIF3m. OseIF3f showed high mRNA levels in immature florets and is highly abundant in developing anthers. Subcellular localization analysis showed that OseIF3f was localized to the cytosol and the endoplasmic reticulum in rice root cells. We further analyzed the biological function of OseIF3f using the double-stranded RNA-mediated interference (RNAi approach. The OseIF3f-RNAi lines grew normally at the vegetative stage but displayed a large reduction in seed production and pollen viability, which is associated with the down-regulation of OseIF3f. Further cytological observations of pollen development revealed that the OseIF3f-RNAi lines showed no obvious abnormalities at the male meiotic stage and the unicellular microspore stage. However, compared to the wild type, OseIF3f-RNAi lines contained a higher percentage of arrested unicellular pollen at the bicellular stage and a higher percentage of arrested unicellular and bicellular pollen, and aborted pollen at the tricellular stage. These results indicate that OseIF3f plays a role in microgametogenesis.

  8. The nonenzymatic subunit of pseutarin C, a prothrombin activator from eastern brown snake (Pseudonaja textilis) venom, shows structural similarity to mammalian coagulation factor V.

    Science.gov (United States)

    Rao, Veena S; Swarup, Sanjay; Kini, R Manjunatha

    2003-08-15

    Pseutarin C is a group C prothrombin activator from the venom of the eastern brown snake Pseudonaja textilis. It is a multi-subunit protein complex consisting of catalytic and nonenzymatic subunits similar to coagulation factor Xa and factor Va, respectively. Here we describe the complete sequence of the nonenzymatic subunit. Based on the partial amino acid sequence of the nonenzymatic subunit, degenerate primers were designed. Using a "walking" strategy based on sequentially designed primers, we determined the complete cDNA sequence of the nonenzymatic subunit. The cDNA encodes a protein of 1461 amino acid residues, which includes a 30-residue signal peptide, a mature protein of 1430 amino acid residues, and a stop codon. cDNA blot analysis showed a single transcript of approximately 4.6 kb. The deduced amino acid sequence shows approximately 50% identity to mammalian factor V and by homology has a similar domain structure consisting of domains A1-A2-B-A3-C1-C2. Interestingly, the B domain of pseutarin C is shorter than that of mammalian factor V (FV). Although most of the proteolytic activation sites are conserved, 2 of 3 proteolytic sites cleaved by activated protein C are mutated, and thus activated protein C is not able to inactivate this procoagulant toxin. The predicted posttranslational modifications, including disulfide bonds, N-glycosylation, phosphorylation, and sulfation, in pseutarin C are significantly different compared with bovine factor V. Thus, our data demonstrate that the nonenzymatic subunit of group C prothrombin activators is structurally similar to mammalian FV.

  9. The Mediator Complex MED15 Subunit Mediates Activation of Downstream Lipid-Related Genes by the WRINKLED1 Transcription Factor.

    Science.gov (United States)

    Kim, Mi Jung; Jang, In-Cheol; Chua, Nam-Hai

    2016-07-01

    The Mediator complex is known to be a master coordinator of transcription by RNA polymerase II, and this complex is recruited by transcription factors (TFs) to target promoters for gene activation or repression. The plant-specific TF WRINKLED1 (WRI1) activates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. However, no Mediator subunit has yet been identified that mediates WRI1 transcriptional activity. Promoter-β-glucuronidase fusion experiments showed that MEDIATOR15 (MED15) is expressed in the same cells in the embryo as WRI1. We found that the Arabidopsis (Arabidopsis thaliana) MED15 subunit of the Mediator complex interacts directly with WRI1 in the nucleus. Overexpression of MED15 or WRI1 increased transcript levels of WRI1 target genes involved in glycolysis and fatty acid biosynthesis; these genes were down-regulated in wild-type or WRI1-overexpressing plants by silencing of MED15 However, overexpression of MED15 in the wri1 mutant also increased transcript levels of WRI1 target genes, suggesting that MED15 also may act with other TFs to activate downstream lipid-related genes. Chromatin immunoprecipitation assays confirmed the association of MED15 with six WRI1 target gene promoters. Additionally, silencing of MED15 resulted in reduced fatty acid content in seedlings and mature seeds, whereas MED15 overexpression increased fatty acid content in both developmental stages. Similar results were found in wri1 mutant and WRI1 overexpression lines. Together, our results indicate that the WRI1/MED15 complex transcriptionally regulates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

  10. Isolation of proteins involved in the replication of adenoviral DNA in vitro

    International Nuclear Information System (INIS)

    Lichy, J.H.; Nagata, K.; Friefeld, B.R.; Enomoto, T.; Field, J.; Guggenheimer, R.A.; Ikeda, J.E.; Horwitz, M.S.; Hurwitz, J.

    1983-01-01

    The simple mechanism of replication of adenoviral DNA has made adenovirus an especially useful model system for studies of eukaryotic replication mechanisms. The availability of this in vitro system that replicates exogenously added Ad DNA-pro has made it possible to characterize the factors involved in replication. The results presented in this paper summarize our further fractionation of the in vitro system. First, the properties of two factors purified from the uninfected nuclear extract are described. Second, the separation of the pTP/Ad Pol complex into subunits and the properties of the isolated subunits are presented. The 140K protein is shown to possess the Ad DNA polymerase activity. The results suggest that the only DNA polymerase required for adenoviral DNA replication in vitro is the 140K Ad DNA polymerase and that this enzyme is probably a viral gene product. 50 references, 10 figures, 3 tables

  11. The polyadenylation factor subunit CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30: A key factor of programmed cell death and a regulator of immunity in arabidopsis

    KAUST Repository

    Bruggeman, Quentin

    2014-04-04

    Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. Indeed, incompatible plant-pathogen interactions are well known to induce the hypersensitive response, a localized cell death. Mutational analyses have identified several key PCD components, and we recently identified the mips1 mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for the key enzyme catalyzing the limiting step of myoinositol synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD, revealing roles for myoinositol or inositol derivatives in the regulation of PCD. Here, we identified a regulator of plant PCD by screening for mutants that display transcriptomic profiles opposing that of the mips1 mutant. Our screen identified the oxt6 mutant, which has been described previously as being tolerant to oxidative stress. In the oxt6 mutant, a transfer DNA is inserted in the CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30 (CPSF30) gene, which encodes a polyadenylation factor subunit homolog. We show that CPSF30 is required for lesion formation in mips1 via SA-dependent signaling, that the prodeath function of CPSF30 is not mediated by changes in the glutathione status, and that CPSF30 activity is required for Pseudomonas syringae resistance. We also show that the oxt6 mutation suppresses cell death in other lesion-mimic mutants, including lesion-simulating disease1, mitogen-activated protein kinase4, constitutive expressor of pathogenesis-related genes5, and catalase2, suggesting that CPSF30 and, thus, the control of messenger RNA 3′ end processing, through the regulation of SA production, is a key component of plant immune responses. © 2014 American Society of Plant Biologists. All rights reserved.

  12. Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strains.

    Science.gov (United States)

    Sukhanova, Maria V; D'Herin, Claudine; Boiteux, Serge; Lavrik, Olga I

    2014-10-01

    To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [(32)P]-labeled photoreactive partial DNA duplexes containing a 3'-ss/ds-junction (3'-junction) or a 5'-ss/ds-junction (5'-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3'-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5'-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5'-junction. The results show that RPAp70 crosslinked to DNA with a 5'-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. The translation initiation factor 3 subunit eIF3K interacts with PML and associates with PML nuclear bodies

    Energy Technology Data Exchange (ETDEWEB)

    Salsman, Jayme; Pinder, Jordan; Tse, Brenda [Department of Pathology, Dalhousie University, P.O. Box 15000, Halifax, Nova Scotia, Canada B3H 4R2 (Canada); Corkery, Dale [Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia (Canada); Dellaire, Graham, E-mail: dellaire@dal.ca [Department of Pathology, Dalhousie University, P.O. Box 15000, Halifax, Nova Scotia, Canada B3H 4R2 (Canada); Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia (Canada)

    2013-10-15

    The promyelocytic leukemia protein (PML) is a tumor suppressor protein that regulates a variety of important cellular processes, including gene expression, DNA repair and cell fate decisions. Integral to its function is the ability of PML to form nuclear bodies (NBs) that serve as hubs for the interaction and modification of over 90 cellular proteins. There are seven canonical isoforms of PML, which encode diverse C-termini generated by alternative pre-mRNA splicing. Recruitment of specific cellular proteins to PML NBs is mediated by protein–protein interactions with individual PML isoforms. Using a yeast two-hybrid screen employing peptide sequences unique to PML isoform I (PML-I), we identified an interaction with the eukaryotic initiation factor 3 subunit K (eIF3K), and in the process identified a novel eIF3K isoform, which we term eIF3K-2. We further demonstrate that eIF3K and PML interact both in vitro via pull-down assays, as well as in vivo within human cells by co-immunoprecipitation and co-immunofluorescence. In addition, eIF3K isoform 2 (eIF3K-2) colocalizes to PML bodies, particularly those enriched in PML-I, while eIF3K isoform 1 associates poorly with PML NBs. Thus, we report eIF3K as the first known subunit of the eIF3 translation pre-initiation complex to interact directly with the PML protein, and provide data implicating alternative splicing of both PML and eIF3K as a possible regulatory mechanism for eIF3K localization at PML NBs. - Highlights: • The PML-I C-terminus, encoded by exon 9, interacts with translation factor eIF3K. • We identify a novel eIF3K isoform that excludes exon 2 (eIF3K-2). • eIF3K-2 preferentially associates with PML bodies enriched in PML-I vs. PML-IV. • Alternative splicing of eIF3K regulates association with PML bodies.

  14. Evolution of DNA replication protein complexes in eukaryotes and Archaea.

    Directory of Open Access Journals (Sweden)

    Nicholas Chia

    Full Text Available BACKGROUND: The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA, replication factor C (RFC, and the minichromosome maintenance (MCM complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best. METHODOLOGY/PRINCIPAL FINDINGS: While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex-all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea. CONCLUSION/SIGNIFICANCE: This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota.

  15. Only Three Factors of Personality Description Are Fully Replicable Across Languages: A Comparison of 14 Trait Taxonomies

    Czech Academy of Sciences Publication Activity Database

    De Raad, B.; Barelds, D.P.H.; Levert, E.; Ostendorf, F.; Mlačić, B.; Di Blas, L.; Hřebíčková, Martina; Szirmák, Z.; Szarota, P.; Perugini, M.; Church, A.T.; Katigbak, M. S.

    2010-01-01

    Roč. 98, č. 1 (2010), s. 160-173 ISSN 0022-3514 R&D Projects: GA ČR GA406/07/1561 Institutional research plan: CEZ:AV0Z70250504 Keywords : trait taxonomy * lexical approach to personality * cross - cultural replicability Subject RIV: AN - Psychology Impact factor: 5.205, year: 2010

  16. Evidence that the primary effect of phosphorylation of eukaryotic initiation factor 2(alpha) in rabbit reticulocyte lysate is inhibition of the release of eukaryotic initiation factor-2.GDP from 60 S ribosomal subunits

    International Nuclear Information System (INIS)

    Gross, M.; Redman, R.; Kaplansky, D.A.

    1985-01-01

    The phosphorylation of eukaryotic initiation factor (eIF) 2 alpha that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin or with poly(I.C) causes inhibition of polypeptide chain initiation by preventing a separate factor (termed RF) from promoting the exchange of GTP for GDP on eIF-2. When lysate was incubated in the presence of hemin and [ 14 C] eIF-2 or [alpha- 32 P]GTP, the authors observed binding of eIF-2 and GDP or GTP to 60 S ribosomal subunits that was slightly greater than that bound to 40 S subunits and little binding to 80 S ribosomes. When incubation was in the absence of hemin or in the presence of hemin plus 0.1 microgram/ml poly(I.C), eIF-2 and GDP binding to 60 S subunits was increased 1.5- to 2-fold, that bound to 80 S ribosomes was almost as great as that bound to 60 S subunits, and that bound to 40 S subunits was unchanged. The data indicate that about 40% of the eIF-2 that becomes bound to 60 S subunits and 80 S ribosomes in the absence of hemin or with poly(I.C) is eIF-2(alpha-P) and suggest that the eIF-2 and GDP bound is probably in the form of a binary complex. The rate of turnover of GDP (presumably eIF-2.GDP) on 60 S subunits and 80 S ribosomes in the absence of hemin is reduced to less than 10% the control rate, because the dissociation of eIF-2.GDP is inhibited. Our findings suggest that eIF-2.GTP binding to and eIF-2.GDP release from 60 S subunits may normally occur and serve to promote subunit joining

  17. Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells

    OpenAIRE

    Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J.; Méndez, Ernesto; Arias, Carlos F.

    2015-01-01

    Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellula...

  18. Externalization and recognition by macrophages of large subunit of eukaryotic translation initiation factor 3 in apoptotic cells

    International Nuclear Information System (INIS)

    Nakai, Yuji; Shiratsuchi, Akiko; Manaka, Junko; Nakayama, Hiroshi; Takio, Koji; Zhang Jianting; Suganuma, Tatsuo; Nakanishi, Yoshinobu

    2005-01-01

    We previously isolated a monoclonal antibody named PH2 that inhibits phosphatidylserine-mediated phagocytosis of apoptotic cells by macrophages [C. Fujii, A. Shiratsuchi, J. Manaka, S. Yonehara, Y. Nakanishi. Cell Death Differ. 8 (2001) 1113-1122]. We report here the identification of the cognate antigen. A protein bound by PH2 in Western blotting was identified as the 170-kDa subunit of eukaryotic translation initiation factor 3 (eIF3 p170/eIF3a). When eIF3a was expressed in a culture cell line as a protein fused to green fluorescence protein, the fusion protein was detected at the cell surface only after the induction of apoptosis. The same phenomenon was seen when the localization of endogenous eIF3a was determined using anti-eIF3a antibody, and eIF3a seemed to be partially degraded during apoptosis. Furthermore, bacterially expressed N-terminal half of eIF3a fused to glutathione S-transferase bound to the surface of macrophages and inhibited phagocytosis of apoptotic cells by macrophages when it was added to phagocytosis reactions. These results collectively suggest that eIF3a translocates to the cell surface upon apoptosis, probably after partial degradation, and bridges apoptotic cells and macrophages to enhance phagocytosis

  19. The acid-labile subunit of human ternary insulin-like growth factor binding protein complex in serum

    DEFF Research Database (Denmark)

    Juul, A; Møller, S; Mosfeldt-Laursen, E

    1998-01-01

    Circulating insulin-like growth factor-I (IGF-I) is predominantly bound in the trimeric complex comprised of IGF binding protein-3 (IGFBP-3) and acid-labile subunit (ALS). Circulating concentrations of IGF-I, IGFBP-3 and ALS are believed to reflect the GH secretory status, but the clinical use...... of ALS determination is not known. We therefore, determined the: 1) hepatosplanchnic release of ALS by liver vein catheterization (n=30); 2) 24-h diurnal variation of ALS (n=8); 3) normal age-related ranges of circulating ALS (n=1158); 4) diagnostic value of ALS in 108 patients with childhood-onset GH...... in adults; and 4) ALS levels were below -2 SD in 57 of 79 GHD patients (sensitivity 72%) and above 2 SD in 22 of 29 patients with normal GH response (specificity 76%), which was similar, compared with the diagnostic utility of IGF-I and IGFBP-3. Finally, our findings indicate that hepatic ALS production...

  20. Insulin-like growth factor I has independent effects on bone matrix formation and cell replication

    International Nuclear Information System (INIS)

    Hock, J.M.; Centrella, M.; Canalis, E.

    1988-01-01

    The effects of insulin-like growth factor-I (IGF-I) and insulin on bone matrix synthesis and bone cell replication were studied in cultured 21-day-old fetal rat calvariae. Histomorphometry techniques were developed to measure the incorporation of [2,3- 3 H]proline and [methyl- 3 H]thymidine into bone matrix and bone cell nuclei, respectively, using autoradiographs of sagittal sections of calvariae cultured with IGF-I, insulin, or vehicle for up to 96 h. To confirm an effect on bone formation, IGF-I was also studied for its effects on [ 3 H]proline incorporation into collagenase-digestible protein (CDP) and noncollagen protein and on [ 3 H]thymidine incorporation into acid-precipitable material (DNA). IGF-I at 10(-9)-10(-7) M significantly increased the rate of bone matrix apposition and CDP after 24 h by 45-50% and increased cell labeling by 8-fold in the osteoprogenitor cell zone, by 4-fold in the osteoblast cell zone, and by 2-fold in the periosteal fibroblast zone. Insulin at 10(-9)-10(-6) M also increased matrix apposition rate and CDP by 40-50%, but increased cell labeling by 2-fold only at a concentration of 10(-7) M or higher and then only in the osteoprogenitor cell zone. When hydroxyurea was added to IGF-I-treated bones, the effects of IGF-I on DNA synthesis were abolished, but the increase in bone matrix apposition induced by IGF-I was only partly diminished. In conclusion, IGF-I stimulates matrix synthesis in calvariae, an effect that is partly, although not completely, dependent on its stimulatory effect on DNA synthesis

  1. Eukaryotic translation initiation factor 3 subunit e controls intracellular calcium homeostasis by regulation of cav1.2 surface expression.

    Directory of Open Access Journals (Sweden)

    Pawel Buda

    Full Text Available Inappropriate surface expression of voltage-gated Ca(2+channels (CaV in pancreatic ß-cells may contribute to the development of type 2 diabetes. First, failure to increase intracellular Ca(2+ concentrations at the sites of exocytosis impedes insulin release. Furthermore, excessive Ca(2+ influx may trigger cytotoxic effects. The regulation of surface expression of CaV channels in the pancreatic β-cells remains unknown. Here, we used real-time 3D confocal and TIRFM imaging, immunocytochemistry, cellular fractionation, immunoprecipitation and electrophysiology to study trafficking of L-type CaV1.2 channels upon β-cell stimulation. We found decreased surface expression of CaV1.2 and a corresponding reduction in L-type whole-cell Ca(2+ currents in insulin-secreting INS-1 832/13 cells upon protracted (15-30 min stimulation. This internalization occurs by clathrin-dependent endocytosis and could be prevented by microtubule or dynamin inhibitors. eIF3e (Eukaryotic translation initiation factor 3 subunit E is part of the protein translation initiation complex, but its effect on translation are modest and effects in ion channel trafficking have been suggested. The factor interacted with CaV1.2 and regulated CaV1.2 traffic bidirectionally. eIF3e silencing impaired CaV1.2 internalization, which resulted in an increased intracellular Ca(2+ load upon stimulation. These findings provide a mechanism for regulation of L-type CaV channel surface expression with consequences for β-cell calcium homeostasis, which will affect pancreatic β-cell function and insulin production.

  2. Suppression of gross chromosomal rearrangements by a new alternative replication factor C complex

    International Nuclear Information System (INIS)

    Banerjee, Soma; Sikdar, Nilabja; Myung, Kyungjae

    2007-01-01

    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

  3. Identification of Poxvirus Genome Uncoating and DNA Replication Factors with Mutually Redundant Roles.

    Science.gov (United States)

    Liu, Baoming; Panda, Debasis; Mendez-Rios, Jorge D; Ganesan, Sundar; Wyatt, Linda S; Moss, Bernard

    2018-04-01

    Genome uncoating is essential for replication of most viruses. For poxviruses, the process is divided into two stages: removal of the envelope, allowing early gene expression, and breaching of the core wall, allowing DNA release, replication, and late gene expression. Subsequent studies showed that the host proteasome and the viral D5 protein, which has an essential role in DNA replication, are required for vaccinia virus (VACV) genome uncoating. In a search for additional VACV uncoating proteins, we noted a report that described a defect in DNA replication and late expression when the gene encoding a 68-kDa ankyrin repeat/F-box protein (68k-ank), associated with the cellular SCF (Skp1, cullin1, F-box-containing complex) ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the 68k-ank deletion mutant exhibited diminished genome uncoating, formation of DNA prereplication sites, and degradation of viral cores as well as an additional, independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect, suggesting the existence of compensating genes. By inserting VACV genes into an MVA 68k-ank deletion mutant, we discovered that M2, a member of the poxvirus immune evasion (PIE) domain superfamily and a regulator of NF-κB, and C5, a member of the BTB/Kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in addition to D5. IMPORTANCE Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm, and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may

  4. A conserved helicase processivity factor is needed for conjugation and replication of an integrative and conjugative element.

    Directory of Open Access Journals (Sweden)

    Jacob Thomas

    Full Text Available Integrative and conjugative elements (ICEs are agents of horizontal gene transfer and have major roles in evolution and acquisition of new traits, including antibiotic resistances. ICEs are found integrated in a host chromosome and can excise and transfer to recipient bacteria via conjugation. Conjugation involves nicking of the ICE origin of transfer (oriT by the ICE-encoded relaxase and transfer of the nicked single strand of ICE DNA. For ICEBs1 of Bacillus subtilis, nicking of oriT by the ICEBs1 relaxase NicK also initiates rolling circle replication. This autonomous replication of ICEBs1 is critical for stability of the excised element in growing cells. We found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication of ICEBs1. Our results indicate that this gene, helP (formerly ydcP, encodes a helicase processivity factor that enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA. HelP was required for both conjugation and replication of ICEBs1, and HelP and NicK were the only ICEBs1 proteins needed for replication from ICEBs1 oriT. Using chromatin immunoprecipitation, we measured association of HelP, NicK, PcrA, and the host-encoded single-strand DNA binding protein Ssb with ICEBs1. We found that NicK was required for association of HelP and PcrA with ICEBs1 DNA. HelP was required for association of PcrA and Ssb with ICEBs1 regions distal, but not proximal, to oriT, indicating that PcrA needs HelP to progress beyond nicked oriT and unwind ICEBs1. In vitro, HelP directly stimulated the helicase activity of the PcrA homologue UvrD. Our findings demonstrate that HelP is a helicase processivity factor needed for efficient unwinding of ICEBs1 for conjugation and replication. Homologues of HelP and PcrA-type helicases are encoded on many known and putative ICEs. We propose that these factors are essential for ICE conjugation, replication, and genetic stability.

  5. A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli.

    Science.gov (United States)

    Smith, Corey L; Matheson, Timothy D; Trombly, Daniel J; Sun, Xiaoming; Campeau, Eric; Han, Xuemei; Yates, John R; Kaufman, Paul D

    2014-09-15

    Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element-containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus. © 2014 Smith et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  6. Epidermal growth factor receptor targeting of replication competent adenovirus enhances cytotoxicity in bladder cancer

    NARCIS (Netherlands)

    van der Poel, HG; Molenaar, B; van Beusechem, VW; Haisma, HJ; Rodriguez, R; Curiel, DT; Gerritsen, WR

    Purpose: We evaluated the delivery and oncolytic potential of targeted replication competent adenoviruses in bladder cancer lines. Materials and Methods: Seven established human bladder cancer tumor lines (5637, SW800, TCCsup, J82, Scaber, T24 and 253J) were studied for the expression of integrins

  7. Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells.

    Science.gov (United States)

    Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J; Méndez, Ernesto; Arias, Carlos F

    2015-10-01

    Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients

  8. Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells

    Science.gov (United States)

    Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J.; Méndez, Ernesto

    2015-01-01

    ABSTRACT Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. IMPORTANCE Astroviruses are common etiological agents of acute gastroenteritis in children and

  9. Physical Factors Affecting in Vitro Replication of Foot and Mouth Disease Virus (Serotype “O”

    Directory of Open Access Journals (Sweden)

    Muhammad Taslim Ghori*, Khushi Muhammad and Masood Rabbani1

    2011-10-01

    Full Text Available Effect of physical factors (temperature, pH and UV light on replicating ability of “O” type of Foot and Mouth Disease (FMD virus on Baby Hamster Kidney (BHK cell line was determined. The freshly grown FMD virus containing 106 units of tissue culture infective dose (TCID50 was divided into aliquots. Each of the 9 virus aliquots was exposed to 37, 57 or 77C for 15, 30 or 45 minutes, respectively. Each of the 5 virus aliquots was mixed with MEM-199 maintenance medium having pH 3, 5, 7, 9, or 11. Similarly, each of the 3 aliquots having 1 mm depth of the medium was exposed to ultraviolet light (252.7 nm wavelength: one foot distance for 15, 30 or 45 minutes. Each of the virus aliquot exposed to either of the temperature, pH or ultraviolet light (UV for either of the interaction time was inoculated to 8 wells of the 96-well cell culture plate containing complete monolayer of BHK cell line. One row of 8 wells served as virus control and other row of 8 wells served as control for monolayer of the BHK-21 cell line. The plates were incubated at 37°C for 48 hours. It was observed that temperature of 57 and 77C inactivated the virus within 15 minutes. The virus when admixed in the MEM-199 maintenance medium having pH 3, 5, 9 or 11, of the medium inactivated the virus while pH 7 did not show any detrimental effect on its survival. The ultraviolet light for 15, 30 or 45 minutes showed undetectable effect on survival of the virus as either of the virus aliquot exposed to the UV light for either of the interaction time showed cytopathogenic effects (CPE. It was concluded that the temperature of 57°C or higher for 15 minutes, acidic pH (below 5 or basic pH (more than 9 may inactivate the FMD virus.

  10. Cryo-EM structure of the archaeal 50S ribosomal subunit in complex with initiation factor 6 and implications for ribosome evolution

    DEFF Research Database (Denmark)

    Greber, Basil J; Boehringer, Daniel; Godinic-Mikulcic, Vlatka

    2012-01-01

    additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter...... between this archaeal ribosome and eukaryotic ribosomes but are mostly absent in bacteria and in some archaeal lineages. Furthermore, the structure reveals that, in spite of highly divergent evolutionary trajectories of the ribosomal particle and the acquisition of novel functions of IF6 in eukaryotes......, the molecular binding of IF6 on the ribosome is conserved between eukaryotes and archaea. The structure also provides a snapshot of the reductive evolution of the archaeal ribosome and offers new insights into the evolution of the translation system in archaea....

  11. Characterization of a Novel Class I Transcription Factor A (CITFA) Subunit That Is Indispensable for Transcription by the Multifunctional RNA Polymerase I of Trypanosoma brucei

    KAUST Repository

    Nguyen, T. N.

    2012-10-26

    Trypanosoma brucei is the only organism known to have evolved a multifunctional RNA polymerase I (pol I) system that is used to express the parasite\\'s ribosomal RNAs, as well as its major cell surface antigens, namely, the variant surface glycoprotein (VSG) and procyclin, which are vital for establishing successful infections in the mammalian host and the tsetse vector, respectively. Thus far, biochemical analyses of the T. brucei RNA pol I transcription machinery have elucidated the subunit structure of the enzyme and identified the class I transcription factor A (CITFA). CITFA binds to RNA pol I promoters, and its CITFA-2 subunit was shown to be absolutely essential for RNA pol I transcription in the parasite. Tandem affinity purification (TAP) of CITFA revealed the subunits CITFA-1 to -6, which are conserved only among kinetoplastid organisms, plus the dynein light chain DYNLL1. Here, by tagging CITFA-6 instead of CITFA-2, a complex was purified that contained all known CITFA subunits, as well as a novel proline-rich protein. Functional studies carried out in vivo and in vitro, as well as a colocalization study, unequivocally demonstrated that this protein is a bona fide CITFA subunit, essential for parasite viability and indispensable for RNA pol I transcription of ribosomal gene units and the active VSG expression site in the mammalian-infective life cycle stage of the parasite. Interestingly, CITFA-7 function appears to be species specific, because expression of an RNA interference (RNAi)-resistant CITFA-7 transgene from Trypanosoma cruzi could not rescue the lethal phenotype of silencing endogenous CITFA-7.

  12. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  13. Risk/protective factors among addicted mothers' offspring: a replication study.

    Science.gov (United States)

    Weissman, M M; McAvay, G; Goldstein, R B; Nunes, E V; Verdeli, H; Wickramaratne, P J

    1999-11-01

    There are few systematic studies of the school-aged offspring of drug-dependent patients, although this information is useful for planning evidence-based prevention programs. We have completed such a study, which we compare to a similar study independently conducted in 1998. In both studies, both the parent and offspring were assessed blindly and independently by direct diagnostic interviews, and parental assessment of offspring was also obtained. The similarity in design and methods between studies provided an opportunity for replication by reanalysis of data. The major findings are a replication in two independently conducted studies of school-aged offspring of opiate- and/or cocaine-addicted mothers of the high rates of any psychiatric disorder (60% in both studies), major depression (20%, 26%), oppositional defiant disorder (ODD) (18%, 23%), conduct disorder (17%, 9%), attention-deficit/hyperactivity disorder (ADHD) (13%, 8%), and substance abuse (5%, 10%) among offspring. Both studies also found high rates of comorbid alcohol abuse, depression, and multiple drugs of abuse in the mothers. We conclude that efforts to replicate findings by analyses of independently conducted studies are an inexpensive way to test the sturdiness of findings that can provide the empirical basis for preventive efforts. Clinically, the data in both studies suggest that both drug dependence and associated psychopathology should be assessed and treated in opiate addicts with young offspring, and the offspring should be monitored for the development of conduct and mood disorders and substance use.

  14. Cryo-EM Structure of the Archaeal 50S Ribosomal Subunit in Complex with Initiation Factor 6 and Implications for Ribosome Evolution

    Science.gov (United States)

    Greber, Basil J.; Boehringer, Daniel; Godinic-Mikulcic, Vlatka; Crnkovic, Ana; Ibba, Michael; Weygand-Durasevic, Ivana; Ban, Nenad

    2013-01-01

    Translation of mRNA into proteins by the ribosome is universally conserved in all cellular life. The composition and complexity of the translation machinery differ markedly between the three domains of life. Organisms from the domain Archaea show an intermediate level of complexity, sharing several additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter thermautotrophicus in complex with archaeal IF6 at 6.6 Å resolution using cryo-electron microscopy (EM). The structure provides detailed architectural insights into the 50S ribosomal subunit from a methanogenic archaeon through identification of the rRNA expansion segments and ribosomal proteins that are shared between this archaeal ribosome and eukaryotic ribosomes but are mostly absent in bacteria and in some archaeal lineages. Furthermore, the structure reveals that, in spite of highly divergent evolutionary trajectories of the ribosomal particle and the acquisition of novel functions of IF6 in eukaryotes, the molecular binding of IF6 on the ribosome is conserved between eukaryotes and archaea. The structure also provides a snapshot of the reductive evolution of the archaeal ribosome and offers new insights into the evolution of the translation system in archaea. PMID:22306461

  15. IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN-stimulated genes.

    Science.gov (United States)

    Xu, Lei; Zhou, Xinying; Wang, Wenshi; Wang, Yijin; Yin, Yuebang; Laan, Luc J W van der; Sprengers, Dave; Metselaar, Herold J; Peppelenbosch, Maikel P; Pan, Qiuwei

    2016-10-01

    IFN regulatory factor 1 (IRF1) is one of the most important IFN-stimulated genes (ISGs) in cellular antiviral immunity. Although hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide, how ISGs counteract HEV infection is largely unknown. This study was conducted to investigate the effect of IRF1 on HEV replication. Multiple cell lines were used in 2 models that harbor HEV. In different HEV cell culture systems, IRF1 effectively inhibited HEV replication. IRF1 did not trigger IFN production, and chromatin immunoprecipitation sequencing data analysis revealed that IRF1 bound to the promoter region of signal transducers and activators of transcription 1 (STAT1). Functional assay confirmed that IRF1 could drive the transcription of STAT1, resulting in elevation of total and phosphorylated STAT1 proteins and further activating the transcription of a panel of downstream antiviral ISGs. By pharmacological inhibitors and RNAi-mediated gene-silencing approaches, we revealed that antiviral function of IRF1 is dependent on the JAK-STAT cascade. Furthermore, induction of ISGs and the anti-HEV effect of IRF1 overlapped that of IFNα, but was potentiated by ribavirin. We demonstrated that IRF1 effectively inhibits HEV replication through the activation of the JAK-STAT pathway, and the subsequent transcription of antiviral ISGs, but independent of IFN production.-Xu, L., Zhou, X., Wang, W., Wang, Y., Yin, Y., van der Laan, L. J. W., Sprengers, D., Metselaar, H. J., Peppelenbosch, M. P., Pan, Q. IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN-stimulated genes. © FASEB.

  16. Sulfolobus Replication Factor C stimulates the activity of DNA Polymerase B1

    DEFF Research Database (Denmark)

    Xing, Xuanxuan; Zhang, Likui; Guo, Li

    2014-01-01

    the hyperthermophilic archaea of the genus Sulfolobus physically interacts with DNA polymerase B1 (PolB1) and enhances both the polymerase and 3'-5' exonuclease activities of PolB1 in an ATP-independent manner. Stimulation of the PolB1 activity by RFC is independent of the ability of RFC to bind DNA but is consistent...... with the ability of RFC to facilitate DNA binding by PolB1 through protein-protein interaction. These results suggest that Sulfolobus RFC may play a role in recruiting DNA polymerase for efficient primer extension, in addition to clamp loading, during DNA replication....

  17. Individual and Contextual Factors Influencing Engagement in Learning Activities after Errors at Work: A Replication Study in a German Retail Bank

    Science.gov (United States)

    Leicher, Veronika; Mulder, Regina H.

    2016-01-01

    Purpose: The purpose of this replication study is to identify relevant individual and contextual factors influencing learning from errors at work and to determine if the predictors for learning activities are the same for the domains of nursing and retail banking. Design/methodology/approach: A cross-sectional replication study was carried out in…

  18. Nuclear respiratory factor 2 regulates the expression of the same NMDA receptor subunit genes as NRF-1: both factors act by a concurrent and parallel mechanism to couple energy metabolism and synaptic transmission.

    Science.gov (United States)

    Priya, Anusha; Johar, Kaid; Wong-Riley, Margaret T T

    2013-01-01

    Neuronal activity and energy metabolism are tightly coupled processes. Previously, we found that nuclear respiratory factor 1 (NRF-1) transcriptionally co-regulates energy metabolism and neuronal activity by regulating all 13 subunits of the critical energy generating enzyme, cytochrome c oxidase (COX), as well as N-methyl-d-aspartate (NMDA) receptor subunits 1 and 2B, GluN1 (Grin1) and GluN2B (Grin2b). We also found that another transcription factor, nuclear respiratory factor 2 (NRF-2 or GA-binding protein) regulates all subunits of COX as well. The goal of the present study was to test our hypothesis that NRF-2 also regulates specific subunits of NMDA receptors, and that it functions with NRF-1 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation of mouse neuroblastoma cells and rat visual cortical tissue, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate Grin1 and Grin2b genes, but not any other NMDA subunit genes. Grin1 and Grin2b transcripts were up-regulated by depolarizing KCl, but silencing of NRF-2 prevented this up-regulation. On the other hand, over-expression of NRF-2 rescued the down-regulation of these subunits by the impulse blocker TTX. NRF-2 binding sites on Grin1 and Grin2b are conserved among species. Our data indicate that NRF-2 and NRF-1 operate in a concurrent and parallel manner in mediating the tight coupling between energy metabolism and neuronal activity at the molecular level. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Fission yeast cdc24(+) encodes a novel replication factor required for chromosome integrity.

    Science.gov (United States)

    Gould, K L; Burns, C G; Feoktistova, A; Hu, C P; Pasion, S G; Forsburg, S L

    1998-07-01

    A mutation within the Schizosaccharomyces pombe cdc24(+) gene was identified previously in a screen for cell division cycle mutants and the cdc24(+) gene was determined to be essential for S phase in this yeast. We have isolated the cdc24(+) gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1. The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD. A cdc24 null mutant was generated by homologous recombination. Haploid cells lacking cdc24(+) are inviable, indicating that cdc24(+) is an essential gene. The transcript of cdc24(+) is present at constant levels throughout the cell cycle. Cells lacking cdc24(+) function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase. Arrest is accompanied by a rapid loss of viability and chromosome breakage. An S. pombe homolog of the replicative DNA helicase DNA2 of S. cerevisiae suppresses cdc24. These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity.

  20. Activation of the Hypoxia Inducible FactorSubunit Pathway in Steatotic Liver Contributes to Formation of Cholesterol Gallstones.

    Science.gov (United States)

    Asai, Yoichiro; Yamada, Tetsuya; Tsukita, Sohei; Takahashi, Kei; Maekawa, Masamitsu; Honma, Midori; Ikeda, Masanori; Murakami, Keigo; Munakata, Yuichiro; Shirai, Yuta; Kodama, Shinjiro; Sugisawa, Takashi; Chiba, Yumiko; Kondo, Yasuteru; Kaneko, Keizo; Uno, Kenji; Sawada, Shojiro; Imai, Junta; Nakamura, Yasuhiro; Yamaguchi, Hiroaki; Tanaka, Kozo; Sasano, Hironobu; Mano, Nariyasu; Ueno, Yoshiyuki; Shimosegawa, Tooru; Katagiri, Hideki

    2017-05-01

    Hypoxia-inducible factorsubunit (HIF1A) is a transcription factor that controls the cellular response to hypoxia and is activated in hepatocytes of patients with nonalcoholic fatty liver disease (NAFLD). NAFLD increases the risk for cholesterol gallstone disease by unclear mechanisms. We studied the relationship between HIF1A and gallstone formation associated with liver steatosis. We performed studies with mice with inducible disruption of Hif1a in hepatocytes via a Cre adenoviral vector (inducible hepatocyte-selective HIF1A knockout [iH-HIFKO] mice), and mice without disruption of Hif1a (control mice). Mice were fed a diet rich in cholesterol and cholate for 1 or 2 weeks; gallbladders were collected and the number of gallstones was determined. Livers and biliary tissues were analyzed by histology, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and immunoblots. We measured concentrations of bile acid, cholesterol, and phospholipid in bile and rates of bile flow. Primary hepatocytes and cholangiocytes were isolated and analyzed. HIF1A was knocked down in Hepa1-6 cells with small interfering RNAs. Liver biopsy samples from patients with NAFLD, with or without gallstones, were analyzed by quantitative reverse-transcription polymerase chain reaction. Control mice fed a diet rich in cholesterol and cholate developed liver steatosis with hypoxia; levels of HIF1A protein were increased in hepatocytes around central veins and 90% of mice developed cholesterol gallstones. Only 20% of the iH-HIFKO mice developed cholesterol gallstones. In iH-HIFKO mice, the biliary lipid concentration was reduced by 36%, compared with control mice, and bile flow was increased by 35%. We observed increased water secretion from hepatocytes into bile canaliculi to mediate these effects, resulting in suppression of cholelithogenesis. Hepatic expression of aquaporin 8 (AQP8) protein was 1.5-fold higher in iH-HIFKO mice than in control mice. Under hypoxic

  1. Identification of a domain within human TAF(I)48, a subunit of Selectivity Factor 1, that interacts with helix 2 of TBP.

    Science.gov (United States)

    Xu, Shuping; Hori, Roderick T

    2004-09-01

    RNA polymerase I transcription in human cells requires Selectivity Factor 1, a multisubunit complex composed of the TATA-box-binding protein (TBP) and three TBP-associated factors (TAFs) called TAF(I)48, TAF(I)63 and TAF(I)110. Each of the Selectivity Factor 1 subunits binds directly to the other three components, but these interactions have not been characterized. This study is the initial identification and analysis of a TBP-binding domain within a Selectivity Factor 1 TAF. The interaction between human TBP and human TAF(I)48 was initially examined using the yeast two-hybrid assay, and a TBP-binding domain was identified in the carboxyl-terminus of human (h)TAF(I)48. Consistent with this result, the hTAF(I)48 carboxyl-terminus was able to bind directly to TBP in protein-protein interaction assays. When mutations were introduced into the hTAF(I)48 carboxyl-terminus, we identified changes in uncharged and positive residues that affect its interaction with TBP. By examining TBP mutants, residues within and adjacent to helix 2 of TBP, previously demonstrated to interact with subunits of other TBP-containing complexes [Transcription Factor IID (TFIID) and TFIIIB] were also found to diminish its affinity for the carboxyl-terminus of hTAF(I)48. The regions of hTAF(I)48 and TBP that interact are compared to those identified within other complexes containing TBP.

  2. Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors.

    Directory of Open Access Journals (Sweden)

    Sachin Gupta

    Full Text Available Type I interferon is known to inhibit HIV-1 replication through the induction of interferon stimulated genes (ISG, including a number of HIV-1 restriction factors. To better understand interferon-mediated HIV-1 restriction, we constructed a constitutively active form of the RIG-I adapter protein MAVS. Constitutive MAVS was generated by fusion of full length MAVS to a truncated form of the Epstein Barr virus protein LMP1 (ΔLMP1. Supernatant from ΔLMP1-MAVS-transfected 293T cells contained high levels of type I interferons and inhibited HIV replication in both TZM-bl and primary human CD4+ T cells. Supernatant from ΔLMP1-MAVS-transfected 293T cells also inhibited replication of VSV-G pseudotyped single cycle SIV in TZM-bl cells, suggesting restriction was post-entry and common to both HIV and SIV. Gene array analysis of ΔLMP1-MAVS-transfected 293T cells and trans-activated CD4+ T cells showed significant upregulation of ISG, including previously characterized HIV restriction factors Viperin, Tetherin, MxB, and ISG56. Interferon blockade studies implicated interferon-beta in this response. In addition to direct viral inhibition, ΔLMP1-MAVS markedly enhanced secretion of IFN-β and IL-12p70 by dendritic cells and the activation and maturation of dendritic cells. Based on this immunostimulatory activity, an adenoviral vector (Ad5 expressing ΔLMP1-MAVS was tested as a molecular adjuvant in an HIV vaccine mouse model. Ad5-Gag antigen combined with Ad5-ΔLMP1-MAVS enhanced control of vaccinia-gag replication in a mouse challenge model, with 4/5 animals showing undetectable virus following challenge. Overall, ΔLMP1-MAVS is a promising reagent to inhibit HIV-1 replication in infected tissues and enhance vaccine-mediated immune responses, while avoiding toxicity associated with systemic type I interferon administration.

  3. Inhibition of Avian Influenza A Virus Replication in Human Cells by Host Restriction Factor TUFM Is Correlated with Autophagy.

    Science.gov (United States)

    Kuo, Shu-Ming; Chen, Chi-Jene; Chang, Shih-Cheng; Liu, Tzu-Jou; Chen, Yi-Hsiang; Huang, Sheng-Yu; Shih, Shin-Ru

    2017-06-13

    Avian influenza A viruses generally do not replicate efficiently in human cells, but substitution of glutamic acid (Glu, E) for lysine (Lys, K) at residue 627 of avian influenza virus polymerase basic protein 2 (PB2) can serve to overcome host restriction and facilitate human infectivity. Although PB2 residue 627 is regarded as a species-specific signature of influenza A viruses, host restriction factors associated with PB2 627 E have yet to be fully investigated. We conducted immunoprecipitation, followed by differential proteomic analysis, to identify proteins associating with PB2 627 K (human signature) and PB2 627 E (avian signature) of influenza A/WSN/1933(H1N1) virus, and the results indicated that Tu elongation factor, mitochondrial (TUFM), had a higher binding affinity for PB2 627 E than PB2 627 K in transfected human cells. Stronger binding of TUFM to avian-signature PB2 590 G/ 591 Q and PB2 627 E in the 2009 swine-origin pandemic H1N1 and 2013 avian-origin H7N9 influenza A viruses was similarly observed. Viruses carrying avian-signature PB2 627 E demonstrated increased replication in TUFM-deficient cells, but viral replication decreased in cells overexpressing TUFM. Interestingly, the presence of TUFM specifically inhibited the replication of PB2 627 E viruses, but not PB2 627 K viruses. In addition, enhanced levels of interaction between TUFM and PB2 627 E were noted in the mitochondrial fraction of infected cells. Furthermore, TUFM-dependent autophagy was reduced in TUFM-deficient cells infected with PB2 627 E virus; however, autophagy remained consistent in PB2 627 K virus-infected cells. The results suggest that TUFM acts as a host restriction factor that impedes avian-signature influenza A virus replication in human cells in a manner that correlates with autophagy. IMPORTANCE An understanding of the mechanisms that influenza A viruses utilize to shift host tropism and the identification of host restriction factors that can limit infection are both

  4. Insights into the Initiation of Eukaryotic DNA Replication.

    Science.gov (United States)

    Bruck, Irina; Perez-Arnaiz, Patricia; Colbert, Max K; Kaplan, Daniel L

    2015-01-01

    The initiation of DNA replication is a highly regulated event in eukaryotic cells to ensure that the entire genome is copied once and only once during S phase. The primary target of cellular regulation of eukaryotic DNA replication initiation is the assembly and activation of the replication fork helicase, the 11-subunit assembly that unwinds DNA at a replication fork. The replication fork helicase, called CMG for Cdc45-Mcm2-7, and GINS, assembles in S phase from the constituent Cdc45, Mcm2-7, and GINS proteins. The assembly and activation of the CMG replication fork helicase during S phase is governed by 2 S-phase specific kinases, CDK and DDK. CDK stimulates the interaction between Sld2, Sld3, and Dpb11, 3 initiation factors that are each required for the initiation of DNA replication. DDK, on the other hand, phosphorylates the Mcm2, Mcm4, and Mcm6 subunits of the Mcm2-7 complex. Sld3 recruits Cdc45 to Mcm2-7 in a manner that depends on DDK, and recent work suggests that Sld3 binds directly to Mcm2-7 and also to single-stranded DNA. Furthermore, recent work demonstrates that Sld3 and its human homolog Treslin substantially stimulate DDK phosphorylation of Mcm2. These data suggest that the initiation factor Sld3/Treslin coordinates the assembly and activation of the eukaryotic replication fork helicase by recruiting Cdc45 to Mcm2-7, stimulating DDK phosphorylation of Mcm2, and binding directly to single-stranded DNA as the origin is melted.

  5. Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus.

    Directory of Open Access Journals (Sweden)

    Vidya P Nair

    2016-04-01

    Full Text Available Hepatitis E virus (HEV causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4. Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp, X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1 and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient

  6. Negative parental attributions mediate associations between risk factors and dysfunctional parenting: A replication and extension.

    Science.gov (United States)

    Beckerman, Marieke; van Berkel, Sheila R; Mesman, Judi; Alink, Lenneke R A

    2018-05-12

    The primary goal of the current study was to replicate our previous study in which was found that negative maternal attributions mediate the association between parenting stress and harsh and abusive discipline. In addition, we investigated this association in fathers, and added observational parenting data. During two home visits mothers and fathers were observed with their children (age 1.5-6.0 years), filled in questionnaires, and completed the Parental Attributions of Child behavior Task (PACT; a computerized attribution task). Similar to our previous study, negative parental attributions mediated the relation between parenting stress and self-reported harsh and abusive parenting for both mothers and fathers. For mothers, this mediation effect was also found in the relation between parenting stress and lower levels of observed supportive parenting in a challenging disciplinary task. In addition, the relation of partner-related stress and abuse risk with harsh, abusive, and (low) supportive parenting were also mediated by maternal negative attributions. When parenting stress, partner-related stress, and abuse risk were studied in one model, only parenting stress remained significant. Results are discussed in terms of the importance of targeting parental attributions for prevention and intervention purposes in families experiencing stress. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Validation-based insertional mutagenesis for identification of Nup214 as a host factor for EV71 replication in RD cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Bei; Zhang, XiaoYu; Zhao, Zhendong, E-mail: timjszzd@163.com

    2013-08-02

    Highlights: •We introduced a new mutagenesis strategy named VBIM to the viral research. •This method can identify either host factors or host restriction factors. •Using VBIM system, we identified Nup214 as a host factor for EV71 replication in RD cells. -- Abstract: Lentiviral validation-based insertional mutagenesis (VBIM) is a sophisticated, forward genetic approach that is used for the investigation of signal transduction in mammalian cells. Using VBIM, we conducted function-based genetic screening for host genes that affect enterovirus 71 (EV71) viral replication. This included host factors that are required for the life cycle of EV71 and host restriction factors that inhibit EV71 replication. Several cell clones, resistant to EV71, were produced using EV71 infection as a selection pressure and the nuclear pore protein 214 (Nup214) was identified as a host factor required for EV71 replication. In SD2-2, the corresponding VBIM lentivirus transformed clone, the expression of endogenous Nup214 was significantly down-regulated by the reverse inserted VBIM promoter. After Cre recombinase-mediated excision of the VBIM promoter, the expression of Nup214 recovered and the clone regained sensitivity to the EV71 infection. Furthermore, over-expression of Nup214 in the cells suggested that Nup214 was promoting EV71 replication. Results of this study indicate that a successful mutagenesis strategy has been established for screening host genes related to viral replication.

  8. Validation-based insertional mutagenesis for identification of Nup214 as a host factor for EV71 replication in RD cells

    International Nuclear Information System (INIS)

    Wang, Bei; Zhang, XiaoYu; Zhao, Zhendong

    2013-01-01

    Highlights: •We introduced a new mutagenesis strategy named VBIM to the viral research. •This method can identify either host factors or host restriction factors. •Using VBIM system, we identified Nup214 as a host factor for EV71 replication in RD cells. -- Abstract: Lentiviral validation-based insertional mutagenesis (VBIM) is a sophisticated, forward genetic approach that is used for the investigation of signal transduction in mammalian cells. Using VBIM, we conducted function-based genetic screening for host genes that affect enterovirus 71 (EV71) viral replication. This included host factors that are required for the life cycle of EV71 and host restriction factors that inhibit EV71 replication. Several cell clones, resistant to EV71, were produced using EV71 infection as a selection pressure and the nuclear pore protein 214 (Nup214) was identified as a host factor required for EV71 replication. In SD2-2, the corresponding VBIM lentivirus transformed clone, the expression of endogenous Nup214 was significantly down-regulated by the reverse inserted VBIM promoter. After Cre recombinase-mediated excision of the VBIM promoter, the expression of Nup214 recovered and the clone regained sensitivity to the EV71 infection. Furthermore, over-expression of Nup214 in the cells suggested that Nup214 was promoting EV71 replication. Results of this study indicate that a successful mutagenesis strategy has been established for screening host genes related to viral replication

  9. Interaction between FMDV Lpro and transcription factor ADNP is required for viral replication

    Science.gov (United States)

    The foot-and-mouth disease virus (FMDV) leader protease (Lpro) inhibits host translation and transcription affecting the expression of several factors involved in innate immunity. In this study, we have identified the host transcription factor ADNP (activity dependent neuroprotective protein) as an ...

  10. Identification of rep-associated factors in herpes simplex virus type 1-induced adeno-associated virus type 2 replication compartments.

    Science.gov (United States)

    Nicolas, Armel; Alazard-Dany, Nathalie; Biollay, Coline; Arata, Loredana; Jolinon, Nelly; Kuhn, Lauriane; Ferro, Myriam; Weller, Sandra K; Epstein, Alberto L; Salvetti, Anna; Greco, Anna

    2010-09-01

    Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase delta was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.

  11. A Novel, Broad-Spectrum Inhibitor of Enterovirus Replication That Targets Host Cell Factor Phosphatidylinositol 4-Kinase IIIβ

    Science.gov (United States)

    van der Schaar, Hilde M.; Leyssen, Pieter; Thibaut, Hendrik J.; de Palma, Armando; van der Linden, Lonneke; Lanke, Kjerstin H. W.; Lacroix, Céline; Verbeken, Erik; Conrath, Katja; MacLeod, Angus M.; Mitchell, Dale R.; Palmer, Nicholas J.; van de Poël, Hervé; Andrews, Martin

    2013-01-01

    Despite their high clinical and socioeconomic impacts, there is currently no approved antiviral therapy for the prophylaxis or treatment of enterovirus infections. Here we report on a novel inhibitor of enterovirus replication, compound 1, 2-fluoro-4-(2-methyl-8-(3-(methylsulfonyl)benzylamino)imidazo[1,2-a]pyrazin-3-yl)phenol. This compound exhibited a broad spectrum of antiviral activity, as it inhibited all tested species of enteroviruses and rhinoviruses, with 50% effective concentrations ranging between 4 and 71 nM. After a lengthy resistance selection process, coxsackievirus mutants resistant to compound 1 were isolated that carried substitutions in their 3A protein. Remarkably, the same substitutions were recently shown to provide resistance to inhibitors of phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ), a lipid kinase that is essential for enterovirus replication, suggesting that compound 1 may also target this host factor. Accordingly, compound 1 directly inhibited PI4KIIIβ in an in vitro kinase activity assay. Furthermore, the compound strongly reduced the PI 4-phosphate levels of the Golgi complex in cells. Rescue of coxsackievirus replication in the presence of compound 1 by a mutant PI4KIIIβ carrying a substitution in its ATP-binding pocket revealed that the compound directly binds the kinase at this site. Finally, we determined that an analogue of compound 1, 3-(3-fluoro-4-methoxyphenyl)-2-methyl-N-(pyridin-4-ylmethyl)imidazo[1,2-a]pyrazin-8-amine, is well tolerated in mice and has a dose-dependent protective activity in a coxsackievirus serotype B4-induced pancreatitis model. PMID:23896472

  12. Distributional Replication

    OpenAIRE

    Beare, Brendan K.

    2009-01-01

    Suppose that X and Y are random variables. We define a replicating function to be a function f such that f(X) and Y have the same distribution. In general, the set of replicating functions for a given pair of random variables may be infinite. Suppose we have some objective function, or cost function, defined over the set of replicating functions, and we seek to estimate the replicating function with the lowest cost. We develop an approach to estimating the cheapest replicating function that i...

  13. Validation of reported genetic risk factors for periodontitis in a large-scale replication study

    NARCIS (Netherlands)

    Schaefer, A.S.; Bochenek, G.; Manke, T.; Nothnagel, M.; Graetz, C.; Thien, A.; Jockel-Schneider, Y.; Harks, I.; Staufenbiel, I.; Wijmenga, C.; Eberhard, J.; Guzeldemir-Akcakanat, E.; Cine, N.; Folwaczny, M.; Noack, B.; Meyle, J.; Eickholz, P.; Trombelli, L.; Scapoli, C.; Nohutcu, R.; Bruckmann, C.; Doerfer, C.; Jepsen, S.; Loos, B.G.; Schreiber, S.

    2013-01-01

    Aim Many studies investigated the role of genetic variants in periodontitis, but few were established as risk factors. We aimed to validate the associations of recent candidate genes in aggressive periodontitis (AgP). Material and Methods We analysed 23 genes in 600 German AgP patients and 1441

  14. Validation of reported genetic risk factors for periodontitis in a large-scale replication study

    NARCIS (Netherlands)

    Schaefer, Arne S.; Bochenek, Gregor; Manke, Thomas; Nothnagel, Michael; Graetz, Christian; Thien, Anneke; Jockel-Schneider, Yvonne; Harks, Inga; Staufenbiel, Ingmar; Wijmenga, Cisca; Eberhard, Joerg; Guzeldemir-Akcakanat, Esra; Cine, Naci; Folwaczny, Mathias; Noack, Barbara; Meyle, Joerg; Eickholz, Peter; Trombelli, Leonardo; Scapoli, Chiara; Nohutcu, Rahime; Bruckmann, Corinna; Doerfer, Christof; Jepsen, Soren; Loos, Bruno G.; Schreiber, Stefan

    Aim Many studies investigated the role of genetic variants in periodontitis, but few were established as risk factors. We aimed to validate the associations of recent candidate genes in aggressive periodontitis (AgP). Material and Methods We analysed 23 genes in 600 German AgP patients and 1441

  15. The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of α2δ subunits is key to trafficking voltage-gated Ca2+ channels

    Science.gov (United States)

    Cantí, C.; Nieto-Rostro, M.; Foucault, I.; Heblich, F.; Wratten, J.; Richards, M. W.; Hendrich, J.; Douglas, L.; Page, K. M.; Davies, A.; Dolphin, A. C.

    2005-01-01

    All auxiliary α2δ subunits of voltage-gated Ca2+ (CaV) channels contain an extracellular Von Willebrand factor-A (VWA) domain that, in α2δ-1 and -2, has a perfect metal-ion-dependent adhesion site (MIDAS). Modeling of the α2δ-2 VWA domain shows it to be highly likely to bind a divalent cation. Mutating the three key MIDAS residues responsible for divalent cation binding resulted in a MIDAS mutant α2δ-2 subunit that was still processed and trafficked normally when it was expressed alone. However, unlike WT α2δ-2, the MIDAS mutant α2δ-2 subunit did not enhance and, in some cases, further diminished CaV1.2, -2.1, and -2.2 currents coexpressed with β1b by using either Ba2+ or Na+ as a permeant ion. Furthermore, expression of the MIDAS mutant α2δ-2 reduced surface expression and strongly increased the perinuclear retention of CaVα1 subunits at the earliest time at which expression was observed in both Cos-7 and NG108–15 cells. Despite the presence of endogenous α2δ subunits, heterologous expression of α2δ-2 in differentiated NG108–15 cells further enhanced the endogenous high-threshold Ca2+ currents, whereas this enhancement was prevented by the MIDAS mutations. Our results indicate that α2δ subunits normally interact with the CaVα1 subunit early in their maturation, before the appearance of functional plasma membrane channels, and an intact MIDAS motif in the α2δ subunit is required to promote trafficking of the α1 subunit to the plasma membrane by an integrin-like switch. This finding provides evidence for a primary role of a VWA domain in intracellular trafficking of a multimeric complex, in contrast to the more usual roles in binding extracellular ligands in other exofacial VWA domains. PMID:16061813

  16. In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA.

    Directory of Open Access Journals (Sweden)

    Janet L Smith

    2015-05-01

    Full Text Available DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq. We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.

  17. Stimulation of casein kinase II by epidermal growth factor: Relationship between the physiological activity of the kinase and the phosphorylation state of its beta subunit

    International Nuclear Information System (INIS)

    Ackerman, P.; Osheroff, N.; Glover, C.V.C.

    1990-01-01

    To determine relationships between the hormonal activation of casein kinase II and its phosphorylation state, epidermal growth factor (EGF)-treated and EGF-naive human A-431 carcinoma cells were cultured in the presence of [ 32 P]orthophosphate. Immunoprecipitation experiments indicated that casein kinase II in the cytosol of EGF-treated cells contained approximately 3-fold more incorporated [ 32 P]phosphate than did its counterpart in untreated cells. Levels of kinase phosphorylation paralleled levels of kinase activity over a wide range of EGF concentrations as well as over a time course of hormone action. Approximately 97% of the incorporated [ 32 P]phosphate was found in the β subunit of casein kinase II. Both activated and hormone-naive kinase contained radioactive phosphoserine and phosphothreonine but no phosphotyronsine. On the basis of proteolytic mapping experiments, EGF treatment of A-431 cells led to an increase in the average [ 32 P]phosphate content (i.e., hyperphosphorylation) of casein kinase II β subunit peptides which were modified prior to hormone treatment. Finally, the effect of alkaline phosphatase on the reaction kinetics of activated casein kinase II indicated that hormonal stimulation of the kinase resulted from the increase in its phosphorylation state

  18. msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding.

    Directory of Open Access Journals (Sweden)

    Anil Raj

    Full Text Available Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede.

  19. msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding.

    Science.gov (United States)

    Raj, Anil; Shim, Heejung; Gilad, Yoav; Pritchard, Jonathan K; Stephens, Matthew

    2015-01-01

    Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede.

  20. A DEAD box protein facilitates HIV-1 replication as a cellular co-factor of Rev

    International Nuclear Information System (INIS)

    Fang Jianhua; Kubota, Satoshi; Yang Bin; Zhou Naiming; Zhang Hui; Godbout, Roseline; Pomerantz, Roger J.

    2004-01-01

    HIV-1 Rev escorts unspliced viral mRNAs out of the nucleus of infected cells, which allows formation of infectious HIV-1 virions. We have identified a putative DEAD box (Asp-Glu-Ala-Asp) RNA helicase, DDX1, as a cellular co-factor of Rev, through yeast and mammalian two-hybrid systems using the N-terminal motif of Rev as 'bait'. DDX1 is not a functional homolog of HIV-1 Rev, but down-regulation of DDX1 resulted in an alternative splicing pattern of Rev-responsive element (RRE)-containing mRNA, and attenuation of Gag p24 antigen production from HLfb rev(-) cells rescued by exogenous Rev. Co-transfection of a DDX1 expression vector with HIV-1 significantly increased viral production. DDX1 binding to Rev, as well as to the RRE, strongly suggest that DDX1 affects Rev function through the Rev-RRE axis. Moreover, down-regulation of DDX1 altered the steady state subcellular distribution of Rev, from nuclear/nucleolar to cytoplasmic dominance. These findings indicate that DDX1 is a critical cellular co-factor for Rev function, which maintains the proper subcellular distribution of this lentiviral regulatory protein. Therefore, alterations in DDX1-Rev interactions could induce HIV-1 persistence and targeting DDX1 may lead to rationally designed and novel anti-HIV-1 strategies and therapeutics

  1. Overexpression of insulin-like growth factor (IGF)-I receptor enhances inhibition of DNA replication in mouse cells exposed to x-rays

    International Nuclear Information System (INIS)

    Wang, Y.; Cheong, N.; Miura, M.; Iliakis, G.

    1997-01-01

    Previous studies from our laboratory provided evidence for the operation of signal transduction pathways involving ras, myc, and staurosporine-sensitive protein kinases in the regulation of DNA replication in irradiated cells. Because ras and myc are also involved in the signal transduction elicited in response to ligand activation of growth factor receptors, we wondered whether growth factor receptors are upstream elements in the regulation of DNA replication in irradiated cells. Here, we report on the role of insulin-like growth factor I receptor (IGF-IR) in the regulation of DNA replication in irradiated cells. We compare radiation-induced inhibition of DNA replication in BALB/c 3T3 cells with that in P6 cells. P6 cells are derived from BALB/c 3T3 cells by transfection with a vector expressing IGF-IR, leading to 30-fold overexpression. We observe a significantly stronger inhibition of DNA replication after irradiation in P6 as compared with BALB/c 3T3 cells at all doses examined. Sedimentation in alkaline sucrose gradients shows that the increased inhibition in P6 cells is due to an increased inhibition of replicon initiation, the main controlling event in DNA replication. Staurosporine at 20 nM reduces radiation-induced inhibition of DNA replication in BALB/c 3T3 cells, but has only a small effect in P6 cells. Caffeine at a concentration of 1 mM, on the other hand, removes over 60% of the inhibition in both cell lines. The results implicate IGF-IR in the regulation of DNA replication in irradiated cells, but also suggest differences between cells of different origins in the proteins involved in the regulating signal transduction pathway. (orig.). With 5 figs

  2. The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication.

    Science.gov (United States)

    de Chassey, Benoît; Aublin-Gex, Anne; Ruggieri, Alessia; Meyniel-Schicklin, Laurène; Pradezynski, Fabrine; Davoust, Nathalie; Chantier, Thibault; Tafforeau, Lionel; Mangeot, Philippe-Emmanuel; Ciancia, Claire; Perrin-Cocon, Laure; Bartenschlager, Ralf; André, Patrice; Lotteau, Vincent

    2013-01-01

    Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

  3. The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication.

    Directory of Open Access Journals (Sweden)

    Benoît de Chassey

    Full Text Available Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1 appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

  4. Mechanisms of bacterial DNA replication restart

    Science.gov (United States)

    Windgassen, Tricia A; Wessel, Sarah R; Bhattacharyya, Basudeb

    2018-01-01

    Abstract Multi-protein DNA replication complexes called replisomes perform the essential process of copying cellular genetic information prior to cell division. Under ideal conditions, replisomes dissociate only after the entire genome has been duplicated. However, DNA replication rarely occurs without interruptions that can dislodge replisomes from DNA. Such events produce incompletely replicated chromosomes that, if left unrepaired, prevent the segregation of full genomes to daughter cells. To mitigate this threat, cells have evolved ‘DNA replication restart’ pathways that have been best defined in bacteria. Replication restart requires recognition and remodeling of abandoned replication forks by DNA replication restart proteins followed by reloading of the replicative DNA helicase, which subsequently directs assembly of the remaining replisome subunits. This review summarizes our current understanding of the mechanisms underlying replication restart and the proteins that drive the process in Escherichia coli (PriA, PriB, PriC and DnaT). PMID:29202195

  5. Replication Catastrophe

    DEFF Research Database (Denmark)

    Toledo, Luis; Neelsen, Kai John; Lukas, Jiri

    2017-01-01

    Proliferating cells rely on the so-called DNA replication checkpoint to ensure orderly completion of genome duplication, and its malfunction may lead to catastrophic genome disruption, including unscheduled firing of replication origins, stalling and collapse of replication forks, massive DNA...... breakage, and, ultimately, cell death. Despite many years of intensive research into the molecular underpinnings of the eukaryotic replication checkpoint, the mechanisms underlying the dismal consequences of its failure remain enigmatic. A recent development offers a unifying model in which the replication...... checkpoint guards against global exhaustion of rate-limiting replication regulators. Here we discuss how such a mechanism can prevent catastrophic genome disruption and suggest how to harness this knowledge to advance therapeutic strategies to eliminate cancer cells that inherently proliferate under...

  6. QTL replication and targeted association highlight the nerve growth factor gene for nonverbal communication deficits in autism spectrum disorders.

    Science.gov (United States)

    Lu, A T-H; Yoon, J; Geschwind, D H; Cantor, R M

    2013-02-01

    Autism Spectrum Disorder (ASD) has a heterogeneous etiology that is genetically complex. It is defined by deficits in communication and social skills and the presence of restricted and repetitive behaviors. Genetic analyses of heritable quantitative traits that correlate with ASD may reduce heterogeneity. With this in mind, deficits in nonverbal communication (NVC) were quantified based on items from the Autism Diagnostic Interview Revised. Our previous analysis of 228 families from the Autism Genetics Research Exchange (AGRE) repository reported 5 potential quantitative trait loci (QTL). Here we report an NVC QTL replication study in an independent sample of 213 AGRE families. One QTL was replicated (Panalysis of 476 haplotype blocks with 708 AGRE families using the Family Based Association Test (FBAT). Blocks in two QTL genes were associated with NVC with a P-value of 0.001. Three associated haplotype blocks were intronic to the Nerve Growth Factor (NGF) gene (P=0.001, 0.001, 0.002), and one was intronic to KCND3 (P=0.001). Individual haplotypes within the associated blocks drove the associations (0.003, 0.0004 and 0.0002) for NGF and 0.0001 for KCND3. Using the same methods, these genes were tested for association with NVC in an independent sample of 1517 families from an Autism Genome Project (AGP). NVC was associated with a haplotype in an adjacent NGF block (P=0.0005) and one 46 kb away from the associated block in KCND3 (0.008). These analyses illustrate the value of QTL and targeted association studies for genetically complex disorders such as ASD. NGF is a promising risk gene for NVC deficits.

  7. Database Replication

    CERN Document Server

    Kemme, Bettina

    2010-01-01

    Database replication is widely used for fault-tolerance, scalability and performance. The failure of one database replica does not stop the system from working as available replicas can take over the tasks of the failed replica. Scalability can be achieved by distributing the load across all replicas, and adding new replicas should the load increase. Finally, database replication can provide fast local access, even if clients are geographically distributed clients, if data copies are located close to clients. Despite its advantages, replication is not a straightforward technique to apply, and

  8. The DNA replication checkpoint directly regulates MBF-dependent G1/S transcription.

    Science.gov (United States)

    Dutta, Chaitali; Patel, Prasanta K; Rosebrock, Adam; Oliva, Anna; Leatherwood, Janet; Rhind, Nicholas

    2008-10-01

    The DNA replication checkpoint transcriptionally upregulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G(1)/S transcriptional program by directly regulating MBF, the G(1)/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G(1)/S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitro phosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Replacement of two potential phosphorylation sites with phosphomimetic amino acids suffices to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggests that checkpoint regulation of the MBF transcription factor is a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G(1)/S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes.

  9. The DNA Replication Checkpoint Directly Regulates MBF-Dependent G1/S Transcription▿

    Science.gov (United States)

    Dutta, Chaitali; Patel, Prasanta K.; Rosebrock, Adam; Oliva, Anna; Leatherwood, Janet; Rhind, Nicholas

    2008-01-01

    The DNA replication checkpoint transcriptionally upregulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G1/S transcriptional program by directly regulating MBF, the G1/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G1/S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitro phosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Replacement of two potential phosphorylation sites with phosphomimetic amino acids suffices to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggests that checkpoint regulation of the MBF transcription factor is a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G1/S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes. PMID:18662996

  10. Repletion of factor XIII following cardiopulmonary bypass using a recombinant A-subunit homodimer. A preliminary report

    DEFF Research Database (Denmark)

    Levy, Jerrold H; Gill, Ravi; Nussmeier, Nancy A

    2009-01-01

    Bleeding following cardiac surgery involving cardiopulmonary bypass (CPB) remains a major concern. Coagulation factor XIII (FXIII) functions as a clot-stabilising factor by cross-linking fibrin. Low post-operative levels of FXIII correlate with increased post-operative blood loss. To evaluate...... preliminary safety and pharmacokinetics of recombinant FXIII (rFXIII-A(2)) in cardiac surgery, patients scheduled for coronary artery bypass grafting were randomised to receive a single dose of either rFXIII-A(2) (11.9, 25, 35 or 50 IU/kg) or placebo in a 4:1 ratio. Study drug was given post-CPB within 10...

  11. Coxsackievirus mutants that can bypass host factor PI4KIIIbeta and the need for high levels of PI4P lipids for replication

    NARCIS (Netherlands)

    van der Schaar, H.M.; van der Linden, L.; Lanke, K.H.W.; Strating, J.R.P.M.; Purstinger, G.; Vries, E. De; de Haan, C.A.; Neyts, J.; Kuppeveld, F.J.M. van

    2012-01-01

    RNA viruses can rapidly mutate and acquire resistance to drugs that directly target viral enzymes, which poses serious problems in a clinical context. Therefore, there is a growing interest in the development of antiviral drugs that target host factors critical for viral replication, since they are

  12. A charged residue at the subunit interface of PCNA promotes trimer formation by destabilizing alternate subunit interactions

    International Nuclear Information System (INIS)

    Freudenthal, Bret D.; Gakhar, Lokesh; Ramaswamy, S.; Washington, M. Todd

    2009-01-01

    Eukaryotic proliferating cell nuclear antigen (PCNA), an essential accessory factor in DNA replication and repair, is a ring-shaped homotrimer. A novel nontrimeric structure of E113G-mutant PCNA protein is reported, which shows that this protein forms alternate subunit interactions. It is concluded that the charged side chain of Glu113 promotes normal trimer formation by destabilizing these alternate subunit interactions. Eukaryotic proliferating cell nuclear antigen (PCNA) is an essential replication accessory factor that interacts with a variety of proteins involved in DNA replication and repair. Each monomer of PCNA has an N-terminal domain A and a C-terminal domain B. In the structure of the wild-type PCNA protein, domain A of one monomer interacts with domain B of a neighboring monomer to form a ring-shaped trimer. Glu113 is a conserved residue at the subunit interface in domain A. Two distinct X-ray crystal structures have been determined of a mutant form of PCNA with a substitution at this position (E113G) that has previously been studied because of its effect on translesion synthesis. The first structure was the expected ring-shaped trimer. The second structure was an unanticipated nontrimeric form of the protein. In this nontrimeric form, domain A of one PCNA monomer interacts with domain A of a neighboring monomer, while domain B of this monomer interacts with domain B of a different neighboring monomer. The B–B interface is stabilized by an antiparallel β-sheet and appears to be structurally similar to the A–B interface observed in the trimeric form of PCNA. The A–A interface, in contrast, is primarily stabilized by hydrophobic interactions. Because the E113G substitution is located on this hydrophobic surface, the A–A interface should be less favorable in the case of the wild-type protein. This suggests that the side chain of Glu113 promotes trimer formation by destabilizing these possible alternate subunit interactions

  13. Replication Research and Special Education

    Science.gov (United States)

    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. Solution structure of the N-terminal domain of a replication restart primosome factor, PriC, in Escherichia coli

    Science.gov (United States)

    Aramaki, Takahiko; Abe, Yoshito; Katayama, Tsutomu; Ueda, Tadashi

    2013-01-01

    In eubacterial organisms, the oriC-independent primosome plays an essential role in replication restart after the dissociation of the replication DNA-protein complex by DNA damage. PriC is a key protein component in the replication restart primosome. Our recent study suggested that PriC is divided into two domains: an N-terminal and a C-terminal domain. In the present study, we determined the solution structure of the N-terminal domain, whose structure and function have remained unknown until now. The revealed structure was composed of three helices and one extended loop. We also observed chemical shift changes in the heteronuclear NMR spectrum and oligomerization in the presence of ssDNA. These abilities may contribute to the PriC-ssDNA complex, which is important for the replication restart primosome. PMID:23868391

  15. A novel, broad-spectrum inhibitor of enterovirus replication that targets host cell factor phosphatidylinositol 4-kinase IIIβ

    NARCIS (Netherlands)

    van der Schaar, H.M.; Leyssen, Pieter; Thibaut, H.J.; de Palma, Armando; van der Linden, Lonneke; Lanke, Kjerstin H.W.; Lacroix, Céline; Verbeken, Erik; Conrath, Katja; Macleod, Angus M; Mitchell, Dale R; Palmer, Nicholas J; van de Poël, Hervé; Andrews, Martin; Neyts, Johan; van Kuppeveld, F.J.M.

    2013-01-01

    Despite their high clinical and socioeconomic impacts, there is currently no approved antiviral therapy for the prophylaxis or treatment of enterovirus infections. Here we report on a novel inhibitor of enterovirus replication, compound 1,

  16. Mechanisms Governing DDK Regulation of the Initiation of DNA Replication

    Directory of Open Access Journals (Sweden)

    Larasati

    2016-12-01

    Full Text Available The budding yeast Dbf4-dependent kinase (DDK complex—comprised of cell division cycle (Cdc7 kinase and its regulatory subunit dumbbell former 4 (Dbf4—is required to trigger the initiation of DNA replication through the phosphorylation of multiple minichromosome maintenance complex subunits 2-7 (Mcm2-7. DDK is also a target of the radiation sensitive 53 (Rad53 checkpoint kinase in response to replication stress. Numerous investigations have determined mechanistic details, including the regions of Mcm2, Mcm4, and Mcm6 phosphorylated by DDK, and a number of DDK docking sites. Similarly, the way in which the Rad53 forkhead-associated 1 (FHA1 domain binds to DDK—involving both canonical and non-canonical interactions—has been elucidated. Recent work has revealed mutual promotion of DDK and synthetic lethal with dpb11-1 3 (Sld3 roles. While DDK phosphorylation of Mcm2-7 subunits facilitates their interaction with Sld3 at origins, Sld3 in turn stimulates DDK phosphorylation of Mcm2. Details of a mutually antagonistic relationship between DDK and Rap1-interacting factor 1 (Rif1 have also recently come to light. While Rif1 is able to reverse DDK-mediated Mcm2-7 complex phosphorylation by targeting the protein phosphatase glycogen 7 (Glc7 to origins, there is evidence to suggest that DDK can counteract this activity by binding to and phosphorylating Rif1.

  17. Regulation of the voltage-gated Ca2+ channel CaVα2δ-1 subunit expression by the transcription factor Egr-1.

    Science.gov (United States)

    González-Ramírez, Ricardo; Martínez-Hernández, Elizabeth; Sandoval, Alejandro; Gómez-Mora, Kimberly; Felix, Ricardo

    2018-04-23

    It is well known that the Ca V α 2 δ auxiliary subunit regulates the density of high voltage-activated Ca 2+ channels in the plasma membrane and that alterations in their functional expression might have implications in the pathophysiology of diverse human diseases such as neuropathic pain. However, little is known concerning the transcriptional regulation of this protein. We previously characterized the promoter of Ca V α 2 δ, and here we report its regulation by the transcription factor Egr-1. Using the neuroblastoma N1E-115 cells, we found that Egr-1 interacts specifically with its binding site in the promoter, affecting the transcriptional regulation of Ca V α 2 δ. Overexpression and knockdown analysis of Egr-1 showed significant changes in the transcriptional activity of the Ca V α 2 δ promoter. Egr-1 also regulated the expression of Ca V α 2 δ at the level of protein. Also, functional studies showed that Egr-1 knockdown significantly decreases Ca 2+ currents in dorsal root ganglion (DRG) neurons, while overexpression of the transcription factor increased Ca 2+ currents in the F11 cell line, a hybrid of DRG and N18TG2 neuroblastoma cells. Studying the effects of Egr-1 on the transcriptional expression of Ca V α 2 δ could help to understand the regulatory mechanisms of this protein in both health and disease. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Functional Characterization of the Role of the N-terminal Domain of the c/Nip1 Subunit of Eukaryotic Initiation Factor 3 (eIF3) in AUG Recognition

    Czech Academy of Sciences Publication Activity Database

    Karásková, Martina; Gunišová, Stanislava; Herrmannová, Anna; Wagner, Susan; Munzarová, Vanda; Valášek, Leoš Shivaya

    2012-01-01

    Roč. 287, č. 34 (2012), s. 28420-28434 ISSN 0021-9258 R&D Projects: GA ČR GAP305/10/0335 Institutional support: RVO:61388971 Keywords : START CODON SELECTION * 40S RIBOSOMAL-SUBUNIT * GCN4 TRANSLATIONAL CONTROL Subject RIV: CE - Biochemistry Impact factor: 4.651, year: 2012

  19. Normal growth spurt and final height despite low levels of all forms of circulating insulin-like growth factor-I in a patient with acid-labile subunit deficiency

    DEFF Research Database (Denmark)

    Domené, Horacio M; Martínez, Alicia S; Frystyk, Jan

    2007-01-01

    BACKGROUND: In a recently described patient with acid-labile subunit (ALS) deficiency, the inability to form ternary complexes resulted in a marked reduction in circulating total insulin-like growth factor (IGF)-I, whereas skeletal growth was only marginally affected. To further study the role of...

  20. Cloning of the DNA-binding subunit of human nuclear factor κB: The level of its mRNA is strongly regulated by phorbol ester or tumor necrosis factor α

    International Nuclear Information System (INIS)

    Meyer, R.; Hatada, E.N.; Bartsch, C.; Scheidereit, C.; Hohmann, H.P.; Haiker, M.; Roethlisberger, U.; Lahm, H.W.; Schlaeger, E.J.; van Loon, A.P.G.M.

    1991-01-01

    The DNA binding subunit of nuclear factor κB (NF-κB), a B-cell protein that interacts with the immunoglobulin κ light-chain gene enhancer, has been purified from nuclei of human HL-60 cells stimulated with tumor necrosis factor α (TNFα), and internal peptide sequences were obtained. Overlapping cDNA clones were isolated and sequenced. The encoded open reading frame of about 105 kDa contained at its N-terminal half all six tryptic peptide sequences, suggesting that the 51-kDa NF-κB protein is processed from a 105-kDa precursor. An in vitro synthesized protein containing most of the N-terminal half of the open reading frame bound specifically to an NF-κB binding site. This region also showed high homology to a domain shared by the Drosophila dorsal gene and the avian and mammalian rel (proto)oncogene products. The level of the 3.8-kilobase mRNA was strongly increased after stimulation with TNFα or phorbol ester. Thus, both factors not only activate NF-κB protein, as described previously, but also induce expression of the gene encoding the DNA-binding subunit of NF-κB

  1. Low LET radiation-induced telomerase catalytic subunit promoter activation is mediated by nuclear factor Kappa B

    International Nuclear Information System (INIS)

    Natarajan, M.; Hong, F.A.; Mohan, S.; Herman, T.S.

    2003-01-01

    Full text: The objective of this study is to understand whether low doses of low LET radiation induces survival advantage in normal cells. As an increase in telomerase activity is associated with longevity and cell proliferation, we examined the telomerase response following gamma-irradiation in normal aortic endothelial cells. Telomeric Repeat Amplification Protocol assay following low LET radiation showed an increase in telomerase enzyme activity as early as 8 h post irradiation and reaches its maximum at 24 h. Subsequent analysis revealed that the increased telomerse enzyme activity is due to increased synthesis resulting from an increased transcription. Examination of transcriptional activation of telomerase reverse transcriptase (TERT) promoter regulation showed an enhanced transcription of the telomerse gene following gamma-irradiation. In our previous reports we documented an increase in NF-kB DNA-binding property following low LET radiation (3). Therefore, to determine whether the activation of NF-kB-signaling is responsible for induced TERT promoter activation, cells transiently transfected with minimal promoter region of TERT containing wild type or mutant NF-kB binding site were examined following low LET radiation. TERT promoter activation was induced in wild type transfected cells whereas, in mutant kB binding site, the activation remained at the basal level similar to that of un-irradiated cells. More significantly, the gamma-ray mediated promoter activation of telomerase gene as well as induce telomerase enzyme activity was abrogated by ectopically expressing the IkBa mutant (IkBa (S32A/S36A)), which blocks NF-kB activation. The results thus suggest that exposure to low LET radiation could induce telomerase activity and the activation is at least, in part, mediated by the transcription factor NF-kB. Sustained activation of telomerase in these cells after low LET radiation may impart extended life span

  2. Genetic variations in insulin-like growth factor binding protein acid labile subunit gene associated with growth traits in beef cattle (Bos taurus) in China.

    Science.gov (United States)

    Liu, Yu; Duan, Xiaoyan; Liu, Xiaolin; Guo, Jiazhong; Wang, Hongliang; Li, Zhixiong; Yang, Jing

    2014-05-01

    The insulin-like growth factor binding protein acid labile subunit (IGFALS) gene encodes a serum protein that binds to IGFs and regulates growth, development, and other physiological processes. We have found that sequencing of the IGFALS gene in Chinese Qinchuan beef cattle (n=300) revealed four SNP loci in exon two of the gene (g1219: T>C, g1893: T>C, g2612: G>A, and g2696: A>G). The SNP g2696: A>G resulted in a change from asparagine to aspartic acid (p. N574D) in the leucine-rich repeat region in the carboxyl-terminal domain of IGFALS. Four SNPs were in low linkage disequilibrium, and 12 different haplotypes were identified in the population. Association analysis suggested that SNP g1219: T>C had a significant association with hip width (PG displayed a significant association with stature (Pgrowth traits of bovine, and may serve as a genetic marker for selection of beef cattle for growth traits, including stature. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Crystal structure of the regulatory subunit of archaeal initiation factor 2B (aIF2B) from hyperthermophilic archaeon Pyrococcus horikoshii OT3: a proposed structure of the regulatory subcomplex of eukaryotic IF2B

    International Nuclear Information System (INIS)

    Kakuta, Yoshimitsu; Tahara, Maino; Maetani, Shigehiro; Yao, Min; Tanaka, Isao; Kimura, Makoto

    2004-01-01

    Eukaryotic translation initiation factor 2B (eIF2B) is the guanine-nucleotide exchange factor for eukaryotic initiation factor 2 (eIF2). eIF2B is a heteropentameric protein composed of α-ε subunits. The α, β, and δ subunits form a regulatory subcomplex, while the γ and ε form a catalytic subcomplex. Archaea possess homologues of α, β, and δ subunits of eIF2B. Here, we report the three-dimensional structure of an archaeal regulatory subunit (aIF2Bα) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 determined by X-ray crystallography at 2.2 A resolution. aIF2Bα consists of two subdomains, an N-domain (residues 1-95) and a C-domain (residues 96-276), connected by a long α-helix (α5: 78-106). The N-domain contains a five helix bundle structure, while the C-domain folds into the α/β structure, thus showing similarity to D-ribose-5-phosphate isomerase structure. The presence of two molecules in the crystallographic asymmetric unit and the gel filtration analysis suggest a dimeric structure of aIF2Bα in solution, interacting with each other by C-domains. Furthermore, the crystallographic 3-fold symmetry generates a homohexameric structure of aIF2Bα; the interaction is primarily mediated by the long α-helix at the N-domains. This structure suggests an architecture of the three subunits, α, β, and δ, in the regulatory subcomplex within eIF2B

  4. Down-regulation of MicroRNAs 222/221 in Acute Myelogenous Leukemia with Deranged Core-Binding Factor Subunits

    Directory of Open Access Journals (Sweden)

    Matteo Brioschi

    2010-11-01

    Full Text Available Core-binding factor leukemia (CBFL is a subgroup of acutemyeloid leukemia (AML characterized by genetic mutations involving the subunits of the core-binding factor (CBF. The leukemogenesis model for CBFL posits that one, or more, gene mutations inducing increased cell proliferation and/or inhibition of apoptosis cooperate with CBF mutations for leukemia development. One of the most commonmutations associated with CBF mutations involves the KIT receptor. A high expression of KIT is a hallmark of a high proportion of CBFL. Previous studies indicate that microRNA (MIR 222/221 targets the 3′ untranslated region of the KIT messenger RNA and our observation that AML1 can bind the MIR-222/221 promoter, we hypothesized that MIR-222/221 represents the link between CBF and KIT. Here, we show that MIR-222/221 expression is upregulated after myeloid differentiation of normal bone marrow AC133+ stem progenitor cells. CBFL blasts with either t(8;21 or inv(16 CBF rearrangements with high expression levels of KIT (CD117 display a significantly lower level of MIR-222/221 expression than non-CBFL blasts. Consistently, we found that the t(8;21 AML1-MTG8 fusion protein binds the MIR-222/221 promoter and induces transcriptional repression of a MIR-222/221-LUC reporter. Because of the highly conserved sequence homology, we demonstrated concomitant MIR-222/221 down-regulation and KIT up-regulation in the 32D/WT1 mouse cell model carrying the AML1-MTG16 fusion protein. This study provides the first hint that CBFL-associated fusion proteins may lead to up-regulation of the KIT receptor by down-regulating MIR-222/221, thus explaining the concomitant occurrence of CBF genetic rearrangements and overexpression of wild type or mutant KIT in AML.

  5. Androgen signaling promotes translation of TMEFF2 in prostate cancer cells via phosphorylation of the α subunit of the translation initiation factor 2.

    Directory of Open Access Journals (Sweden)

    Ryan F Overcash

    Full Text Available The type I transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2, is expressed mainly in brain and prostate. Expression of TMEFF2 is deregulated in prostate cancer, suggesting a role in this disease, but the molecular mechanism(s involved in this effect are not clear. Although androgens promote tmeff2 transcription, androgen delivery to castrated animals carrying CWR22 xenografts increases TMEFF2 protein levels in the absence of mRNA changes, suggesting that TMEFF2 may also be post-transcriptionally regulated. Here we show that translation of TMEFF2 is regulated by androgens. Addition of physiological concentrations of dihydrotestosterone (DHT to prostate cancer cell lines increases translation of endogenous TMEFF2 or transfected TMEFF2-Luciferase fusions, and this effect requires the presence of upstream open reading frames (uORFs in the 5'-untranslated region (5'-UTR of TMEFF2. Using chemical and siRNA inhibition of the androgen receptor (AR, we show that the androgen effect on TMEFF2 translation is mediated by the AR. Importantly, DHT also promotes phosphorylation of the α subunit of the translation initiation factor 2 (eIF2α in an AR-dependent manner, paralleling the effect on TMEFF2 translation. Moreover, endoplasmic reticulum (ER stress conditions, which promote eIF2α phosphorylation, also stimulate TMEFF2 translation. These results indicate that androgen signaling promotes eIF2α phosphorylation and subsequent translation of TMEFF2 via a mechanism that requires uORFs in the 5'-UTR of TMEFF2.

  6. Engineered zinc-finger transcription factors inhibit the replication and transcription of HBV in vitro and in vivo.

    Science.gov (United States)

    Luo, Wei; Wang, Junxia; Xu, Dengfeng; Bai, Huili; Zhang, Yangli; Zhang, Yuhong; Li, Xiaosong

    2018-04-01

    In the present study, an artificial zinc-finger transcription factor eukaryotic expression vector specifically recognizing and binding to the hepatitis B virus (HBV) enhancer (Enh) was constructed, which inhibited the replication and expression of HBV DNA. The HBV EnhI‑specific pcDNA3.1‑artificial transcription factor (ATF) vector was successfully constructed, and then transformed or injected into HepG2.2.15 cells and HBV transgenic mice, respectively. The results demonstrated that the HBV EnhI (1,070‑1,234 bp)‑specific ATF significantly inhibited the replication and transcription of HBV DNA in vivo and in vitro. The HBV EnhI‑specific ATF may be a meritorious component of progressive combination therapies for eliminating HBV DNA in infected patients. A radical cure for chronic HBV infection may become feasible by using this bioengineering technology.

  7. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the central zinc-binding domain of the human Mcm10 DNA-replication factor

    International Nuclear Information System (INIS)

    Jung, Nam Young; Bae, Won Jin; Chang, Jeong Ho; Kim, Young Chang; Cho, Yunje

    2008-01-01

    Mcm10 is a highly conserved nuclear protein that plays a key role in the initiation and elongation processes of DNA replication by providing a physical link between the Mcm2–7 complex and DNA polymerases. In this study, the central domain of human Mcm10 was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 3350. The initiation of eukaryotic DNA replication requires the tightly controlled assembly of a set of replication factors. Mcm10 is a highly conserved nuclear protein that plays a key role in the initiation and elongation processes of DNA replication by providing a physical link between the Mcm2–7 complex and DNA polymerases. The central domain, which contains the CCCH zinc-binding motif, is most conserved within Mcm10 and binds to DNA and several proteins, including proliferative cell nuclear antigen. In this study, the central domain of human Mcm10 was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 3350. An X-ray diffraction data set was collected to a resolution of 2.6 Å on a synchrotron beamline. The crystals formed belonged to space group R3, with unit-cell parameters a = b = 99.5, c = 133.0 Å. According to Matthews coefficient calculations, the crystals were predicted to contain six MCM10 central domain molecules in the asymmetric unit

  8. Loss-of-function nuclear factor κB subunit 1 (NFKB1) variants are the most common monogenic cause of common variable immunodeficiency in Europeans.

    Science.gov (United States)

    Tuijnenburg, Paul; Lango Allen, Hana; Burns, Siobhan O; Greene, Daniel; Jansen, Machiel H; Staples, Emily; Stephens, Jonathan; Carss, Keren J; Biasci, Daniele; Baxendale, Helen; Thomas, Moira; Chandra, Anita; Kiani-Alikhan, Sorena; Longhurst, Hilary J; Seneviratne, Suranjith L; Oksenhendler, Eric; Simeoni, Ilenia; de Bree, Godelieve J; Tool, Anton T J; van Leeuwen, Ester M M; Ebberink, Eduard H T M; Meijer, Alexander B; Tuna, Salih; Whitehorn, Deborah; Brown, Matthew; Turro, Ernest; Thrasher, Adrian J; Smith, Kenneth G C; Thaventhiran, James E; Kuijpers, Taco W

    2018-03-02

    The genetic cause of primary immunodeficiency disease (PID) carries prognostic information. We conducted a whole-genome sequencing study assessing a large proportion of the NIHR BioResource-Rare Diseases cohort. In the predominantly European study population of principally sporadic unrelated PID cases (n = 846), a novel Bayesian method identified nuclear factor κB subunit 1 (NFKB1) as one of the genes most strongly associated with PID, and the association was explained by 16 novel heterozygous truncating, missense, and gene deletion variants. This accounted for 4% of common variable immunodeficiency (CVID) cases (n = 390) in the cohort. Amino acid substitutions predicted to be pathogenic were assessed by means of analysis of structural protein data. Immunophenotyping, immunoblotting, and ex vivo stimulation of lymphocytes determined the functional effects of these variants. Detailed clinical and pedigree information was collected for genotype-phenotype cosegregation analyses. Both sporadic and familial cases demonstrated evidence of the noninfective complications of CVID, including massive lymphadenopathy (24%), unexplained splenomegaly (48%), and autoimmune disease (48%), features prior studies correlated with worse clinical prognosis. Although partial penetrance of clinical symptoms was noted in certain pedigrees, all carriers have a deficiency in B-lymphocyte differentiation. Detailed assessment of B-lymphocyte numbers, phenotype, and function identifies the presence of an increased CD21 low B-cell population. Combined with identification of the disease-causing variant, this distinguishes between healthy subjects, asymptomatic carriers, and clinically affected cases. We show that heterozygous loss-of-function variants in NFKB1 are the most common known monogenic cause of CVID, which results in a temporally progressive defect in the formation of immunoglobulin-producing B cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Probing the interaction between the histone methyltransferase/deacetylase subunit RBBP4/7 and the transcription factor BCL11A in epigenetic complexes.

    Science.gov (United States)

    Moody, Rebecca Reed; Lo, Miao-Chia; Meagher, Jennifer L; Lin, Chang-Ching; Stevers, Nicholas O; Tinsley, Samantha L; Jung, Inkyung; Matvekas, Aleksas; Stuckey, Jeanne A; Sun, Duxin

    2018-02-09

    The transcription factor BCL11A has recently been reported to be a driving force in triple-negative breast cancer (TNBC), contributing to the maintenance of a chemoresistant breast cancer stem cell (BCSC) population. Although BCL11A was shown to suppress γ-globin and p21 and to induce MDM2 expression in the hematopoietic system, its downstream targets in TNBC are still unclear. For its role in transcriptional repression, BCL11A was found to interact with several corepressor complexes; however, the mechanisms underlying these interactions remain unknown. Here, we reveal that BCL11A interacts with histone methyltransferase (PRC2) and histone deacetylase (NuRD and SIN3A) complexes through their common subunit, RBBP4/7. In fluorescence polarization assays, we show that BCL11A competes with histone H3 for binding to the negatively charged top face of RBBP4. To define that interaction, we solved the crystal structure of RBBP4 in complex with an N-terminal peptide of BCL11A (residues 2-16, BCL11A(2-16)). The crystal structure identifies novel interactions between BCL11A and the side of the β-propeller of RBBP4 that are not seen with histone H3. We next show that BCL11A(2-16) pulls down RBBP4, RBBP7, and other components of PRC2, NuRD, and SIN3A from the cell lysate of the TNBC cell line SUM149. Furthermore, we demonstrate the therapeutic potential of targeting the RBBP4-BCL11A binding by showing that a BCL11A peptide can decrease aldehyde dehydrogenase-positive BCSCs and mammosphere formation capacity in SUM149. Together, our findings have uncovered a previously unidentified mechanism that BCL11A may use to recruit epigenetic complexes to regulate transcription and promote tumorigenesis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. The 25 kDa subunit of cleavage factor Im Is a RNA-binding protein that interacts with the poly(A polymerase in Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Marisol Pezet-Valdez

    Full Text Available In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25 from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety X domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25 was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the EhPgp5 mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A polymerase (EhPAP that is responsible for the synthesis of the poly(A tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in E. histolytica. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A polymerase, another member of the pre-mRNA 3' end processing machinery in this protozoan parasite.

  11. The Wheat Mediator Subunit TaMED25 Interacts with the Transcription Factor TaEIL1 to Negatively Regulate Disease Resistance against Powdery Mildew1

    Science.gov (United States)

    Zhang, Tianren; Jia, Jizeng; Sun, Jiaqiang

    2016-01-01

    Powdery mildew, caused by the biotrophic fungal pathogen Blumeria graminis f. sp. tritici, is a major limitation for the production of bread wheat (Triticum aestivum). However, to date, the transcriptional regulation of bread wheat defense against powdery mildew remains largely unknown. Here, we report the function and molecular mechanism of the bread wheat Mediator subunit 25 (TaMED25) in regulating the bread wheat immune response signaling pathway. Three homoalleles of TaMED25 from bread wheat were identified and mapped to chromosomes 5A, 5B, and 5D, respectively. We show that knockdown of TaMED25 by barley stripe mosaic virus-induced gene silencing reduced bread wheat susceptibility to the powdery mildew fungus during the compatible plant-pathogen interaction. Moreover, our results indicate that MED25 may play a conserved role in regulating bread wheat and barley (Hordeum vulgare) susceptibility to powdery mildew. Similarly, bread wheat ETHYLENE INSENSITIVE3-LIKE1 (TaEIL1), an ortholog of Arabidopsis (Arabidopsis thaliana) ETHYLENE INSENSITIVE3, negatively regulates bread wheat resistance against powdery mildew. Using various approaches, we demonstrate that the conserved activator-interacting domain of TaMED25 interacts physically with the separate amino- and carboxyl-terminal regions of TaEIL1, contributing to the transcriptional activation activity of TaEIL1. Furthermore, we show that TaMED25 and TaEIL1 synergistically activate ETHYLENE RESPONSE FACTOR1 (TaERF1) transcription to modulate bread wheat basal disease resistance to B. graminis f. sp. tritici by repressing the expression of pathogenesis-related genes and deterring the accumulation of reactive oxygen species. Collectively, we identify the TaMED25-TaEIL1-TaERF1 signaling module as a negative regulator of bread wheat resistance to powdery mildew. PMID:26813794

  12. A network of hydrophobic residues impeding helix alphaC rotation maintains latency of kinase Gcn2, which phosphorylates the alpha subunit of translation initiation factor 2.

    Science.gov (United States)

    Gárriz, Andrés; Qiu, Hongfang; Dey, Madhusudan; Seo, Eun-Joo; Dever, Thomas E; Hinnebusch, Alan G

    2009-03-01

    Kinase Gcn2 is activated by amino acid starvation and downregulates translation initiation by phosphorylating the alpha subunit of translation initiation factor 2 (eIF2alpha). The Gcn2 kinase domain (KD) is inert and must be activated by tRNA binding to the adjacent regulatory domain. Previous work indicated that Saccharomyces cerevisiae Gcn2 latency results from inflexibility of the hinge connecting the N and C lobes and a partially obstructed ATP-binding site in the KD. Here, we provide strong evidence that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining helix alphaC rotation in the KD in a manner relieved during amino acid starvation by tRNA binding and autophosphorylation of Thr-882 in the activation loop. Thus, we show that mutationally disrupting the hydrophobic network in various ways constitutively activates eIF2alpha phosphorylation in vivo and bypasses the requirement for a key tRNA binding motif (m2) and Thr-882 in Gcn2. In particular, replacing Leu-856 with any nonhydrophobic residue activates Gcn2, while substitutions with various hydrophobic residues maintain kinase latency. We further provide strong evidence that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856 hydrophobic network or enhance hinge flexibility eliminate the need for the conserved salt bridge at the parallel dimer interface, implying that KD dimerization facilitates the reorientation of alphaC and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of alphaC are mutually reinforcing conformational transitions stimulated by tRNA binding and secured by the ensuing autophosphorylation of T882 for stable kinase activation.

  13. Dual Role for Hsc70 in the Biogenesis and Regulation of the Heme-Regulated Kinase of the α Subunit of Eukaryotic Translation Initiation Factor 2

    Science.gov (United States)

    Uma, Sheri; Thulasiraman, Vanitha; Matts, Robert L.

    1999-01-01

    The heme-regulated kinase of the α subunit of eukaryotic initiation factor 2 (HRI) is activated in rabbit reticulocyte lysate (RRL) in response to a number of environmental conditions, including heme deficiency, heat shock, and oxidative stress. Activation of HRI causes an arrest of initiation of protein synthesis. Recently, we have demonstrated that the heat shock cognate protein Hsc70 negatively modulates the activation of HRI in RRL in response to these environmental conditions. Hsc70 is also known to be a critical component of the Hsp90 chaperone machinery in RRL, which plays an obligatory role for HRI to acquire and maintain a conformation that is competent to activate. Using de novo-synthesized HRI in synchronized pulse-chase translations, we have examined the role of Hsc70 in the regulation of HRI biogenesis and activation. Like Hsp90, Hsc70 interacted with nascent HRI and HRI that was matured to a state which was competent to undergo stimulus-induced activation (mature-competent HRI). Interaction of HRI with Hsc70 was required for the transformation of HRI, as the Hsc70 antagonist clofibric acid inhibited the folding of HRI into a mature-competent conformation. Unlike Hsp90, Hsc70 also interacted with transformed HRI. Clofibric acid disrupted the interaction of Hsc70 with transformed HRI that had been matured and transformed in the absence of the drug. Disruption of Hsc70 interaction with transformed HRI in heme-deficient RRL resulted in its hyperactivation. Furthermore, activation of HRI in response to heat shock or denatured proteins also resulted in a similar blockage of Hsc70 interaction with transformed HRI. These results indicate that Hsc70 is required for the folding and transformation of HRI into an active kinase but is subsequently required to negatively attenuate the activation of transformed HRI. PMID:10454533

  14. The Wheat Mediator Subunit TaMED25 Interacts with the Transcription Factor TaEIL1 to Negatively Regulate Disease Resistance against Powdery Mildew.

    Science.gov (United States)

    Liu, Jie; Zhang, Tianren; Jia, Jizeng; Sun, Jiaqiang

    2016-03-01

    Powdery mildew, caused by the biotrophic fungal pathogen Blumeria graminis f. sp. tritici, is a major limitation for the production of bread wheat (Triticum aestivum). However, to date, the transcriptional regulation of bread wheat defense against powdery mildew remains largely unknown. Here, we report the function and molecular mechanism of the bread wheat Mediator subunit 25 (TaMED25) in regulating the bread wheat immune response signaling pathway. Three homoalleles of TaMED25 from bread wheat were identified and mapped to chromosomes 5A, 5B, and 5D, respectively. We show that knockdown of TaMED25 by barley stripe mosaic virus-induced gene silencing reduced bread wheat susceptibility to the powdery mildew fungus during the compatible plant-pathogen interaction. Moreover, our results indicate that MED25 may play a conserved role in regulating bread wheat and barley (Hordeum vulgare) susceptibility to powdery mildew. Similarly, bread wheat ETHYLENE INSENSITIVE3-LIKE1 (TaEIL1), an ortholog of Arabidopsis (Arabidopsis thaliana) ETHYLENE INSENSITIVE3, negatively regulates bread wheat resistance against powdery mildew. Using various approaches, we demonstrate that the conserved activator-interacting domain of TaMED25 interacts physically with the separate amino- and carboxyl-terminal regions of TaEIL1, contributing to the transcriptional activation activity of TaEIL1. Furthermore, we show that TaMED25 and TaEIL1 synergistically activate ETHYLENE RESPONSE FACTOR1 (TaERF1) transcription to modulate bread wheat basal disease resistance to B. graminis f. sp. tritici by repressing the expression of pathogenesis-related genes and deterring the accumulation of reactive oxygen species. Collectively, we identify the TaMED25-TaEIL1-TaERF1 signaling module as a negative regulator of bread wheat resistance to powdery mildew. © 2016 American Society of Plant Biologists. All Rights Reserved.

  15. miR-200c targets nuclear factor IA to suppress HBV replication and gene expression via repressing HBV Enhancer I activity.

    Science.gov (United States)

    Tian, Hui; He, Zhenkun

    2018-03-01

    Hepatitis B virus (HBV) chronic infection is a health problem in the worldwide, with a underlying higher risk of liver cirrhosis and hepaticocellular carcinoma. A number of studies indicate that microRNAs (miRNAs) play vital roles in HBV replication. This study was designed to explore the potential molecular mechanism of miR-200c in HBV replication. The expression of miR-200c, nuclear factor IA (NFIA) mRNA, HBV DNA, and HBV RNA (pregenomic RNA (pgRNA), and total RNA) were measured by qRCR. The levels of HBsAg and HBeAg were detected by ELISA. NFIA expression at protein level was measured by western blot. The direct interaction between miR-200c and NFIA were identified by Targetscan software and Dual-Luciferase reporter analysis. Enhance I activity were detected by Dual-Luciferase reporter assay. miR-200c expression was prominently reduced in pHBV1.3-tranfected Huh7 and in stable HBV-producing cell line (HepG2.2.15). The enforced expression of miR-200c significantly suppressed HBV replication, as demonstrated by the reduced levels of HBV protein (HBsAg and HBeAg) and, DNA and RNA (pgRNA and total RNA) levels. NFIA was proved to be a target of miR-200c and NFIA overexpression notably stimulated HBV replication. In addition, the inhibitory effect of miR-200c on HBV Enhance I activity was abolished following restoration of NFIA. miR-200c repressed HBV replication by directly targeting NFIA, which might provide a novel therapeutic target for HBV infection. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  16. Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

    Science.gov (United States)

    Beresova, Lucie; Vesela, Eva; Chamrad, Ivo; Voller, Jiri; Yamada, Masayuki; Furst, Tomas; Lenobel, Rene; Chroma, Katarina; Gursky, Jan; Krizova, Katerina; Mistrik, Martin; Bartek, Jiri

    2016-12-02

    Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

  17. International Expansion through Flexible Replication

    DEFF Research Database (Denmark)

    Jonsson, Anna; Foss, Nicolai Juul

    2011-01-01

    Business organizations may expand internationally by replicating a part of their value chain, such as a sales and marketing format, in other countries. However, little is known regarding how such “international replicators” build a format for replication, or how they can adjust it in order to ada......, etc.) are replicated in a uniform manner across stores, and change only very slowly (if at all) in response to learning (“flexible replication”). We conclude by discussing the factors that influence the approach to replication adopted by an international replicator.......Business organizations may expand internationally by replicating a part of their value chain, such as a sales and marketing format, in other countries. However, little is known regarding how such “international replicators” build a format for replication, or how they can adjust it in order to adapt...

  18. Robust Replication Control Is Generated by Temporal Gaps between Licensing and Firing Phases and Depends on Degradation of Firing Factor Sld2

    Directory of Open Access Journals (Sweden)

    Karl-Uwe Reusswig

    2016-10-01

    Full Text Available Temporal separation of DNA replication initiation into licensing and firing phases ensures the precise duplication of the genome during each cell cycle. Cyclin-dependent kinase (CDK is known to generate this separation by activating firing factors and at the same time inhibiting licensing factors but may not be sufficient to ensure robust separation at transitions between both phases. Here, we show that a temporal gap separates the inactivation of firing factors from the re-activation of licensing factors during mitosis in budding yeast. We find that gap size critically depends on phosphorylation-dependent degradation of the firing factor Sld2 mediated by CDK, DDK, Mck1, and Cdc5 kinases and the ubiquitin-ligases Dma1/2. Stable mutants of Sld2 minimize the gap and cause increased genome instability in an origin-dependent manner when combined with deregulation of other replication regulators or checkpoint mechanisms. Robust separation of licensing and firing phases therefore appears indispensable to safeguard genome stability.

  19. The Cac2 subunit is essential for productive histone binding and nucleosome assembly in CAF-1

    Energy Technology Data Exchange (ETDEWEB)

    Mattiroli, Francesca; Gu, Yajie; Balsbaugh, Jeremy L.; Ahn, Natalie G.; Luger, Karolin

    2017-04-18

    Nucleosome assembly following DNA replication controls epigenome maintenance and genome integrity. Chromatin assembly factor 1 (CAF-1) is the histone chaperone responsible for histone (H3-H4)2 deposition following DNA synthesis. Structural and functional details for this chaperone complex and its interaction with histones are slowly emerging. Using hydrogen-deuterium exchange coupled to mass spectrometry, combined with in vitro and in vivo mutagenesis studies, we identified the regions involved in the direct interaction between the yeast CAF-1 subunits, and mapped the CAF-1 domains responsible for H3-H4 binding. The large subunit, Cac1 organizes the assembly of CAF-1. Strikingly, H3-H4 binding is mediated by a composite interface, shaped by Cac1-bound Cac2 and the Cac1 acidic region. Cac2 is indispensable for productive histone binding, while deletion of Cac3 has only moderate effects on H3-H4 binding and nucleosome assembly. These results define direct structural roles for yeast CAF-1 subunits and uncover a previously unknown critical function of the middle subunit in CAF-1.

  20. Total and free insulin-like growth factor I, insulin-like growth factor binding protein 3 and acid-labile subunit reflect clinical activity in acromegaly

    DEFF Research Database (Denmark)

    Sneppen, S B; Lange, Merete Wolder; Pedersen, L M

    2001-01-01

    The aim was to evaluate, markers of disease activity in acromegaly in relation to perceived disease activity. Thirty-seven consecutively treated, acromegalic patients, classified by clinical symptoms as inactive (n=16), slightly active (n=10) and active (n=11), entered the study. When evaluating......-like growth factor binding protein-3 (IGFBP-3) with PV(pos) of 0.69 and 0.71 and PV(neg) of 0.91 and 0.92 respectively. We conclude that free IGF-I is more closely related than total IGF-I to perceived disease activity and is as such useful when evaluating previously treated acromegaly for disease activity...

  1. Inhibition of human T cell leukemia virus type 2 replication by the suppressive action of class II transactivator and nuclear factor Y.

    Science.gov (United States)

    Tosi, Giovanna; Pilotti, Elisabetta; Mortara, Lorenzo; De Lerma Barbaro, Andrea; Casoli, Claudio; Accolla, Roberto S

    2006-08-22

    The master regulator of MHC-II gene transcription, class II transactivator (CIITA), acts as a potent inhibitor of human T cell leukemia virus type 2 (HTLV-2) replication by blocking the activity of the viral Tax-2 transactivator. Here, we show that this inhibitory effect takes place at the nuclear level and maps to the N-terminal 1-321 region of CIITA, where we identified a minimal domain, from positions 64-144, that is strictly required to suppress Tax-2 function. Furthermore, we show that Tax-2 specifically cooperates with cAMP response element binding protein-binding protein (CBP) and p300, but not with p300/CBP-associated factor, to enhance transcription from the viral promoter. This finding represents a unique difference with respect to Tax-1, which uses all three coactivators to transactivate the human T cell leukemia virus type 1 LTR. Direct sequestering of CBP or p300 is not the primary mechanism by which CIITA causes suppression of Tax-2. Interestingly, we found that the transcription factor nuclear factor Y, which interacts with CIITA to increase transcription of MHC-II genes, exerts a negative regulatory action on the Tax-2-mediated HTLV-2 LTR transactivation. Thus, CIITA may inhibit Tax-2 function, at least in part, through nuclear factor Y. These findings demonstrate the dual defensive role of CIITA against pathogens: it increases the antigen-presenting function for viral determinants and suppresses HTLV-2 replication in infected cells.

  2. Placental macrophage contact potentiates the complete replicative cycle of human cytomegalovirus in syncytiotrophoblast cells: role of interleukin-8 and transforming growth factor-beta1.

    Science.gov (United States)

    Bácsi, A; Aranyosi, J; Beck, Z; Ebbesen, P; Andirkó, I; Szabó, J; Lampé, L; Kiss, J; Gergely, L; Tóth, F D

    1999-10-01

    Although syncytiotrophoblast (ST) cells can be infected by human cytomegalovirus (HCMV), in vitro studies have indicated that ST cells do not support the complete viral reproductive cycle, or HCMV replication may occur in less than 3% of ST cells. The present study tested the possibility that placental macrophages might enhance activation of HCMV carried in ST cells and, further, that infected ST cells would be capable of transmitting virus to neighboring macrophages. For this purpose, we studied HCMV replication in ST cells grown alone or cocultured with uninfected placental macrophages. Our results demonstrated that HCMV gene expression in ST cells was markedly upregulated by coculture with macrophages, resulting in release of substantial amounts of infectious virus from HCMV-infected ST cells. After having become permissive for viral replication, ST cells delivered HCMV to the cocultured macrophages, as evidenced by detection of virus-specific antigens in these cells. The stimulatory effect of coculture on HCMV gene expression in ST cells was mediated by marked interleukin-8 (IL-8) and transforming growth factor-beta1 (TGF-beta1) release from macrophages, an effect caused by contact between the different placental cells. Our findings indicate an interactive role for the ST layer and placental macrophages in the dissemination of HCMV among placental tissue. Eventually, these interactions may contribute to the transmission of HCMV from mother to the fetus.

  3. Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

    Directory of Open Access Journals (Sweden)

    Caroline M Li

    Full Text Available We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE. In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40 origin of DNA replication and the viral large tumor antigen (T-antigen protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA, DNA topoisomerase I (topo I, DNA polymerase δ (Pol δ, DNA polymerase ɛ (Pol ɛ, replication protein A (RPA and replication factor C (RFC. Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

  4. Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

    Science.gov (United States)

    Li, Caroline M; Miao, Yunan; Lingeman, Robert G; Hickey, Robert J; Malkas, Linda H

    2016-01-01

    We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

  5. Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

    OpenAIRE

    Vassin, Vitaly M.; Wold, Marc S.; Borowiec, James A.

    2004-01-01

    Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2D) or alanine (RPA2A). Although RPA2D was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2D mutant was selectively unable to associate with re...

  6. DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi's sarcoma-associated herpesvirus latent replication

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Seho [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of); Lim, Chunghun [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of); Lee, Jae Young [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of); Song, Yoon-Jae [Department of Life Science, Kyungwon University, Seongnam-Si, Kyeonggi-Do 461-701 (Korea, Republic of); Park, Junsoo [Division of Biological Science and Technology, Yonsei University, Wonju 220-100 (Korea, Republic of); Choe, Joonho [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of); Seo, Taegun, E-mail: tseo@dongguk.edu [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of)

    2010-04-16

    During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.

  7. DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi's sarcoma-associated herpesvirus latent replication

    International Nuclear Information System (INIS)

    Cha, Seho; Lim, Chunghun; Lee, Jae Young; Song, Yoon-Jae; Park, Junsoo; Choe, Joonho; Seo, Taegun

    2010-01-01

    During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.

  8. Identification of a new adapter protein that may link the common beta subunit of the receptor for granulocyte/macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 to phosphatidylinositol 3-kinase.

    Science.gov (United States)

    Jücker, M; Feldman, R A

    1995-11-17

    Binding of human granulocyte/macrophage colony-stimulating factor (hGM-CSF) to its receptor induces the rapid activation of phosphatidylinositol-3 kinase (PI 3-kinase). As hGM-CSF receptor (hGMR) does not contain a consensus sequence for binding of PI 3-kinase, hGMR must use a distinct mechanism for its association with and activation of PI 3-kinase. Here, we describe the identification of a tyrosine-phosphorylated protein of 76-85 kDa (p80) that associates with the common beta subunit of hGMR and with the SH2 domains of the p85 subunit of PI 3-kinase in hGM-CSF-stimulated cells. Src/Yes and Lyn were tightly associated with the p80.PI 3-kinase complex, suggesting that p80 and other phosphotyrosyl proteins present in the complex were phosphorylated by Src family kinases. Tyrosine phosphorylation of p80 was only detected in hGM-CSF or human interleukin-3-stimulated cells, suggesting that activation of p80 might be specific for signaling via the common beta subunit. We postulate that p80 functions as an adapter protein that may participate in linking the hGM-CSF receptor to the PI 3-kinase signaling pathway.

  9. Replication of a Modified Factor Structure for the Eating Disorder Examination-Questionnaire: Extension to Clinical Eating Disorder and Non-clinical Samples in Portugal.

    Science.gov (United States)

    Machado, Paulo P P; Grilo, Carlos M; Crosby, Ross D

    2018-01-01

    Psychometric investigations of the Eating Disorder Examination-Questionnaire (EDE-Q) have generally not supported the original scale structure. The present study tested an alternative brief factor structure in two large Portuguese samples: (1) a non-clinical sample of N = 4117 female students and (2) a treatment-seeking sample of N = 609 patients diagnosed with eating disorders. Confirmatory factor analysis revealed a poor fit for the original EDE-Q structure in both the non-clinical and the clinical samples but revealed a good fit for the alternative 7-item 3-factor structure (dietary restraint, shape/weight overvaluation and body dissatisfaction). Factor loadings were invariant across samples and across the different specific eating disorder diagnoses in the clinical sample. These confirmatory factor analysis findings, which replicate findings from studies with diverse predominately overweight/obese samples, supported a modified 7-item, 3-factor structure for the EDE-Q. The reliable findings across different non-clinical and clinical eating disorder groups provide confidence regarding the potential utility of this brief version. Copyright © 2017 John Wiley & Sons, Ltd and Eating Disorders Association. Copyright © 2017 John Wiley & Sons, Ltd and Eating Disorders Association.

  10. Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery.

    Science.gov (United States)

    Murphy, Anar K; Fitzgerald, Michael; Ro, Teresa; Kim, Jee Hyun; Rabinowitsch, Ariana I; Chowdhury, Dipanjan; Schildkraut, Carl L; Borowiec, James A

    2014-08-18

    Phosphorylation of replication protein A (RPA) by Cdk2 and the checkpoint kinase ATR (ATM and Rad3 related) during replication fork stalling stabilizes the replisome, but how these modifications safeguard the fork is not understood. To address this question, we used single-molecule fiber analysis in cells expressing a phosphorylation-defective RPA2 subunit or lacking phosphatase activity toward RPA2. Deregulation of RPA phosphorylation reduced synthesis at forks both during replication stress and recovery from stress. The ability of phosphorylated RPA to stimulate fork recovery is mediated through the PALB2 tumor suppressor protein. RPA phosphorylation increased localization of PALB2 and BRCA2 to RPA-bound nuclear foci in cells experiencing replication stress. Phosphorylated RPA also stimulated recruitment of PALB2 to single-strand deoxyribonucleic acid (DNA) in a cell-free system. Expression of mutant RPA2 or loss of PALB2 expression led to significant DNA damage after replication stress, a defect accentuated by poly-ADP (adenosine diphosphate) ribose polymerase inhibitors. These data demonstrate that phosphorylated RPA recruits repair factors to stalled forks, thereby enhancing fork integrity during replication stress. © 2014 Murphy et al.

  11. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    Science.gov (United States)

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  12. Interleukin-7 induces HIV replication in primary naive T cells through a nuclear factor of activated T cell (NFAT)-dependent pathway

    International Nuclear Information System (INIS)

    Managlia, Elizabeth Z.; Landay, Alan; Al-Harthi, Lena

    2006-01-01

    Interleukin (IL)-7 plays several roles critical to T cell maturation, survival, and homeostasis. Because of these functions, IL-7 is under investigation as an immune-modulator for therapeutic use in lymphopenic clinical conditions, including HIV. We reported that naive T cells, typically not permissive to HIV, can be productively infected when pre-treated with IL-7. We evaluated the mechanism by which IL-7-mediates this effect. IL-7 potently up-regulated the transcriptional factor NFAT, but had no effect on NFκB. Blocking NFAT activity using a number of reagents, such as Cyclosporin A, FK-506, or the NFAT-specific inhibitor known as VIVIT peptide, all markedly reduced IL-7-mediated induction of HIV replication in naive T cells. Additional neutralization of cytokines present in IL-7-treated cultures and/or those that have NFAT-binding sequences within their promotors indicated that IL-10, IL-4, and most significantly IFNγ, all contribute to IL-7-induction of HIV productive replication in naive T cells. These data clarify the mechanism by which IL-7 can overcome the block to HIV productive infection in naive T cells, despite their quiescent cell status. These findings are relevant to the treatment of HIV disease and understanding HIV pathogenesis in the naive CD4+ T cell compartment, especially in light of the vigorous pursuit of IL-7 as an in vivo immune modulator

  13. The multi-replication protein A (RPA) system--a new perspective.

    Science.gov (United States)

    Sakaguchi, Kengo; Ishibashi, Toyotaka; Uchiyama, Yukinobu; Iwabata, Kazuki

    2009-02-01

    Replication protein A (RPA) complex has been shown, using both in vivo and in vitro approaches, to be required for most aspects of eukaryotic DNA metabolism: replication, repair, telomere maintenance and homologous recombination. Here, we review recent data concerning the function and biological importance of the multi-RPA complex. There are distinct complexes of RPA found in the biological kingdoms, although for a long time only one type of RPA complex was believed to be present in eukaryotes. Each complex probably serves a different role. In higher plants, three distinct large and medium subunits are present, but only one species of the smallest subunit. Each of these protein subunits forms stable complexes with their respective partners. They are paralogs as complex. Humans possess two paralogs and one analog of RPA. The multi-RPA system can be regarded as universal in eukaryotes. Among eukaryotic kingdoms, paralogs, orthologs, analogs and heterologs of many DNA synthesis-related factors, including RPA, are ubiquitous. Convergent evolution seems to be ubiquitous in these processes. Using recent findings, we review the composition and biological functions of RPA complexes.

  14. Database Replication Prototype

    OpenAIRE

    Vandewall, R.

    2000-01-01

    This report describes the design of a Replication Framework that facilitates the implementation and com-parison of database replication techniques. Furthermore, it discusses the implementation of a Database Replication Prototype and compares the performance measurements of two replication techniques based on the Atomic Broadcast communication primitive: pessimistic active replication and optimistic active replication. The main contributions of this report can be split into four parts....

  15. Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex.

    Science.gov (United States)

    Hiraga, Shin-Ichiro; Alvino, Gina M; Chang, Fujung; Lian, Hui-Yong; Sridhar, Akila; Kubota, Takashi; Brewer, Bonita J; Weinreich, Michael; Raghuraman, M K; Donaldson, Anne D

    2014-02-15

    Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

  16. The role of IREB2 and transforming growth factor beta-1 genetic variants in COPD: a replication case-control study

    LENUS (Irish Health Repository)

    Chappell, Sally L

    2011-02-14

    Abstract Background Genetic factors are known to contribute to COPD susceptibility and these factors are not fully understood. Conflicting results have been reported for many genetic studies of candidate genes based on their role in the disease. Genome-wide association studies in combination with expression profiling have identified a number of new candidates including IREB2. A meta-analysis has implicated transforming growth factor beta-1 (TGFbeta1) as a contributor to disease susceptibility. Methods We have examined previously reported associations in both genes in a collection of 1017 white COPD patients and 912 non-diseased smoking controls. Genotype information was obtained for seven SNPs in the IREB2 gene, and for four SNPs in the TGFbeta1 gene. Allele and genotype frequencies were compared between COPD cases and controls, and odds ratios were calculated. The analysis was adjusted for age, sex, smoking and centre, including interactions of age, sex and smoking with centre. Results Our data replicate the association of IREB2 SNPs in association with COPD for SNP rs2568494, rs2656069 and rs12593229 with respective adjusted p-values of 0.0018, 0.0039 and 0.0053. No significant associations were identified for TGFbeta1. Conclusions These studies have therefore confirmed that the IREB2 locus is a contributor to COPD susceptibility and suggests a new pathway in COPD pathogenesis invoking iron homeostasis.

  17. A systematic analysis of host factors reveals a Med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication.

    Science.gov (United States)

    Griffiths, Samantha J; Koegl, Manfred; Boutell, Chris; Zenner, Helen L; Crump, Colin M; Pica, Francesca; Gonzalez, Orland; Friedel, Caroline C; Barry, Gerald; Martin, Kim; Craigon, Marie H; Chen, Rui; Kaza, Lakshmi N; Fossum, Even; Fazakerley, John K; Efstathiou, Stacey; Volpi, Antonio; Zimmer, Ralf; Ghazal, Peter; Haas, Jürgen

    2013-01-01

    Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to

  18. A systematic analysis of host factors reveals a Med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication.

    Directory of Open Access Journals (Sweden)

    Samantha J Griffiths

    Full Text Available Herpes simplex virus type 1 (HSV-1 is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi screen with a druggable genome small interfering RNA (siRNA library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome

  19. A new MCM modification cycle regulates DNA replication initiation.

    Science.gov (United States)

    Wei, Lei; Zhao, Xiaolan

    2016-03-01

    The MCM DNA helicase is a central regulatory target during genome replication. MCM is kept inactive during G1, and it initiates replication after being activated in S phase. During this transition, the only known chemical change to MCM is the gain of multisite phosphorylation that promotes cofactor recruitment. Because replication initiation is intimately linked to multiple biological cues, additional changes to MCM can provide further regulatory points. Here, we describe a yeast MCM SUMOylation cycle that regulates replication. MCM subunits undergo SUMOylation upon loading at origins in G1 before MCM phosphorylation. MCM SUMOylation levels then decline as MCM phosphorylation levels rise, thus suggesting an inhibitory role of MCM SUMOylation during replication. Indeed, increasing MCM SUMOylation impairs replication initiation, partly through promoting the recruitment of a phosphatase that decreases MCM phosphorylation and activation. We propose that MCM SUMOylation counterbalances kinase-based regulation, thus ensuring accurate control of replication initiation.

  20. Viral and Host Factors Required for Avian H5N1 Influenza A Virus Replication in Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Hong Zhang

    2013-06-01

    Full Text Available Following the initial and sporadic emergence into humans of highly pathogenic avian H5N1 influenza A viruses in Hong Kong in 1997, we have come to realize the potential for avian influenza A viruses to be transmitted directly from birds to humans. Understanding the basic viral and cellular mechanisms that contribute to infection of mammalian species with avian influenza viruses is essential for developing prevention and control measures against possible future human pandemics. Multiple physical and functional cellular barriers can restrict influenza A virus infection in a new host species, including the cell membrane, the nuclear envelope, the nuclear environment, and innate antiviral responses. In this review, we summarize current knowledge on viral and host factors required for avian H5N1 influenza A viruses to successfully establish infections in mammalian cells. We focus on the molecular mechanisms underpinning mammalian host restrictions, as well as the adaptive mutations that are necessary for an avian influenza virus to overcome them. It is likely that many more viral and host determinants remain to be discovered, and future research in this area should provide novel and translational insights into the biology of influenza virus-host interactions.

  1. An Alternative Form of Replication Protein A Prevents Viral Replication in Vitro*

    OpenAIRE

    Mason, Aaron C.; Haring, Stuart J.; Pryor, John M.; Staloch, Cathy A.; Gan, Tze Fei; Wold, Marc S.

    2009-01-01

    Replication protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism. The “canonical” RPA is composed of three subunits (RPA1, RPA2, and RPA3); however, there is a human homolog to the RPA2 subunit, called RPA4, that can substitute for RPA2 in complex formation. We demonstrate that the resulting “alternative” RPA (aRPA) complex has solution and DNA binding properties indistinguishable from the c...

  2. Genetic structure of the snakehead murrel, Channa striata (channidae) based on the cytochrome c oxidase subunit I gene: Influence of historical and geomorphological factors.

    Science.gov (United States)

    Jamsari, Amirul Firdaus Jamaluddin; Jamaluddin, Jamsari Amirul Firdaus; Pau, Tan Min; Siti-Azizah, Mohd Nor

    2011-01-01

    Nucleotide sequences of a partial cytochrome c oxidase subunit I gene were used to assess the manner in which historical processes and geomorphological effects may have influenced genetic structuring and phylogeographic patterns in Channa striata. Assaying was based on individuals from twelve populations in four river systems, which were separated into two regions, the eastern and western, of the biodiversely rich state of Perak in central Peninsular Malaysia. In 238 specimens, a total of 368-bp sequences with ten polymorphic sites and eleven unique haplotypes were detected. Data on all the twelve populations revealed incomplete divergence due to past historical coalescence and the short period of separation. Nevertheless, SAMOVA and F(ST) revealed geographical structuring existed to a certain extent in both regions. For the eastern region, the data also showed that the upstream populations were genetically significantly different compared to the mid- and downstream ones. It is inferred that physical barriers and historical processes played a dominant role in structuring the genetic dispersal of the species. A further inference is that the Grik, Tanjung Rambutan and Sungkai are potential candidates for conservation and aquaculture programmes since they contained most of the total diversity in this area.

  3. Genetic structure of the snakehead murrel, Channa striata (channidae based on the cytochrome c oxidase subunit I gene: influence of historical and geomorphological factors

    Directory of Open Access Journals (Sweden)

    Jamsari Amirul Firdaus Jamaluddin

    2011-01-01

    Full Text Available Nucleotide sequences of a partial cytochrome c oxidase subunit I gene were used to assess the manner in which historical processes and geomorphological effects may have influenced genetic structuring and phylogeographic patterns in Channa striata. Assaying was based on individuals from twelve populations in four river systems, which were separated into two regions, the eastern and western, of the biodiversely rich state of Perak in central Peninsular Malaysia. In 238 specimens, a total of 368-bp sequences with ten polymorphic sites and eleven unique haplotypes were detected. Data on all the twelve populations revealed incomplete divergence due to past historical coalescence and the short period of separation. Nevertheless, SAMOVA and F ST revealed geographical structuring existed to a certain extent in both regions. For the eastern region, the data also showed that the upstream populations were genetically significantly different compared to the mid- and downstream ones. It is inferred that physical barriers and historical processes played a dominant role in structuring the genetic dispersal of the species. A further inference is that the Grik, Tanjung Rambutan and Sungkai are potential candidates for conservation and aquaculture programmes since they contained most of the total diversity in this area.

  4. Prelife catalysts and replicators

    OpenAIRE

    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 acti...

  5. Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation.

    Science.gov (United States)

    Deegan, Tom D; Yeeles, Joseph Tp; Diffley, John Fx

    2016-05-02

    The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  6. HIV-1 replication through hHR23A-mediated interaction of Vpr with 26S proteasome.

    Directory of Open Access Journals (Sweden)

    Ge Li

    2010-06-01

    Full Text Available HIV-1 Vpr is a virion-associated protein. Its activities link to viral pathogenesis and disease progression of HIV-infected patients. In vitro, Vpr moderately activates HIV-1 replication in proliferating T cells, but it is required for efficient viral infection and replication in vivo in non-dividing cells such as macrophages. How exactly Vpr contributes to viral replication remains elusive. We show here that Vpr stimulates HIV-1 replication at least in part through its interaction with hHR23A, a protein that binds to 19S subunit of the 26S proteasome and shuttles ubiquitinated proteins to the proteasome for degradation. The Vpr-proteasome interaction was initially discovered in fission yeast, where Vpr was shown to associate with Mts4 and Mts2, two 19S-associated proteins. The interaction of Vpr with the 19S subunit of the proteasome was further confirmed in mammalian cells where Vpr associates with the mammalian orthologues of fission yeast Mts4 and S5a. Consistently, depletion of hHR23A interrupts interaction of Vpr with proteasome in mammalian cells. Furthermore, Vpr promotes hHR23A-mediated protein-ubiquitination, and down-regulation of hHR23A using RNAi significantly reduced viral replication in non-proliferating MAGI-CCR5 cells and primary macrophages. These findings suggest that Vpr-proteasome interaction might counteract certain host restriction factor(s to stimulate viral replication in non-dividing cells.

  7. Host factors in nidovirus replication

    NARCIS (Netherlands)

    Wilde, Adriaan Hugo de

    2013-01-01

    The interplay between nidoviruses and the infected host cell was investigated. Arterivirus RNA-synthesising activity was shown to depend on intact membranes and on a cytosolic host protein which does not cosediment with the RTC. Furthermore, the immunosuppressant drug cyclosporin A (CsA) blocks

  8. Functional characterization of Bombyx mori nucleopolyhedrovirus late gene transcription and genome replication factors in the non-permissive insect cell line SF-21

    International Nuclear Information System (INIS)

    Berretta, Marcelo F.; Deshpande, Mandar; Crouch, Erin A.; Passarelli, A. Lorena

    2006-01-01

    We compared the abilities of late gene transcription and DNA replication machineries of the baculoviruses Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV) in SF-21 cells, an insect-derived cell line permissive for AcMNPV infection. It has been well established that 19 AcMNPV late expression factors (lefs) stimulate substantial levels of late gene promoter activity in SF-21 cells. Thus, we constructed a set of clones containing the BmNPV homologs of the AcMNPV lefs under control of the constitutive Drosophila heat shock 70 protein promoter and tested their ability to activate an AcMNPV late promoter-reporter gene cassette in SF-21 cells. We tested the potential of individual or predicted functional groups of BmNPV lefs to successfully replace the corresponding AcMNPV gene(s) in transient late gene expression assays. We found that most, but not all, BmNPV lefs were able to either fully or partially substitute for the corresponding AcMNPV homolog in the context of the remaining AcMNPV lefs with the exception of BmNPV p143, ie-2, and p35. BmNPV p143 was unable to support late gene expression or be imported into the nucleus of cells in the presence of the AcMNPV or the BmNPV LEF-3, a P143 nuclear shuttling factor. Our results suggest that host-specific factors may affect the function of homologous proteins

  9. INTRINSIC REGULATION OF HEMOGLOBIN EXPRESSION BY VARIABLE SUBUNIT INTERFACE STRENGTHS

    Science.gov (United States)

    Manning, James M.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, Lois R.

    2012-01-01

    SUMMARY The expression of the six types of human hemoglobin subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the “extrinsic” component of regulation). Here we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the “intrinsic” component of regulation). The adult hemoglobin dimers have the strongest subunit interfaces and the embryonic hemoglobins are the weakest with fetal hemoglobins of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these hemoglobin types form functional O2-binding tetramers consistent with gene switching. PMID:22129306

  10. Psychometric characteristics of the chronic Otitis media questionnaire 12 (COMQ - 12): stability of factor structure and replicability shown by the Serbian version.

    Science.gov (United States)

    Bukurov, Bojana; Arsovic, Nenad; Grujicic, Sandra Sipetic; Haggard, Mark; Spencer, Helen; Marinkovic, Jelena Eric

    2017-10-23

    Recently, demand for and supply of short-form patient-reported outcome measures (PROMs) have risen throughout the world healthcare. Our contribution to meeting that demand has been translating and culturally adapting the Chronic Otitis Media Questionnaire-12 (COMQ-12) for adults into Serbian and enhancing its psychometric base on the relatively large Serbian COM caseload. Chronic otitis media can seriously affect quality of life progressively and in long-term, and it remains the major source of hearing problems in the developing world. The translated questionnaire was given twice to 60 adult patients with chronic otitis media of three types (inactive, active mucosal and active squamous disease) and to 60 healthy volunteers. Both patients and volunteers also filled the generic Short-Form 36 questionnaire (SF-36). Conventional statistical procedures were used in strategically driven development of scoring. Additionally, item responses were scaled by linear mapping against the provisional total score. Generalizability, detailed factor interpretation and supportability of scores were criteria, for the best compromise factor solution. Test-retest reliability was very high (0.924 to 0.989, depending on score). The a priori content dimensions of the questionnaire were strongly supported by 3-factor exploratory and confirmatory factor analyses for content validity, separating (i) ear symptoms from (ii) hearing problems, from (iii) daily activity restriction plus healthcare uptake. The 3-factor structure was furthermore highly stable on replication. The very large effect sizes when contrasting patients with healthy volunteers, and active with inactive disease established construct validity for the total score. A strong association with disease activity and a moderate one with generic health-related quality of life (HRQoL), the SF-36, supported construct validity for two of three factors extracted (ear symptoms, and impact on daily activities plus healthcare uptake). Given

  11. Data replicating the factor structure and reliability of commonly used measures of resilience: The Connor–Davidson Resilience Scale, Resilience Scale, and Scale of Protective Factors

    Directory of Open Access Journals (Sweden)

    A.N. Madewell

    2016-09-01

    Full Text Available The data presented in this article are related to the article entitled “Assessing Resilience in Emerging Adulthood: The Resilience Scale (RS, Connor Davidson Resilience Scale (CD-RISC, and Scale of Protective Factors (SPF” (Madewell and Ponce-Garcia, 2016 [1]. The data were collected from a sample of 451 college students from three universities located in the Southwestern region of the United States: 374 from a large public university and 67 from two smaller regional universities. The data from the three universities did not significantly differ in terms of demographics. The data represent participant responses on six measurements to include the Resilience Scale-25 (RS-25, Resilience Scale-14 (RS-14, Connor Davidson Resilience Scale-25 (CD-RISC-25, Connor Davidson Resilience Scale-10 (CD-RISC-10, Scale of Protective Factors-24 (SPF-24, and the Life Stressor Checklist Revised (LSC-R. Keywords: Scale of Protective Factors, Resilience Scale, Connor–Davidson Resilience Scale, Emerging adulthood, Confirmatory factor analysis

  12. DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

    Science.gov (United States)

    Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

    2016-07-26

    DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

  13. Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

    Science.gov (United States)

    Waraky, Ahmed; Lin, Yingbo; Warsito, Dudi; Haglund, Felix; Aleem, Eiman; Larsson, Olle

    2017-11-03

    We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases ( e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G 1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Highly conserved small subunit residues influence rubisco large subunit catalysis.

    Science.gov (United States)

    Genkov, Todor; Spreitzer, Robert J

    2009-10-30

    The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

  15. Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis

    DEFF Research Database (Denmark)

    Natsume, Toyoaki; Nishimura, Kohei; Minocherhomji, Sheroy

    2017-01-01

    stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks...

  16. Replication of LCE3C-LCE3B CNV as a risk factor for psoriasis and analysis of interaction with other genetic risk factors.

    Science.gov (United States)

    Hüffmeier, Ulrike; Bergboer, Judith G M; Becker, Tim; Armour, John A; Traupe, Heiko; Estivill, Xavier; Riveira-Munoz, Eva; Mössner, Rotraut; Reich, Kristian; Kurrat, Werner; Wienker, Thomas F; Schalkwijk, Joost; Zeeuwen, Patrick L J M; Reis, André

    2010-04-01

    Recently, a deletion of two late cornified envelope (LCE) genes within the epidermal differentiation complex on chromosome 1 was shown to be overrepresented in 1,426 psoriasis vulgaris (PsV) patients of European ancestry. In this study, we report a confirmation of this finding in 1,354 PsV patients and 937 control individuals of German origin. We found an allele frequency of the deletion of 70.9% in PsV patients and of 64.9% in control individuals (chi(2)=17.44, P=2.97 x 10(-5), odds ratio (95% confidence interval)=1.31 (1.15-1.48)). The overall copy number of the two LCE genes had no influence on the age of onset, but we observed a dosage effect at the genotype level. There was no evidence of statistically significant interaction with copy number of the beta-defensin cluster on 8p23.1 or with an IL-23R pathway variant in a combined data set of German and Dutch individuals, whereas evidence for interaction with the PSORS1 risk allele in German individuals was marginal and did not remain significant after correction for multiple testing. Our study confirms the recently published finding that the deletion of the two LCE genes is a susceptibility factor for PsV with dosage effect, while, because of power limitation, no final conclusion regarding interaction with other PsV risk factors can be made at this stage.

  17. DNA replication at the single-molecule level

    NARCIS (Netherlands)

    Stratmann, S.A.; Oijen, A.M. van

    2014-01-01

    A cell can be thought of as a highly sophisticated micro factory: in a pool of billions of molecules – metabolites, structural proteins, enzymes, oligonucleotides – multi-subunit complexes assemble to perform a large number of basic cellular tasks, such as DNA replication, RNA/protein synthesis or

  18. Histochemical detection of GM1 ganglioside using cholera toxin-B subunit. Evaluation of critical factors optimal for in situ detection with special emphasis to acetone pre-extraction

    Science.gov (United States)

    Petr, T.; Šmíd, V.; Šmídová, J.; Hůlková, H.; Jirkovská, M.; Elleder, M.; Muchová, L.; Vítek, L.; Šmíd, F.

    2010-01-01

    A comparison of histochemical detection of GM1 ganglioside in cryostat sections using cholera toxin B-subunit after fixation with 4% formaldehyde and dry acetone gave tissue-dependent results. In the liver no pre-treatment showed detectable differences related to GM1 reaction products, while studies in the brain showed the superiority of acetone pre-extraction (followed by formaldehyde), which yielded sharper images compared with the diffuse, blurred staining pattern associated with formaldehyde. Therefore, the aim of our study was to define the optimal conditions for the GM1 detection using cholera toxin B-subunit. Ganglioside extractability with acetone, the ever neglected topic, was tested comparing anhydrous acetone with acetone containing admixture of water. TLC analysis of acetone extractable GM1 ganglioside from liver sections did not exceed 2% of the total GM1 ganglioside content using anhydrous acetone at −20°C, and 4% at room temperature. The loss increased to 30.5% using 9:1 acetone/water. Similarly, photometric analysis of lipid sialic acid, extracted from dried liver homogenates with anhydrous acetone, showed the loss of gangliosides into acetone 3.0±0.3% only. The loss from dried brain homogenate was 9.5±1.1%. Thus, anhydrous conditions (dry tissue samples and anhydrous acetone) are crucial factors for optimal in situ ganglioside detection using acetone pre-treatment. This ensures effective physical fixation, especially in tissues rich in polar lipids (precipitation, prevention of in situ diffusion), and removal of cholesterol, which can act as a hydrophobic blocking barrier. PMID:20558344

  19. Histochemical detection of GM1 ganglioside using cholera toxin-B subunit. Evaluation of critical factors optimal for in situ detection with special emphasis to acetone pre-extraction

    Directory of Open Access Journals (Sweden)

    T. Petr

    2010-05-01

    Full Text Available A comparison of histochemical detection of GM1 ganglioside in cryostat sections using cholera toxin B-subunit after fixation with 4% formaldehyde and dry acetone gave tissue-dependent results. In the liver no pre-treatment showed detectable differences related to GM1 reaction products, while studies in the brain showed the superiority of acetone pre-extraction (followed by formaldehyde, which yielded sharper images compared with the diffuse, blurred staining pattern associated with formaldehyde. Therefore, the aim of our study was to define the optimal conditions for the GM1 detection using cholera toxin B-subunit. Ganglioside extractability with acetone, the ever neglected topic, was tested comparing anhydrous acetone with acetone containing admixture of water. TLC analysis of acetone extractable GM1 ganglioside from liver sections did not exceed 2% of the total GM1 ganglioside content using anhydrous acetone at -20°C, and 4% at room temperature. The loss increased to 30.5% using 9:1 acetone/water. Similarly, photometric analysis of lipid sialic acid, extracted from dried liver homogenates with anhydrous acetone, showed the loss of gangliosides into acetone 3.0±0.3% only. The loss from dried brain homogenate was 9.5±1.1%. Thus, anhydrous conditions (dry tissue samples and anhydrous acetone are crucial factors for optimal in situ ganglioside detection using acetone pre-treatment. This ensures effective physical fixation, especially in tissues rich in polar lipids (precipitation, prevention of in situ diffusion, and removal of cholesterol, which can act as a hydrophobic blocking barrier.

  20. Dynamic and nucleolin-dependent localization of human cytomegalovirus UL84 to the periphery of viral replication compartments and nucleoli.

    Science.gov (United States)

    Bender, Brian J; Coen, Donald M; Strang, Blair L

    2014-10-01

    Protein-protein and protein-nucleic acid interactions within subcellular compartments are required for viral genome replication. To understand the localization of the human cytomegalovirus viral replication factor UL84 relative to other proteins involved in viral DNA synthesis and to replicating viral DNA in infected cells, we created a recombinant virus expressing a FLAG-tagged version of UL84 (UL84FLAG) and used this virus in immunofluorescence assays. UL84FLAG localization differed at early and late times of infection, transitioning from diffuse distribution throughout the nucleus to exclusion from the interior of replication compartments, with some concentration at the periphery of replication compartments with newly labeled DNA and the viral DNA polymerase subunit UL44. Early in infection, UL84FLAG colocalized with the viral single-stranded DNA binding protein UL57, but colocalization became less prominent as infection progressed. A portion of UL84FLAG also colocalized with the host nucleolar protein nucleolin at the peripheries of both replication compartments and nucleoli. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a dramatic elimination of UL84FLAG from replication compartments and other parts of the nucleus and its accumulation in the cytoplasm. Reciprocal coimmunoprecipitation of viral proteins from infected cell lysates revealed association of UL84, UL44, and nucleolin. These results indicate that UL84 localization during infection is dynamic, which is likely relevant to its functions, and suggest that its nuclear and subnuclear localization is highly dependent on direct or indirect interactions with nucleolin. Importance: The protein-protein interactions among viral and cellular proteins required for replication of the human cytomegalovirus (HCMV) DNA genome are poorly understood. We sought to understand how an enigmatic HCMV protein critical for virus replication, UL84, localizes relative to other viral and cellular

  1. A novel form of the RelA nuclear factor kappaB subunit is induced by and forms a complex with the proto-oncogene c-Myc.

    Science.gov (United States)

    Chapman, Neil R; Webster, Gill A; Gillespie, Peter J; Wilson, Brian J; Crouch, Dorothy H; Perkins, Neil D

    2002-01-01

    Members of both Myc and nuclear factor kappaB (NF-kappaB) families of transcription factors are found overexpressed or inappropriately activated in many forms of human cancer. Furthermore, NF-kappaB can induce c-Myc gene expression, suggesting that the activities of these factors are functionally linked. We have discovered that both c-Myc and v-Myc can induce a previously undescribed, truncated form of the RelA(p65) NF-kappaB subunit, RelA(p37). RelA(p37) encodes the N-terminal DNA binding and dimerization domain of RelA(p65) and would be expected to function as a trans-dominant negative inhibitor of NF-kappaB. Surprisingly, we found that RelA(p37) no longer binds to kappaB elements. This result is explained, however, by the observation that RelA(p37), but not RelA(p65), forms a high-molecular-mass complex with c-Myc. These results demonstrate a previously unknown functional and physical interaction between RelA and c-Myc with many significant implications for our understanding of the role that both proteins play in the molecular events underlying tumourigenesis. PMID:12027803

  2. A NuRD Complex from Xenopus laevis Eggs Is Essential for DNA Replication during Early Embryogenesis

    Directory of Open Access Journals (Sweden)

    Christo P. Christov

    2018-02-01

    Full Text Available DNA replication in the embryo of Xenopus laevis changes dramatically at the mid-blastula transition (MBT, with Y RNA-independent random initiation switching to Y RNA-dependent initiation at specific origins. Here, we identify xNuRD, an MTA2-containing assemblage of the nucleosome remodeling and histone deacetylation complex NuRD, as an essential factor in pre-MBT Xenopus embryos that overcomes a functional requirement for Y RNAs during DNA replication. Human NuRD complexes have a different subunit composition than xNuRD and do not support Y RNA-independent initiation of DNA replication. Blocking or immunodepletion of xNuRD inhibits DNA replication initiation in isolated nuclei in vitro and causes inhibition of DNA synthesis, developmental delay, and embryonic lethality in early embryos. xNuRD activity declines after the MBT, coinciding with dissociation of the complex and emergence of Y RNA-dependent initiation. Our data thus reveal an essential role for a NuRD complex as a DNA replication factor during early Xenopus development.

  3. Insulin-like growth factor (IGF)-I binding to a cell membrane associated IGF binding protein-3 acid-labile subunit complex in human anterior pituitary gland

    NARCIS (Netherlands)

    Wilczak, N; Kuhl, N; Chesik, D; Geerts, A; Luiten, P; De Keyser, J

    The binding characteristics of [(125) I]insulin-like growth factor (IGF)-I were studied in human brain and pituitary gland. Competition binding studies with DES(1-3)IGF-I and R-3 -IGF-I, which display high affinity for the IGF-I receptor and low affinity for IGF binding proteins (IGFBPs), were

  4. Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-limited acres.

    Science.gov (United States)

    Nelson, Donald E; Repetti, Peter P; Adams, Tom R; Creelman, Robert A; Wu, Jingrui; Warner, David C; Anstrom, Don C; Bensen, Robert J; Castiglioni, Paolo P; Donnarummo, Meghan G; Hinchey, Brendan S; Kumimoto, Roderick W; Maszle, Don R; Canales, Roger D; Krolikowski, Katherine A; Dotson, Stanton B; Gutterson, Neal; Ratcliffe, Oliver J; Heard, Jacqueline E

    2007-10-16

    Commercially improved crop performance under drought conditions has been challenging because of the complexity of the trait and the multitude of factors that influence yield. Here we report the results of a functional genomics approach that identified a transcription factor from the nuclear factor Y (NF-Y) family, AtNF-YB1, which acts through a previously undescribed mechanism to confer improved performance in Arabidopsis under drought conditions. An orthologous maize transcription factor, ZmNF-YB2, is shown to have an equivalent activity. Under water-limited conditions, transgenic maize plants with increased ZmNF-YB2 expression show tolerance to drought based on the responses of a number of stress-related parameters, including chlorophyll content, stomatal conductance, leaf temperature, reduced wilting, and maintenance of photosynthesis. These stress adaptations contribute to a grain yield advantage to maize under water-limited environments. The application of this technology has the potential to significantly impact maize production systems that experience drought.

  5. COPI is required for enterovirus 71 replication.

    Directory of Open Access Journals (Sweden)

    Jianmin Wang

    Full Text Available 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.

  6. The Assembly Factor SDHAF2 Is Dispensable for Flavination of the Catalytic Subunit of Mitochondrial Complex II in Breast Cancer Cells

    Czech Academy of Sciences Publication Activity Database

    Bezawork-Geleta, A.; Dong, L.; Rohlena, Jakub; Neužil, Jiří

    2016-01-01

    Roč. 291, č. 41 (2016), s. 21414-21420 ISSN 0021-9258 R&D Projects: GA ČR GA15-02203S; GA ČR(CZ) GA16-22823S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : cancer biology * mitochondrial respiratory chain complex * SUCCINATE-UBIQUINONE OXIDOREDUCTASE Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.125, year: 2016

  7. Regulation of beta cell replication

    DEFF Research Database (Denmark)

    Lee, Ying C; Nielsen, Jens Høiriis

    2008-01-01

    Beta cell mass, at any given time, is governed by cell differentiation, neogenesis, increased or decreased cell size (cell hypertrophy or atrophy), cell death (apoptosis), and beta cell proliferation. Nutrients, hormones and growth factors coupled with their signalling intermediates have been...... suggested to play a role in beta cell mass regulation. In addition, genetic mouse model studies have indicated that cyclins and cyclin-dependent kinases that determine cell cycle progression are involved in beta cell replication, and more recently, menin in association with cyclin-dependent kinase...... inhibitors has been demonstrated to be important in beta cell growth. In this review, we consider and highlight some aspects of cell cycle regulation in relation to beta cell replication. The role of cell cycle regulation in beta cell replication is mostly from studies in rodent models, but whether...

  8. Direct non transcriptional role of NF-Y in DNA replication.

    Science.gov (United States)

    Benatti, Paolo; Belluti, Silvia; Miotto, Benoit; Neusiedler, Julia; Dolfini, Diletta; Drac, Marjorie; Basile, Valentina; Schwob, Etienne; Mantovani, Roberto; Blow, J Julian; Imbriano, Carol

    2016-04-01

    NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Transcriptional regulators of Na, K-ATPase subunits

    Directory of Open Access Journals (Sweden)

    Zhiqin eLi

    2015-10-01

    Full Text Available The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

  10. Only one ATP-binding DnaX subunit is required for initiation complex formation by the Escherichia coli DNA polymerase III holoenzyme.

    Science.gov (United States)

    Wieczorek, Anna; Downey, Christopher D; Dallmann, H Garry; McHenry, Charles S

    2010-09-17

    The DnaX complex (DnaX(3)δδ'χ psi) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, β(2), onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. β(2) binding (determined functionally) is diminished 12-30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for β(2). DNA synthesis activity can be restored by increased concentrations of β(2). In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances β(2) loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.

  11. Surface micro topography replication in injection moulding

    DEFF Research Database (Denmark)

    Arlø, Uffe Rolf

    Thermoplastic injection moulding is a widely used industrial process that involves surface generation by replication. The surface topography of injection moulded plastic parts can be important for aesthetical or technical reasons. With the emergence of microengineering and nanotechnology additional...... importance of surface topography follows. In general the replication is not perfect and the topography of the plastic part differs from the inverse topography of the mould cavity. It is desirable to be able to control the degree of replication perfection or replication quality. This requires an understanding...... of the physical mechanisms of replication. Such understanding can lead to improved process design and facilitate in-line process quality control with respect to surface properties. The purpose of the project is to identify critical factors that affect topography replication quality and to obtain an understanding...

  12. Functions of alternative Replication Protein A (aRPA) in initiation and elongation

    OpenAIRE

    Mason, Aaron C.; Roy, Rupa; Simmons, Daniel T.; Wold, Marc S.

    2010-01-01

    Replication protein A (RPA) is a single-stranded DNA-binding complex that is essential for DNA replication, repair and recombination in eukaryotic cells. In addition to this canonical complex, we have recently characterized an alternative Replication Protein A complex (aRPA) that is unique to primates. aRPA is composed of three subunits: RPA1 and RPA3, also present in canonical RPA, and a primate-specific subunit RPA4, homologous to canonical RPA2. aRPA has biochemical properties similar to t...

  13. Uncertain Context Factors in ERP Project Estimation are an Asset: Insights from a Semi-Replication Case Study in a Financial Services Firm

    NARCIS (Netherlands)

    Daneva, Maia

    This paper reports on the findings of a case study in a company in the financial services sector in which we replicated the use of a previously published approach to systematically balance the contextual uncertainties in the estimation of Enterprise Resource Planning (ERP) projects. The approach is

  14. Mutations in Encephalomyocarditis Virus 3A Protein Uncouple the Dependency of Genome Replication on Host Factors Phosphatidylinositol 4-Kinase IIIα and Oxysterol-Binding Protein

    NARCIS (Netherlands)

    Dorobantu, Cristina M|info:eu-repo/dai/nl/372622283; Albulescu, Lucian|info:eu-repo/dai/nl/369492382; Lyoo, Heyrhyoung|info:eu-repo/dai/nl/412352931; van Kampen, Mirjam; De Francesco, Raffaele; Lohmann, Volker; Harak, Christian; van der Schaar, Hilde M|info:eu-repo/dai/nl/318007568; Strating, Jeroen R P M|info:eu-repo/dai/nl/298979594; Gorbalenya, Alexander E; van Kuppeveld, Frank J M|info:eu-repo/dai/nl/156614723

    2016-01-01

    Positive-strand RNA [(+)RNA] viruses are true masters of reprogramming host lipid trafficking and synthesis to support virus genome replication. Via their membrane-associated 3A protein, picornaviruses of the genus Enterovirus (e.g., poliovirus, coxsackievirus, and rhinovirus) subvert Golgi

  15. DNA replication and cancer

    DEFF Research Database (Denmark)

    Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

    2016-01-01

    A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways...... causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy....

  16. Platelet antiheparin activity. The isolation and characterisation of platelet factor 4 released from thrombin-aggregated washed human platelets and its dissociation into subunits and the isolation of membrane-bound antiheparin activity.

    Science.gov (United States)

    Moore, S; Pepper, D S; Cash, J D

    1975-02-27

    Platelet factor 4 was isolated by gel filtration from the soluble release products of thrombin-aggregated washed human platelets as a proteoglycan-platelet factor 4 complex of molecular weight 358 000, Stokes radius (r-s) of 14.0 nm, sedimentation coefficient (s) of 7.1 S and frictional ratio (f/f-o) of 3.04. The complex was dissociated at high ionic strength (I equals 0.75) and the proteoglycan separated from platelet factor 4 by gel filtration. Platelet factor 4 had a molecular weight of 27 100, r-s of 2.52 nm, s of 2.4 S and f/f-o of 1.26, was insoluble under physiological conditions but readily soluble at pH 3. Under these conditions platelet factor 4 dissociated into four subunits with a molecular weight of 6900, r-s of 1.92 nm, s of 0.8 S, and f/f-o of 1.52. Qualitative N-terminal amino acid analysis showed the presence of glutamic acid or glutamine as the major end group. Platelet factor 4 was compared with protamine sulphate, which has similar biological properties, by electrophoresis at pH 2.2, in which both migrated as single bands but with differing mobility, and by amino acid analysis which showed a more normal distribution of residues than occurred in protamine sulphate. Of the basic amino acids platelet factor 4 (molecular weight 27 100) contained 5.97% arginine, 3.18% histidine, and 12.31% lysine compared to protamine sulphate with 64.2% arginine, 0.6% lysine and no histidine. A partial specific volume (v) of 0.747 was calculated for platelet factor 4 from its amino acid analysis. A membrane fraction with antiheparin activity, an isopycnic density of 1.090-1.110 and r-s of 15-35 nm, was also isolated by sucrose density gradient centrifugation from the ultrasonicated insoluble platelet residue remaining after thrombin-induced aggregation of washed human platelets. Trypsin treatment of the membrane fraction neither solubilised nor destroyed the activity.

  17. Interaction of the retinoblastoma protein with Orc1 and its recruitment to human origins of DNA replication.

    Directory of Open Access Journals (Sweden)

    Ramiro Mendoza-Maldonado

    Full Text Available BACKGROUND: The retinoblastoma protein (Rb is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1 specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. CONCLUSIONS/SIGNIFICANCE: The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of genes required for cell cycle progression to the activation of DNA replication.

  18. Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the Minichromosome Maintenance (MCM) Complex*

    Science.gov (United States)

    Douglas, Max E.

    2016-01-01

    Mcm10 is required for the initiation of eukaryotic DNA replication and contributes in some unknown way to the activation of the Cdc45-MCM-GINS (CMG) helicase. How Mcm10 is localized to sites of replication initiation is unclear, as current models indicate that direct binding to minichromosome maintenance (MCM) plays a role, but the details and functional importance of this interaction have not been determined. Here, we show that purified Mcm10 can bind both DNA-bound double hexamers and soluble single hexamers of MCM. The binding of Mcm10 to MCM requires the Mcm10 C terminus. Moreover, the binding site for Mcm10 on MCM includes the Mcm2 and Mcm6 subunits and overlaps that for the loading factor Cdt1. Whether Mcm10 recruitment to replication origins depends on CMG helicase assembly has been unclear. We show that Mcm10 recruitment occurs via two modes: low affinity recruitment in the absence of CMG assembly (“G1-like”) and high affinity recruitment when CMG assembly takes place (“S-phase-like”). Mcm10 that cannot bind directly to MCM is defective in both modes of recruitment and is unable to support DNA replication. These findings indicate that Mcm10 is localized to replication initiation sites by directly binding MCM through the Mcm10 C terminus. PMID:26719337

  19. Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the Minichromosome Maintenance (MCM) Complex.

    Science.gov (United States)

    Douglas, Max E; Diffley, John F X

    2016-03-11

    Mcm10 is required for the initiation of eukaryotic DNA replication and contributes in some unknown way to the activation of the Cdc45-MCM-GINS (CMG) helicase. How Mcm10 is localized to sites of replication initiation is unclear, as current models indicate that direct binding to minichromosome maintenance (MCM) plays a role, but the details and functional importance of this interaction have not been determined. Here, we show that purified Mcm10 can bind both DNA-bound double hexamers and soluble single hexamers of MCM. The binding of Mcm10 to MCM requires the Mcm10 C terminus. Moreover, the binding site for Mcm10 on MCM includes the Mcm2 and Mcm6 subunits and overlaps that for the loading factor Cdt1. Whether Mcm10 recruitment to replication origins depends on CMG helicase assembly has been unclear. We show that Mcm10 recruitment occurs via two modes: low affinity recruitment in the absence of CMG assembly ("G1-like") and high affinity recruitment when CMG assembly takes place ("S-phase-like"). Mcm10 that cannot bind directly to MCM is defective in both modes of recruitment and is unable to support DNA replication. These findings indicate that Mcm10 is localized to replication initiation sites by directly binding MCM through the Mcm10 C terminus. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.

    Science.gov (United States)

    Mulero, Maria Carmen; Shahabi, Shandy; Ko, Myung Soo; Schiffer, Jamie M; Huang, De-Bin; Wang, Vivien Ya-Fan; Amaro, Rommie E; Huxford, Tom; Ghosh, Gourisankar

    2018-05-22

    Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.

  1. NF-κB p65 Subunit Is Modulated by Latent Transforming Growth Factor-β Binding Protein 2 (LTBP2 in Nasopharyngeal Carcinoma HONE1 and HK1 Cells.

    Directory of Open Access Journals (Sweden)

    Rebecca Kan

    Full Text Available NF-κB is a well-characterized transcription factor, widely known as a key player in tumor-derived inflammation and cancer development. Herein, we present the functional and molecular relevance of the canonical NF-κB p65 subunit in nasopharyngeal carcinoma (NPC. Loss- and gain-of-function approaches were utilized to reveal the functional characteristics of p65 in propagating tumor growth, tumor-associated angiogenesis, and epithelial-to-mesenchymal transition in NPC cells. Extracellular inflammatory stimuli are critical factors that trigger the NF-κB p65 signaling; hence, we investigated the components of the tumor microenvironment that might potentially influence the p65 signaling pathway. This led to the identification of an extracellular matrix (ECM protein that was previously reported as a candidate tumor suppressor in NPC. Our studies on the Latent Transforming Growth Factor-β Binding Protein 2 (LTBP2 protein provides substantial evidence that it can modulate the p65 transcriptional activity. Re-expression of LTBP2 elicits tumor suppressive effects that parallel the inactivation of p65 in NPC cells. LTBP2 was able to reduce phosphorylation of p65 at Serine 536, inhibit nuclear localization of active phosphorylated p65, and impair the p65 DNA-binding ability. This results in a consequential down-regulation of p65-related gene expression. Therefore, the data suggest that the overall up-regulation of p65 expression and the loss of this candidate ECM tumor suppressor are milestone events contributing to NPC development.

  2. Who Needs Replication?

    Science.gov (United States)

    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…

  3. Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6 causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

    Directory of Open Access Journals (Sweden)

    Takao Sasado

    Full Text Available Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68. CPSF6 is a component of the Cleavage Factor Im complex (CFIm which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3'UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3'UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3'UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

  4. Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

    Science.gov (United States)

    Sasado, Takao; Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

    2017-01-01

    Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3'UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3'UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3'UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

  5. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    Directory of Open Access Journals (Sweden)

    Natalie R Lazar Adler

    Full Text Available Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA. Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE. A single mutant (bpaC was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA, those attenuated for virulence and net intracellular replication (BpaE, the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA. Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors

  6. Amplification of the Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 lytic origin of DNA replication is dependent upon a cis-acting AT-rich region and an ORF50 response element and the trans-acting factors ORF50 (K-Rta) and K8 (K-bZIP)

    International Nuclear Information System (INIS)

    AuCoin, David P.; Colletti, Kelly S.; Cei, Sylvia A.; Papouskova, Iva; Tarrant, Margaret; Pari, Gregory S.

    2004-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), has significant sequence homology to Epstein-Barr virus (EBV). In cell culture, HHV8 is primarily latent, and viral genes associated with lytic replication are not expressed. Two lytic origins of DNA replication (oriLyt) are present within the HHV8 genome and are composed of an AT-rich region adjacent to GC-rich DNA sequences. We have now identified essential cis- and trans-acting elements required for oriLyt-dependent DNA replication. The transient replication assay was used to show that two AT-rich elements, three consensus AP1 transcription factor-binding sites, an ORF50 response element (RE), and a consensus TATA box motif are essential for efficient origin-dependent DNA replication. Transient transfection of luciferase reporter constructs indicated that the downstream region of the HHV8 oriLyt responds to ORF50 and suggests that part of the oriLyt may be an enhancer/promoter. In addition, a transient cotransfection-replication assay elucidated the set of trans-acting factors required for lytic DNA replication. These factors consist of homologues to the core replication proteins: ORF6 (ssDNA binding protein), ORF9 (DNA polymerase), ORF40-41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), and ORF59 (polymerase processivity factor) common to all herpesviruses along with ORF50 (K-Rta) and K8 (K-bZIP)

  7. Registered Replication Report

    DEFF Research Database (Denmark)

    Bouwmeester, S.; Verkoeijen, P. P.J.L.; Aczel, B.

    2017-01-01

    and colleagues. The results of studies using time pressure have been mixed, with some replication attempts observing similar patterns (e.g., Rand et al., 2014) and others observing null effects (e.g., Tinghög et al., 2013; Verkoeijen & Bouwmeester, 2014). This Registered Replication Report (RRR) assessed...... the size and variability of the effect of time pressure on cooperative decisions by combining 21 separate, preregistered replications of the critical conditions from Study 7 of the original article (Rand et al., 2012). The primary planned analysis used data from all participants who were randomly assigned...

  8. Rescue from replication stress during mitosis.

    Science.gov (United States)

    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.

  9. The replication recipe : What makes for a convincing replication?

    NARCIS (Netherlands)

    Brandt, M.J.; IJzerman, H.; Dijksterhuis, Ap; Farach, Frank J.; Geller, Jason; Giner-Sorolla, Roger; Grange, James A.; Perugini, Marco; Spies, Jeffrey R.; van 't Veer, Anna

    Psychological scientists have recently started to reconsider the importance of close replications in building a cumulative knowledge base; however, there is no consensus about what constitutes a convincing close replication study. To facilitate convincing close replication attempts we have developed

  10. The Replication Recipe: What makes for a convincing replication?

    NARCIS (Netherlands)

    Brandt, M.J.; IJzerman, H.; Dijksterhuis, A.J.; Farach, F.J.; Geller, J.; Giner-Sorolla, R.; Grange, J.A.; Perugini, M.; Spies, J.R.; Veer, A. van 't

    2014-01-01

    Psychological scientists have recently started to reconsider the importance of close replications in building a cumulative knowledge base; however, there is no consensus about what constitutes a convincing close replication study. To facilitate convincing close replication attempts we have developed

  11. Upon Infection the Cellular WD Repeat-containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication.

    Science.gov (United States)

    Ma, Dzwokai; George, Cyril X; Nomburg, Jason; Pfaller, Christian K; Cattaneo, Roberto; Samuel, Charles E

    2017-12-13

    Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5 deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication. IMPORTANCE Measles virus is a human pathogen that remains a global concern with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that

  12. The Role of the Transcriptional Response to DNA Replication Stress.

    Science.gov (United States)

    Herlihy, Anna E; de Bruin, Robertus A M

    2017-03-02

    During DNA replication many factors can result in DNA replication stress. The DNA replication stress checkpoint prevents the accumulation of replication stress-induced DNA damage and the potential ensuing genome instability. A critical role for post-translational modifications, such as phosphorylation, in the replication stress checkpoint response has been well established. However, recent work has revealed an important role for transcription in the cellular response to DNA replication stress. In this review, we will provide an overview of current knowledge of the cellular response to DNA replication stress with a specific focus on the DNA replication stress checkpoint transcriptional response and its role in the prevention of replication stress-induced DNA damage.

  13. The Role of the Transcriptional Response to DNA Replication Stress

    Science.gov (United States)

    Herlihy, Anna E.; de Bruin, Robertus A.M.

    2017-01-01

    During DNA replication many factors can result in DNA replication stress. The DNA replication stress checkpoint prevents the accumulation of replication stress-induced DNA damage and the potential ensuing genome instability. A critical role for post-translational modifications, such as phosphorylation, in the replication stress checkpoint response has been well established. However, recent work has revealed an important role for transcription in the cellular response to DNA replication stress. In this review, we will provide an overview of current knowledge of the cellular response to DNA replication stress with a specific focus on the DNA replication stress checkpoint transcriptional response and its role in the prevention of replication stress-induced DNA damage. PMID:28257104

  14. Molecular cloning of a second subunit of the receptor for human granulocyte - macrophage colony-stimulating factor (GM-CSF): Reconstitution of a high-affinity GM-CSF receptor

    International Nuclear Information System (INIS)

    Hayashida, Kazuhiro; Kitamura, Toshio; Gorman, D.M.; Miyajima, Atsushi; Arai, Kenichi; Yokota, Takashi

    1990-01-01

    Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, the authors obtained a monologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte - macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of 125 I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells - i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. They therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the α and β subunits of the GM-CSF receptor, respectively

  15. Eukaryotic DNA Replication Fork.

    Science.gov (United States)

    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.

  16. Modeling DNA Replication.

    Science.gov (United States)

    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)

  17. Characterization of fimbrial subunits from Bordetella species

    NARCIS (Netherlands)

    Mooi, F.R.; Heide, H.G.J. van der; Avest, A.R. ter; Welinder, K.G.; Livey, I.; Zeijst, B.A.M. van der; Gaastra, W.

    Using antisera raised against serotype 2 and 3 fimbrial subunits from Bordetella pertussis, serologically related polypeptides were detected in Bordetella bronchiseptica, Bordetella parapertussis and Bordetella avium strains. The two B. pertussis fimbrial subunits, and three of the serologically

  18. Structural disorder in proteins of the rhabdoviridae replication complex.

    Science.gov (United States)

    Leyrat, Cédric; Gérard, Francine C A; de Almeida Ribeiro, Euripedes; Ivanov, Ivan; Ruigrok, Rob W H; Jamin, Marc

    2010-08-01

    Rhabdoviridae are single stranded negative sense RNA viruses. The viral RNA condensed by the nucleoprotein (N), the phosphoprotein (P) and the large subunit (L) of the RNA-dependent RNA polymerase are the viral components of the transcription/replication machineries. Both P and N contain intrinsically disordered regions (IDRs) that play different roles in the virus life cycle. Here, we describe the modular organization of P based on recent structural, biophysical and bioinformatics data. We show how flexible loops in N participate in the attachment of P to the N-RNA template by an induced-fit mechanism. Finally, we discuss the roles of IDRs in the mechanism of replication/transcription, and propose a new model for the interaction of the L subunit with its N-RNA template.

  19. Optical tweezers reveal how proteins alter replication

    Science.gov (United States)

    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

  20. Therapeutic potential of Mediator complex subunits in metabolic diseases.

    Science.gov (United States)

    Ranjan, Amol; Ansari, Suraiya A

    2018-01-01

    The multisubunit Mediator is an evolutionary conserved transcriptional coregulatory complex in eukaryotes. It is needed for the transcriptional regulation of gene expression in general as well as in a gene specific manner. Mediator complex subunits interact with different transcription factors as well as components of RNA Pol II transcription initiation complex and in doing so act as a bridge between gene specific transcription factors and general Pol II transcription machinery. Specific interaction of various Mediator subunits with nuclear receptors (NRs) and other transcription factors involved in metabolism has been reported in different studies. Evidences indicate that ligand-activated NRs recruit Mediator complex for RNA Pol II-dependent gene transcription. These NRs have been explored as therapeutic targets in different metabolic diseases; however, they show side-effects as targets due to their overlapping involvement in different signaling pathways. Here we discuss the interaction of various Mediator subunits with transcription factors involved in metabolism and whether specific interaction of these transcription factors with Mediator subunits could be potentially utilized as therapeutic strategy in a variety of metabolic diseases. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  1. Surface Micro Topography Replication in Injection Moulding

    DEFF Research Database (Denmark)

    Arlø, Uffe Rolf; Hansen, Hans Nørgaard; Kjær, Erik Michael

    2005-01-01

    The surface micro topography of injection moulded plastic parts can be important for aesthetical and technical reasons. The quality of replication of mould surface topography onto the plastic surface depends among other factors on the process conditions. A study of this relationship has been...... carried out with rough EDM (electrical discharge machining) mould surfaces, a PS grade, and by applying established three-dimensional topography parameters. Significant quantitative relationships between process parameters and topography parameters were established. It further appeared that replication...

  2. Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication.

    Science.gov (United States)

    Zou, Wei; Wang, Zekun; Xiong, Min; Chen, Aaron Yun; Xu, Peng; Ganaie, Safder S; Badawi, Yomna; Kleiboeker, Steve; Nishimune, Hiroshi; Ye, Shui Qing; Qiu, Jianming

    2018-03-01

    Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication. IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly

  3. α-4 subunit of nicotinic acetylcholine receptor polymorphisms exhibit ...

    African Journals Online (AJOL)

    Background: Smoking behavior is influenced by both genetic and environmental factors. Nicotine is the major addictive substance in cigarettes. Nicotinic acetylcholine receptors (nAChRs) are thought to play an important role in nicotine addiction of smokers. One of the genes, α-4 subunit of nicotinic acetylcholine receptor ...

  4. Targeting DNA Replication Stress for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Jun Zhang

    2016-08-01

    Full Text Available 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.

  5. Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

    Directory of Open Access Journals (Sweden)

    Keegan J. Baldauf

    2015-03-01

    Full Text Available Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT, which consists of two subunits: the A subunit (CTA and the B subunit (CTB. CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction.

  6. Replication studies in longevity

    DEFF Research Database (Denmark)

    Varcasia, O; Garasto, S; Rizza, T

    2001-01-01

    In Danes we replicated the 3'APOB-VNTR gene/longevity association study previously carried out in Italians, by which the Small alleles (less than 35 repeats) had been identified as frailty alleles for longevity. In Danes, neither genotype nor allele frequencies differed between centenarians and 20...

  7. Commercial Building Partnerships Replication and Diffusion

    Energy Technology Data Exchange (ETDEWEB)

    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.

  8. Protection of Mice from Lethal Vaccinia Virus Infection by Vaccinia Virus Protein Subunits with a CpG Adjuvant

    Directory of Open Access Journals (Sweden)

    Sarah Reeman

    2017-12-01

    Full Text Available Smallpox vaccination carries a high risk of adverse events in recipients with a variety of contra-indications for live vaccines. Although alternative non-replicating vaccines have been described in the form of replication-deficient vaccine viruses, DNA vaccines, and subunit vaccines, these are less efficacious than replicating vaccines in animal models. DNA and subunit vaccines in particular have not been shown to give equivalent protection to the traditional replicating smallpox vaccine. We show here that combinations of the orthopoxvirus A27, A33, B5 and L1 proteins give differing levels of protection when administered in different combinations with different adjuvants. In particular, the combination of B5 and A27 proteins adjuvanted with CpG oligodeoxynucleotides (ODN gives a level of protection in mice that is equivalent to the Lister traditional vaccine in a lethal vaccinia virus challenge model.

  9. From structure to mechanism—understanding initiation of DNA replication

    Science.gov (United States)

    Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L. Maximilian; Schneider, Sarah; Speck, Christian

    2017-01-01

    DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2–7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. PMID:28717046

  10. Trovafloxacin-induced replication stress sensitizes HepG2 cells to tumor necrosis factor-alpha-induced cytotoxicity mediated by extracellular signal-regulated kinase and ataxia telangiectasia and Rad3-related

    International Nuclear Information System (INIS)

    Beggs, Kevin M.; Maiuri, Ashley R.; Fullerton, Aaron M.; Poulsen, Kyle L.; Breier, Anna B.; Ganey, Patricia E.; Roth, Robert A.

    2015-01-01

    Use of the fluoroquinolone antibiotic trovafloxacin (TVX) was restricted due to idiosyncratic, drug-induced liver injury (IDILI). Previous studies demonstrated that tumor necrosis factor-alpha (TNF) and TVX interact to cause death of hepatocytes in vitro that was associated with prolonged activation of c-Jun N-terminal kinase (JNK), activation of caspases 9 and 3, and DNA damage. The purpose of this study was to explore further the mechanism by which TVX interacts with TNF to cause cytotoxicity. Treatment with TVX caused cell cycle arrest, enhanced expression of p21 and impaired proliferation, but cell death only occurred after cotreatment with TVX and TNF. Cell death involved activation of extracellular signal-related kinase (ERK), which in turn activated caspase 3 and ataxia telangiectasia and Rad3-related (ATR), both of which contributed to cytotoxicity. Cotreatment of HepG2 cells with TVX and TNF caused double-strand breaks in DNA, and ERK contributed to this effect. Inhibition of caspase activity abolished the DNA strand breaks. The data suggest a complex interaction of TVX and TNF in which TVX causes replication stress, and the downstream effects are exacerbated by TNF, leading to hepatocellular death. These results raise the possibility that IDILI from TVX results from MAPK and ATR activation in hepatocytes initiated by interaction of cytokine signaling with drug-induced replication stress

  11. Essential and non-essential DNA replication genes in the model halophilic Archaeon, Halobacterium sp. NRC-1

    Directory of Open Access Journals (Sweden)

    DasSarma Shiladitya

    2007-06-01

    Full Text Available Abstract Background Information transfer systems in Archaea, including many components of the DNA replication machinery, are similar to those found in eukaryotes. Functional assignments of archaeal DNA replication genes have been primarily based upon sequence homology and biochemical studies of replisome components, but few genetic studies have been conducted thus far. We have developed a tractable genetic system for knockout analysis of genes in the model halophilic archaeon, Halobacterium sp. NRC-1, and used it to determine which DNA replication genes are essential. Results Using a directed in-frame gene knockout method in Halobacterium sp. NRC-1, we examined nineteen genes predicted to be involved in DNA replication. Preliminary bioinformatic analysis of the large haloarchaeal Orc/Cdc6 family, related to eukaryotic Orc1 and Cdc6, showed five distinct clades of Orc/Cdc6 proteins conserved in all sequenced haloarchaea. Of ten orc/cdc6 genes in Halobacterium sp. NRC-1, only two were found to be essential, orc10, on the large chromosome, and orc2, on the minichromosome, pNRC200. Of the three replicative-type DNA polymerase genes, two were essential: the chromosomally encoded B family, polB1, and the chromosomally encoded euryarchaeal-specific D family, polD1/D2 (formerly called polA1/polA2 in the Halobacterium sp. NRC-1 genome sequence. The pNRC200-encoded B family polymerase, polB2, was non-essential. Accessory genes for DNA replication initiation and elongation factors, including the putative replicative helicase, mcm, the eukaryotic-type DNA primase, pri1/pri2, the DNA polymerase sliding clamp, pcn, and the flap endonuclease, rad2, were all essential. Targeted genes were classified as non-essential if knockouts were obtained and essential based on statistical analysis and/or by demonstrating the inability to isolate chromosomal knockouts except in the presence of a complementing plasmid copy of the gene. Conclusion The results showed that ten

  12. Subunit Stoichiometry of Human Muscle Chloride Channels

    OpenAIRE

    Fahlke, Christoph; Knittle, Timothy; Gurnett, Christina A.; Campbell, Kevin P.; George, Alfred L.

    1997-01-01

    Voltage-gated Cl? channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl? channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their ...

  13. Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing

    International Nuclear Information System (INIS)

    Kvissel, Anne-Katrine; Orstavik, Sigurd; Eikvar, Sissel; Brede, Gaute; Jahnsen, Tore; Collas, Philippe; Akusjaervi, Goeran; Skalhegg, Bjorn Steen

    2007-01-01

    Protein kinase A (PKA) is a holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Stimulation by cAMP dissociates the holoenzyme and causes translocation to the nucleus of a fraction of the C subunit. Apart from transcription regulation, little is known about the function of the C subunit in the nucleus. In the present report, we show that both Cα and Cβ are localized to spots in the mammalian nucleus. Double immunofluorescence analysis of splicing factor SC35 with the C subunit indicated that these spots are splicing factor compartments (SFCs). Using the E1A in vivo splicing assay, we found that catalytically active C subunits regulate alternative splicing and phosphorylate several members of the SR-protein family of splicing factors in vitro. Furthermore, nuclear C subunits co-localize with the C subunit-binding protein homologous to AKAP95, HA95. HA95 also regulates E1A alternative splicing in vivo, apparently through its N-terminal domain. Localization of the C subunit to SFCs and the E1A splicing pattern were unaffected by cAMP stimulation. Our findings demonstrate that the nuclear PKA C subunit co-locates with HA95 in SFCs and regulates pre-mRNA splicing, possibly through a cAMP-independent mechanism

  14. Caregiving and Developmental Factors Differentiating Young At-Risk Urban Children Showing Resilient Versus Stress-Affected Outcomes: A Replication and Extension.

    Science.gov (United States)

    Wyman, Peter A.; And Others

    1999-01-01

    Tested hypotheses from an organizational-developmental model for childhood resilience among 7- to 9-year olds. Found that caregiving factors and early development differentiated children with resilient and stress-affected adaptations. Variables reflecting emotionally responsive, competent parenting were direct, proximal predictors of resilience…

  15. Porcine granulocyte-colony stimulating factor (G-CSF) delivered via replication-defective adenovirus induces a sustained increase in circulating peripheral blood neutrophils

    Science.gov (United States)

    The use of immunomodulators is a promising area for biotherapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease, particularly during periods of peak disease incidence. Cytokines, including granulocyte colony-stimulating factor (G-CSF), are one class of compounds that...

  16. Arabidopsis cotyledon chloroplast biogenesis factor CYO1 uses glutathione as an electron donor and interacts with PSI (A1 and A2) and PSII (CP43 and CP47) subunits.

    Science.gov (United States)

    Muranaka, Atsuko; Watanabe, Shunsuke; Sakamoto, Atsushi; Shimada, Hiroshi

    2012-08-15

    CYO1 is required for thylakoid biogenesis in cotyledons of Arabidopsis thaliana. To elucidate the enzymatic characteristics of CYO1, we analyzed the protein disulfide isomerase (PDI) activity of CYO1 using dieosin glutathione disulfide (Di-E-GSSG) as a substrate. The reductase activity of CYO1 increased as a function of Di-E-GSSG, with an apparent K(m) of 824nM and K(cat) of 0.53min(-1). PDI catalyzes dithiol/disulfide interchange reactions, and the cysteine residues in PDI proteins are very important. To analyze the significance of the cysteine residues for the PDI activity of CYO1, we estimated the kinetic parameters of point-mutated CYO1 proteins. C117S, C124S, C135S, and C156S had higher values for K(m) than did wild-type CYO1. C158S had a similar K(m) but a higher K(cat), and C138S and C161S had similar K(m) values but lower K(cat) values than did wild-type CYO1. These results suggested that the cysteine residues at positions 138 and 161 were important for PDI activity. Low PDI activity of CYO1 was observed when NADPH or NADH was used as an electron donor. However, PDI activity was observed with CYO1 and glutathione, suggesting that glutathione may serve as a reducing agent for CYO1 in vivo. Based on analysis with the split-ubiquitin system, CYO1 interacted with the A1 and A2 subunits of PSI and the CP43 and CP47 subunits of PSII. Thus, CYO1 may accelerate the folding of cysteine residue--containing PSI and PSII subunits by repeatedly breaking and creating disulfide bonds. Copyright © 2012 Elsevier GmbH. All rights reserved.

  17. Mechanisms of DNA replication termination.

    Science.gov (United States)

    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.

  18. Role of the Rubisco Small Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert Joseph [Univ. of Nebraska, Lincoln, NE (United States)

    2016-11-05

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. However, it is a slow enzyme, and O2 competes with CO2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO2. If carboxylation could be increased or oxygenation decreased, an increase in net CO2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants, and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO2/O2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a

  19. BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation

    DEFF Research Database (Denmark)

    Feng, Yunpeng; Vlassis, Arsenios; Roques, Céline

    2016-01-01

    implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD finger-containing protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1...

  20. Chromatin replication and epigenome maintenance

    DEFF Research Database (Denmark)

    Alabert, Constance; Groth, Anja

    2012-01-01

    Stability and function of eukaryotic genomes are closely linked to chromatin structure and organization. During cell division the entire genome must be accurately replicated and the chromatin landscape reproduced on new DNA. Chromatin and nuclear structure influence where and when DNA replication...... initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA...

  1. Individual variation and hormonal modulation of a sodium channel beta subunit in the electric organ correlate with variation in a social signal.

    Science.gov (United States)

    Liu, He; Wu, Ming-Ming; Zakon, Harold H

    2007-09-01

    The sodium channel beta1 subunit affects sodium channel gating and surface density, but little is known about the factors that regulate beta1 expression or its participation in the fine control of cellular excitability. In this study we examined whether graded expression of the beta1 subunit contributes to the gradient in sodium current inactivation, which is tightly controlled and directly related to a social behavior, the electric organ discharge (EOD), in a weakly electric fish Sternopygus macrurus. We found the mRNA and protein levels of beta1 in the electric organ both correlate with EOD frequency. We identified a novel mRNA splice form of this gene and found the splicing preference for this novel splice form also correlates with EOD frequency. Androgen implants lowered EOD frequency and decreased the beta1 mRNA level but did not affect splicing. Coexpression of each splice form in Xenopus oocytes with either the human muscle sodium channel gene, hNav1.4, or a Sternopygus ortholog, smNav1.4b, sped the rate of inactivation of the sodium current and shifted the steady-state inactivation toward less negative membrane potentials. The translational product of the novel mRNA splice form lacks a previously identified important tyrosine residue but still functions normally. The properties of the fish alpha and coexpressed beta1 subunits in the oocyte replicate those of the electric organ's endogenous sodium current. These data highlight the role of ion channel beta subunits in regulating cellular excitability.

  2. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

    Boldyreff, B; Piontek, K; Schmidt-Spaniol, I

    1991-01-01

    cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies....

  3. 28 CFR 51.6 - Political subunits.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All political...

  4. Regulated eukaryotic DNA replication origin firing with purified proteins.

    Science.gov (United States)

    Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

    2015-03-26

    Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

  5. Replication of High Fetal Alcohol Spectrum Disorders Prevalence Rates, Child Characteristics, and Maternal Risk Factors in a Second Sample of Rural Communities in South Africa.

    Science.gov (United States)

    May, Philip A; De Vries, Marlene M; Marais, Anna-Susan; Kalberg, Wendy O; Buckley, David; Adnams, Colleen M; Hasken, Julie M; Tabachnick, Barbara; Robinson, Luther K; Manning, Melanie A; Bezuidenhout, Heidre; Adam, Margaret P; Jones, Kenneth L; Seedat, Soraya; Parry, Charles D H; Hoyme, H Eugene

    2017-05-12

    Background : Prevalence and characteristics of fetal alcohol syndrome (FAS) and total fetal alcohol spectrum disorders (FASD) were studied in a second sample of three South African rural communities to assess change. Methods : Active case ascertainment focused on children with height, weight and/or head circumference ≤25th centile and randomly-selected children. Final diagnoses were based on dysmorphology, neurobehavioral scores, and maternal risk interviews. Results : Cardinal facial features, head circumference, and total dysmorphology scores differentiated specific FASD diagnostic categories in a somewhat linear fashion but all FASD traits were significantly worse than those of randomly-selected controls. Neurodevelopmental delays were significantly worse for children with FASD than controls. Binge alcohol use was clearly documented as the proximal maternal risk factor for FASD, and significant distal risk factors were: low body mass, education, and income; high gravidity, parity, and age at birth of the index child. FAS rates continue to extremely high in these communities at 9-129 per 1000 children. Total FASD affect 196-276 per 1000 or 20-28% of the children in these communities. Conclusions : Very high rates of FASD persist in these general populations where regular, heavy drinking, often in a binge fashion, co-occurs with low socioeconomic conditions.

  6. Origin Licensing Requires ATP Binding and Hydrolysis by the MCM Replicative Helicase

    Science.gov (United States)

    Coster, Gideon; Frigola, Jordi; Beuron, Fabienne; Morris, Edward P.; Diffley, John F.X.

    2014-01-01

    Summary Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs. PMID:25087873

  7. Optical replication techniques for image slicers

    Czech Academy of Sciences Publication Activity Database

    Schmoll, J.; Robertson, D.J.; Dubbeldam, C.M.; Bortoletto, F.; Pína, L.; Hudec, René; Prieto, E.; Norrie, C.; Ramsay- Howat, S.

    2006-01-01

    Roč. 50, 4-5 (2006), s. 263-266 ISSN 1387-6473 Institutional research plan: CEZ:AV0Z10030501 Keywords : smart focal planes * image slicers * replication Subject RIV: BN - Astronomy, Celestial Mechanics, Astrophysics Impact factor: 1.914, year: 2006

  8. HuR and Ago2 Bind the Internal Ribosome Entry Site of Enterovirus 71 and Promote Virus Translation and Replication.

    Directory of Open Access Journals (Sweden)

    Jing-Yi Lin

    Full Text Available EV71 (enterovirus 71 RNA contains an internal ribosomal entry site (IRES that directs cap-independent initiation of translation. IRES-dependent translation requires the host's translation initiation factors and IRES-associated trans-acting factors (ITAFs. We reported recently that mRNA decay factor AUF1 is a negative-acting ITAF that binds IRES stem-loop II. We also reported that the small RNA-processing enzyme Dicer produces at least four small RNAs (vsRNAs from the EV71 IRES. One of these, vsRNA1, derived from IRES stem-loop II, reduces IRES activity and virus replication. Since its mechanism of action is unknown, we hypothesized that it might control association of ITAFs with the IRES. Here, we identified the mRNA stability factor HuR and the RISC subunit Argonaute 2 (Ago2 as two ITAFs that bind stem-loop II. In contrast to AUF1, HuR and Ago2 promote EV71 IRES activity and virus replication. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with stem-loop II. This presents a possible mechanism by which vsRNA1 could control viral translation and replication.

  9. Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

    Science.gov (United States)

    Kurat, Christoph F; Yeeles, Joseph T P; Patel, Harshil; Early, Anne; Diffley, John F X

    2017-01-05

    The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Modeling inhomogeneous DNA replication kinetics.

    Directory of Open Access Journals (Sweden)

    Michel G Gauthier

    Full Text Available 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.

  11. Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes.

    Science.gov (United States)

    Li, Hao; van der Linden, Wouter A; Verdoes, Martijn; Florea, Bogdan I; McAllister, Fiona E; Govindaswamy, Kavitha; Elias, Joshua E; Bhanot, Purnima; Overkleeft, Herman S; Bogyo, Matthew

    2014-08-15

    The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

  12. Effect of adjuvants on responses to skin immunization by microneedles coated with influenza subunit vaccine.

    Directory of Open Access Journals (Sweden)

    William C Weldon

    Full Text Available Recent studies have demonstrated the effectiveness of vaccine delivery to the skin by vaccine-coated microneedles; however there is little information on the effects of adjuvants using this approach for vaccination. Here we investigate the use of TLR ligands as adjuvants with skin-based delivery of influenza subunit vaccine. BALB/c mice received 1 µg of monovalent H1N1 subunit vaccine alone or with 1 µg of imiquimod or poly(I:C individually or in combination via coated microneedle patches inserted into the skin. Poly(I:C adjuvanted subunit influenza vaccine induced similar antigen-specific immune responses compared to vaccine alone when delivered to the skin by microneedles. However, imiquimod-adjuvanted vaccine elicited higher levels of serum IgG2a antibodies and increased hemagglutination inhibition titers compared to vaccine alone, suggesting enhanced induction of functional antibodies. In addition, imiquimod-adjuvanted vaccine induced a robust IFN-γ cellular response. These responses correlated with improved protection compared to influenza subunit vaccine alone, as well as reduced viral replication and production of pro-inflammatory cytokines in the lungs. The finding that microneedle delivery of imiquimod with influenza subunit vaccine induces improved immune responses compared to vaccine alone supports the use of TLR7 ligands as adjuvants for skin-based influenza vaccines.

  13. SUMO and KSHV Replication

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Pei-Ching [Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 112, Taiwan (China); Kung, Hsing-Jien, E-mail: hkung@nhri.org.tw [Institute for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan (China); Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616 (United States); UC Davis Cancer Center, University of California, Davis, CA 95616 (United States); Division of Molecular and Genomic Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan (China)

    2014-09-29

    Small Ubiquitin-related MOdifier (SUMO) modification was initially identified as a reversible post-translational modification that affects the regulation of diverse cellular processes, including signal transduction, protein trafficking, chromosome segregation, and DNA repair. Increasing evidence suggests that the SUMO system also plays an important role in regulating chromatin organization and transcription. It is thus not surprising that double-stranded DNA viruses, such as Kaposi’s sarcoma-associated herpesvirus (KSHV), have exploited SUMO modification as a means of modulating viral chromatin remodeling during the latent-lytic switch. In addition, SUMO regulation allows the disassembly and assembly of promyelocytic leukemia protein-nuclear bodies (PML-NBs), an intrinsic antiviral host defense, during the viral replication cycle. Overcoming PML-NB-mediated cellular intrinsic immunity is essential to allow the initial transcription and replication of the herpesvirus genome after de novo infection. As a consequence, KSHV has evolved a way as to produce multiple SUMO regulatory viral proteins to modulate the cellular SUMO environment in a dynamic way during its life cycle. Remarkably, KSHV encodes one gene product (K-bZIP) with SUMO-ligase activities and one gene product (K-Rta) that exhibits SUMO-targeting ubiquitin ligase (STUbL) activity. In addition, at least two viral products are sumoylated that have functional importance. Furthermore, sumoylation can be modulated by other viral gene products, such as the viral protein kinase Orf36. Interference with the sumoylation of specific viral targets represents a potential therapeutic strategy when treating KSHV, as well as other oncogenic herpesviruses. Here, we summarize the different ways KSHV exploits and manipulates the cellular SUMO system and explore the multi-faceted functions of SUMO during KSHV’s life cycle and pathogenesis.

  14. The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

    Science.gov (United States)

    Wieczorek, Anna; McHenry, Charles S

    2006-05-05

    The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

  15. Development and preclinical evaluation of safety and immunogenicity of an oral ETEC vaccine containing inactivated E. coli bacteria overexpressing colonization factors CFA/I, CS3, CS5 and CS6 combined with a hybrid LT/CT B subunit antigen, administered alone and together with dmLT adjuvant.

    Science.gov (United States)

    Holmgren, J; Bourgeois, L; Carlin, N; Clements, J; Gustafsson, B; Lundgren, A; Nygren, E; Tobias, J; Walker, R; Svennerholm, A-M

    2013-05-07

    A first-generation oral inactivated whole-cell enterotoxigenic Escherichia coli (ETEC) vaccine, comprising formalin-killed ETEC bacteria expressing different colonization factor (CF) antigens combined with cholera toxin B subunit (CTB), when tested in phase III studies did not significantly reduce overall (generally mild) ETEC diarrhea in travelers or children although it reduced more severe ETEC diarrhea in travelers by almost 80%. We have now developed a novel more immunogenic ETEC vaccine based on recombinant non-toxigenic E. coli strains engineered to express increased amounts of CF antigens, including CS6 as well as an ETEC-based B subunit protein (LCTBA), and the optional combination with a nontoxic double-mutant heat-labile toxin (LT) molecule (dmLT) as an adjuvant. Two test vaccines were prepared under GMP: (1) A prototype E. coli CFA/I-only formalin-killed whole-cell+LCTBA vaccine, and (2) A "complete" inactivated multivalent ETEC-CF (CFA/I, CS3, CS5 and CS6 antigens) whole-cell+LCTBA vaccine. These vaccines, when given intragastrically alone or together with dmLT in mice, were well tolerated and induced strong intestinal-mucosal IgA antibody responses as well as serum IgG and IgA responses to each of the vaccine CF antigens as well as to LT B subunit (LTB). Both mucosal and serum responses were further enhanced (adjuvanted) when the vaccines were co-administered with dmLT. We conclude that the new multivalent oral ETEC vaccine, both alone and especially in combination with the dmLT adjuvant, shows great promise for further testing in humans. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Genome-wide Control of Heterochromatin Replication by the Telomere Capping Protein TRF2.

    Science.gov (United States)

    Mendez-Bermudez, Aaron; Lototska, Liudmyla; Bauwens, Serge; Giraud-Panis, Marie-Josèphe; Croce, Olivier; Jamet, Karine; Irizar, Agurtzane; Mowinckel, Macarena; Koundrioukoff, Stephane; Nottet, Nicolas; Almouzni, Genevieve; Teulade-Fichou, Mare-Paule; Schertzer, Michael; Perderiset, Mylène; Londoño-Vallejo, Arturo; Debatisse, Michelle; Gilson, Eric; Ye, Jing

    2018-05-03

    Hard-to-replicate regions of chromosomes (e.g., pericentromeres, centromeres, and telomeres) impede replication fork progression, eventually leading, in the event of replication stress, to chromosome fragility, aging, and cancer. Our knowledge of the mechanisms controlling the stability of these regions is essentially limited to telomeres, where fragility is counteracted by the shelterin proteins. Here we show that the shelterin subunit TRF2 ensures progression of the replication fork through pericentromeric heterochromatin, but not centromeric chromatin. In a process involving its N-terminal basic domain, TRF2 binds to pericentromeric Satellite III sequences during S phase, allowing the recruitment of the G-quadruplex-resolving helicase RTEL1 to facilitate fork progression. We also show that TRF2 is required for the stability of other heterochromatic regions localized throughout the genome, paving the way for future research on heterochromatic replication and its relationship with aging and cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. DNA Replication Profiling Using Deep Sequencing.

    Science.gov (United States)

    Saayman, Xanita; Ramos-Pérez, Cristina; Brown, Grant W

    2018-01-01

    Profiling of DNA replication during progression through S phase allows a quantitative snap-shot of replication origin usage and DNA replication fork progression. We present a method for using deep sequencing data to profile DNA replication in S. cerevisiae.

  18. Hydroxyurea-Induced Replication Stress

    Directory of Open Access Journals (Sweden)

    Kenza Lahkim Bennani-Belhaj

    2010-01-01

    Full Text Available Bloom's syndrome (BS displays one of the strongest known correlations between chromosomal instability and a high risk of cancer at an early age. BS cells combine a reduced average fork velocity with constitutive endogenous replication stress. However, the response of BS cells to replication stress induced by hydroxyurea (HU, which strongly slows the progression of replication forks, remains unclear due to publication of conflicting results. Using two different cellular models of BS, we showed that BLM deficiency is not associated with sensitivity to HU, in terms of clonogenic survival, DSB generation, and SCE induction. We suggest that surviving BLM-deficient cells are selected on the basis of their ability to deal with an endogenous replication stress induced by replication fork slowing, resulting in insensitivity to HU-induced replication stress.

  19. DATABASE REPLICATION IN HETEROGENOUS PLATFORM

    OpenAIRE

    Hendro Nindito; Evaristus Didik Madyatmadja; Albert Verasius Dian Sano

    2014-01-01

    The application of diverse database technologies in enterprises today is increasingly a common practice. To provide high availability and survavibality of real-time information, a database replication technology that has capability to replicate databases under heterogenous platforms is required. The purpose of this research is to find the technology with such capability. In this research, the data source is stored in MSSQL database server running on Windows. The data will be replicated to MyS...

  20. Subunit architecture and functional modular rearrangements of the transcriptional mediator complex.

    Science.gov (United States)

    Tsai, Kuang-Lei; Tomomori-Sato, Chieri; Sato, Shigeo; Conaway, Ronald C; Conaway, Joan W; Asturias, Francisco J

    2014-06-05

    The multisubunit Mediator, comprising ∼30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA-binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Acetylcholine Receptor: Complex of Homologous Subunits

    Science.gov (United States)

    Raftery, Michael A.; Hunkapiller, Michael W.; Strader, Catherine D.; Hood, Leroy E.

    1980-06-01

    The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.

  2. APOBEC3 Interference during Replication of Viral Genomes

    Directory of Open Access Journals (Sweden)

    Luc Willems

    2015-06-01

    Full Text Available Co-evolution of viruses and their hosts has reached a fragile and dynamic equilibrium that allows viral persistence, replication and transmission. In response, infected hosts have developed strategies of defense that counteract the deleterious effects of viral infections. In particular, single-strand DNA editing by Apolipoprotein B Editing Catalytic subunits proteins 3 (APOBEC3s is a well-conserved mechanism of mammalian innate immunity that mutates and inactivates viral genomes. In this review, we describe the mechanisms of APOBEC3 editing during viral replication, the viral strategies that prevent APOBEC3 activity and the consequences of APOBEC3 modulation on viral fitness and host genome integrity. Understanding the mechanisms involved reveals new prospects for therapeutic intervention.

  3. Subunit–subunit interactions are weakened in mutant forms of acetohydroxy acid synthase insensitive to valine inhibition

    Czech Academy of Sciences Publication Activity Database

    Kyselková, Martina; Janata, Jiří; Ságová-Marečková, M.; Kopecký, J.

    2010-01-01

    Roč. 192, č. 3 (2010), s. 195-200 ISSN 0302-8933 R&D Projects: GA MŠk 2B08064 Institutional research plan: CEZ:AV0Z50200510 Keywords : Streptomyces cinnamonensis * Acetohydroxy acid synthase * Subunit-subunit interaction Subject RIV: EE - Microbiology, Virology Impact factor: 1.754, year: 2010

  4. The telomerase reverse transcriptase subunit from the dimorphic fungus Ustilago maydis.

    Directory of Open Access Journals (Sweden)

    Dolores Bautista-España

    Full Text Available In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1 contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.

  5. The Crystal Structure of PF-8, the DNA Polymerase Accessory Subunit from Kaposi's Sarcoma-Associated Herpesvirus

    Energy Technology Data Exchange (ETDEWEB)

    Baltz, Jennifer L.; Filman, David J.; Ciustea, Mihai; Silverman, Janice Elaine Y.; Lautenschlager, Catherine L.; Coen, Donald M.; Ricciardi, Robert P.; Hogle, James M.; (UPENN)

    2009-12-01

    Kaposi's sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi's sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 {angstrom}. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predicted to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8's mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.

  6. Replication of bacteriophage lambda DNA

    International Nuclear Information System (INIS)

    Tsurimoto, T.; Matsubara, K.

    1983-01-01

    In this paper results of studies on the mechanism of bacteriophage lambda replication using molecular biological and biochemical approaches are reported. The purification of the initiator proteins, O and P, and the role of the O and P proteins in the initiation of lambda DNA replication through interactions with specific DNA sequences are described. 47 references, 15 figures

  7. Pattern replication by confined dewetting

    NARCIS (Netherlands)

    Harkema, S.; Schäffer, E.; Morariu, M.D.; Steiner, U

    2003-01-01

    The dewetting of a polymer film in a confined geometry was employed in a pattern-replication process. The instability of dewetting films is pinned by a structured confining surface, thereby replicating its topographic pattern. Depending on the surface energy of the confining surface, two different

  8. Charter School Replication. Policy Guide

    Science.gov (United States)

    Rhim, Lauren Morando

    2009-01-01

    "Replication" is the practice of a single charter school board or management organization opening several more schools that are each based on the same school model. The most rapid strategy to increase the number of new high-quality charter schools available to children is to encourage the replication of existing quality schools. This policy guide…

  9. LHCb experience with LFC replication

    International Nuclear Information System (INIS)

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

    2008-01-01

    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

  10. LHCb experience with LFC replication

    CERN Document Server

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

    2008-01-01

    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.

  11. Mechanisms and regulation of DNA replication initiation in eukaryotes.

    Science.gov (United States)

    Parker, Matthew W; Botchan, Michael R; Berger, James M

    2017-04-01

    Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.

  12. Separation of replication and transcription domains in nucleoli.

    Science.gov (United States)

    Smirnov, E; Borkovec, J; Kováčik, L; Svidenská, S; Schröfel, A; Skalníková, M; Švindrych, Z; Křížek, P; Ovesný, M; Hagen, G M; Juda, P; Michalová, K; Cardoso, M C; Cmarko, D; Raška, I

    2014-12-01

    In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Suppression of Coronavirus Replication by Cyclophilin Inhibitors

    Directory of Open Access Journals (Sweden)

    Takashi Sasaki

    2013-05-01

    Full Text Available Coronaviruses infect a variety of mammalian and avian species and cause serious diseases in humans, cats, mice, and birds in the form of severe acute respiratory syndrome (SARS, feline infectious peritonitis (FIP, mouse hepatitis, and avian infectious bronchitis, respectively. No effective vaccine or treatment has been developed for SARS-coronavirus or FIP virus, both of which cause lethal diseases. It has been reported that a cyclophilin inhibitor, cyclosporin A (CsA, could inhibit the replication of coronaviruses. CsA is a well-known immunosuppressive drug that binds to cellular cyclophilins to inhibit calcineurin, a calcium-calmodulin-activated serine/threonine-specific phosphatase. The inhibition of calcineurin blocks the translocation of nuclear factor of activated T cells from the cytosol into the nucleus, thus preventing the transcription of genes encoding cytokines such as interleukin-2. Cyclophilins are peptidyl-prolyl isomerases with physiological functions that have been described for many years to include chaperone and foldase activities. Also, many viruses require cyclophilins for replication; these include human immunodeficiency virus, vesicular stomatitis virus, and hepatitis C virus. However, the molecular mechanisms leading to the suppression of viral replication differ for different viruses. This review describes the suppressive effects of CsA on coronavirus replication.

  14. The hunt for origins of DNA replication in multicellular eukaryotes

    DEFF Research Database (Denmark)

    Urban, J. M.; Foulk, M. S.; Casella, Cinzia

    2015-01-01

    Origins of DNA replication (ORIs) occur at defined regions in the genome. Although DNA sequence defines the position of ORIs in budding yeast, the factors for ORI specification remain elusive in metazoa. Several methods have been used recently to map ORIs in metazoan genomes with the hope...... that features for ORI specification might emerge. These methods are reviewed here with analysis of their advantages and shortcomings. The various factors that may influence ORI selection for initiation of DNA replication are discussed....

  15. NACSA Charter School Replication Guide: The Spectrum of Replication Options. Authorizing Matters. Replication Brief 1

    Science.gov (United States)

    O'Neill, Paul

    2010-01-01

    One of the most important and high-profile issues in public education reform today is the replication of successful public charter school programs. With more than 5,000 failing public schools in the United States, there is a tremendous need for strong alternatives for parents and students. Replicating successful charter school models is an…

  16. Identification of the ENT1 antagonists dipyridamole and dilazep as amplifiers of oncolytic herpes simplex virus-1 replication.

    Science.gov (United States)

    Passer, Brent J; Cheema, Tooba; Zhou, Bingsen; Wakimoto, Hiroaki; Zaupa, Cecile; Razmjoo, Mani; Sarte, Jason; Wu, Shulin; Wu, Chin-lee; Noah, James W; Li, Qianjun; Buolamwini, John K; Yen, Yun; Rabkin, Samuel D; Martuza, Robert L

    2010-05-15

    Oncolytic herpes simplex virus-1 (oHSV) vectors selectively replicate in tumor cells, where they kill through oncolysis while sparing normal cells. One of the drawbacks of oHSV vectors is their limited replication and spread to neighboring cancer cells. Here, we report the outcome of a high-throughput chemical library screen to identify small-molecule compounds that augment the replication of oHSV G47Delta. Of the 2,640-screened bioactives, 6 compounds were identified and subsequently validated for enhanced G47Delta replication. Two of these compounds, dipyridamole and dilazep, interfered with nucleotide metabolism by potently and directly inhibiting the equilibrative nucleoside transporter-1 (ENT1). Replicative amplification promoted by dipyridamole and dilazep were dependent on HSV mutations in ICP6, the large subunit of ribonucleotide reductase. Our results indicate that ENT1 antagonists augment oHSV replication in tumor cells by increasing cellular ribonucleoside activity. (c)2010 AACR.

  17. The mitochondrial ribosomal protein of the large subunit, Afo1p, determines cellular longevity through mitochondrial back-signaling via TOR1.

    Science.gov (United States)

    Heeren, Gino; Rinnerthaler, Mark; Laun, Peter; von Seyerl, Phyllis; Kössler, Sonja; Klinger, Harald; Hager, Matthias; Bogengruber, Edith; Jarolim, Stefanie; Simon-Nobbe, Birgit; Schüller, Christoph; Carmona-Gutierrez, Didac; Breitenbach-Koller, Lore; Mück, Christoph; Jansen-Dürr, Pidder; Criollo, Alfredo; Kroemer, Guido; Madeo, Frank; Breitenbach, Michael

    2009-07-13

    Yeast mother cell-specific aging constitutes a model of replicative aging as it occurs in stem cell populations of higher eukaryotes. Here, we present a new long-lived yeast deletion mutation,afo1 (for aging factor one), that confers a 60% increase in replicative lifespan. AFO1/MRPL25 codes for a protein that is contained in the large subunit of the mitochondrial ribosome. Double mutant experiments indicate that the longevity-increasing action of the afo1 mutation is independent of mitochondrial translation, yet involves the cytoplasmic Tor1p as well as the growth-controlling transcription factor Sfp1p. In their final cell cycle, the long-lived mutant cells do show the phenotypes of yeast apoptosis indicating that the longevity of the mutant is not caused by an inability to undergo programmed cell death. Furthermore, the afo1 mutation displays high resistance against oxidants. Despite the respiratory deficiency the mutant has paradoxical increase in growth rate compared to generic petite mutants. A comparison of the single and double mutant strains for afo1 and fob1 shows that the longevity phenotype of afo1 is independent of the formation of ERCs (ribosomal DNA minicircles). AFO1/MRPL25 function establishes a new connection between mitochondria, metabolism and aging.

  18. The Subunit Principle in Scar Face Revision.

    Science.gov (United States)

    Elshahat, Ahmed; Lashin, Riham

    2017-06-01

    Facial scaring is considered one of the most difficult cosmetic problems for any plastic surgeon to solve. The condition is more difficult if the direction of the scar is not parallel to relaxed skin tension lines. Attempts to manage this difficult situation included revisions using geometric designs, Z plasties or W plasties to camouflage the straight line visible scaring. The use of long-lasting resorbable sutures was tried too. Recently, the use of botulinum toxin during revision improved the results. Fractional CO2 lasers, microfat grafts, and platelet-rich plasma were added to the armamentarium. The scar is least visible if placed in the junction between the facial subunits. The aim of this study is to investigate the use of the subunit principle to improve the results of scar revision. Four patients were included in this study. Tissue expansion of the intact part of the subunit allowed shifting the scar to the junction between the affected subunit and the adjacent one. Tissue expansion, delivery of the expanders, and advancement of the flaps were successful in all patients. The fact that this is a 2-stage procedure and sacrifices some of the intact skin from the affected facial subunit, makes this technique reserved to patients with ugly facial scars who are ambitious to improve their appearance.

  19. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.

    Science.gov (United States)

    Burgers, P M; Kornberg, A; Sakakibara, Y

    1981-09-01

    An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold.

  20. Non‐Canonical Replication Initiation: You’re Fired!

    Directory of Open Access Journals (Sweden)

    Bazilė Ravoitytė

    2017-01-01

    Full Text Available The division of prokaryotic and eukaryotic cells produces two cells that inherit a perfect copy of the genetic material originally derived from the mother cell. The initiation of canonical DNA replication must be coordinated to the cell cycle to ensure the accuracy of genome duplication. Controlled replication initiation depends on a complex interplay of cis‐acting DNA sequences, the so‐called origins of replication (ori, with trans‐acting factors involved in the onset of DNA synthesis. The interplay of cis‐acting elements and trans‐acting factors ensures that cells initiate replication at sequence‐specific sites only once, and in a timely order, to avoid chromosomal endoreplication. However, chromosome breakage and excessive RNA:DNA hybrid formation can cause breakinduced (BIR or transcription‐initiated replication (TIR, respectively. These non‐canonical replication events are expected to affect eukaryotic genome function and maintenance, and could be important for genome evolution and disease development. In this review, we describe the difference between canonical and non‐canonical DNA replication, and focus on mechanistic differences and common features between BIR and TIR. Finally, we discuss open issues on the factors and molecular mechanisms involved in TIR.

  1. From structure to mechanism-understanding initiation of DNA replication.

    Science.gov (United States)

    Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L Maximilian; Schneider, Sarah; Speck, Christian

    2017-06-01

    DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. © 2017 Riera et al.; Published by Cold Spring Harbor Laboratory Press.

  2. Murine leukemia virus (MLV replication monitored with fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Bittner Alexandra

    2004-12-01

    Full Text Available Abstract Background Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. Results We inserted the coding sequences for green fluorescent protein (GFP into the proline-rich region (PRR of the ecotropic envelope protein (Env and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. Conclusions Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.

  3. Chromatin maturation depends on continued DNA-replication

    International Nuclear Information System (INIS)

    Schlaeger, E.J.; Puelm, W.; Knippers, R.

    1983-01-01

    The structure of [ 3 H]thymidine pulse-labeled chromatin in lymphocytes differs from that of non-replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin. These structural features of replicating chromatin rapidly disappear when the [ 3 H]thymidine pulse is followed by a chase in the presence of an excess of non-radioactive thymidine. However, when the rate of DNA replication is reduced, as in cycloheximide-treated lymphocytes, chromatin maturation is retarded. No chromatin maturation is observed when nuclei from pulse-labeled lymphocytes are incubated in vitro in the absence of DNA precursors. In contrast, when these nuclei are incubated under conditions known to be optimal for DNA replication, the structure of replicating chromatin is efficiently converted to that of 'mature', non-replicating chromatin. The authors conclude that the properties of nascent DNA and/or the distance from the replication fork are important factors in chromatin maturation. (Auth.)

  4. Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

    Science.gov (United States)

    Stoeck, Ina Karen; Lee, Ji-Young; Tabata, Keisuke; Romero-Brey, Inés; Paul, David; Schult, Philipp; Lohmann, Volker; Kaderali, Lars; Bartenschlager, Ralf

    2018-01-01

    Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell

  5. Elg1 forms an alternative RFC complex important for DNA replication and genome integrity

    NARCIS (Netherlands)

    Bellaoui, Mohammed; Chang, Michael; Ou, Jiongwen; Xu, Hong; Boone, Charles; Brown, Grant W

    2003-01-01

    Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5. This complex is distinct from the DNA replication RFC, the DNA

  6. MCM interference during licensing of DNA replication in Xenopus egg extracts-Possible Role of a C-terminal region of MCM3.

    Science.gov (United States)

    Mimura, Satoru; Kubota, Yumiko; Takisawa, Haruhiko

    2018-01-01

    The minichromosome maintenance (MCM) complex, consisting of six subunits, Mcm2-7, is loaded onto replication origins through loading factors (origin recognition complex [ORC], Cdc6, and Cdt1) and forms an MCM double hexamer that licenses the initiation of DNA replication. Previous studies with Xenopus egg extracts showed that loading factors, especially Cdc6, dissociate from chromatin on MCM loading, but the molecular mechanism and physiological significance remain largely unknown. Using a cell-free system for MCM loading onto plasmid DNA in Xenopus egg extracts, we found that MCM loaded onto DNA prevents DNA binding of the loading factors ORC, Cdc6, and Cdt1. We further report that a peptide of the C-terminal region of MCM3 (MCM3-C), previously implicated in the initial association with ORC/Cdc6 in budding yeast, prevents ORC/Cdc6/Cdt1 binding to DNA in the absence of MCM loading. ATP-γ-S suppresses inhibitory activities of both the MCM loaded onto DNA and the MCM3-C peptide. Other soluble factors in the extract, but neither MCM nor Cdt1, are required for the activity. Conservation of the amino acid sequences of MCM3-C and its activity in vertebrates implies a novel negative autoregulatory mechanism that interferes with MCM loading in the vicinity of licensed origins to ensure proper origin licensing.

  7. The DNA Replication Checkpoint Directly Regulates MBF-Dependent G1/S Transcription▿

    OpenAIRE

    Dutta, Chaitali; Patel, Prasanta K.; Rosebrock, Adam; Oliva, Anna; Leatherwood, Janet; Rhind, Nicholas

    2008-01-01

    The DNA replication checkpoint transcriptionally upregulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G1/S transcriptional program by directly regulating MBF, the G1/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G1/S transcriptional program du...

  8. Immunochemical aspects of crotoxim and its subunits

    International Nuclear Information System (INIS)

    Nakazone, A.K.

    1979-01-01

    Crotamine and crotoxin with the subunits - phospholipase A and crotapotin - were obtained by purification from Crotalus durissus terrificus venom. Interaction studies of the subunits using crotalic antiserum, indicated that: crotoxin is formed of crotapotin and phospholipase A with the molar ratio of 1 to 1; using crotapotin 125 I the presence of a soluble complex was shown with the same antiserum. Immunological precipitation reactions demonstrated that crotapotin is antigenic: crotapotin and phospholipase A presented similar antigenic determinants; crotoxin antiserum reacted with each one of the submits; when the subunits are mixed to form synthetic crotoxin some antigenic determinants are masked in the process of interaction. Crotamine, interacted with crotapotin 1:1, without hidden antigenic determinants crotapotin antigenic site seems to be formed by, at least, one lysine. Enzimatical activity of phospholipase A apreared to be dependent on some reaction conditions when its arginine residues are blocked. Tyrosines of phospholipase A are more susceptible to labelling with 131 I than crotapotin. Gama irradiation of aqueous solutions of the subunits produced modifications in the ultraviolet spectra. A decrease of the enzymatic activity occured as a function of radiation dosis. Immunological activities of crotapotin and phospholipase A were not altered [pt

  9. Escherichia coli DinB inhibits replication fork progression without significantly inducing the SOS response.

    Science.gov (United States)

    Mori, Tetsuya; Nakamura, Tatsuro; Okazaki, Naoto; Furukohri, Asako; Maki, Hisaji; Akiyama, Masahiro Tatsumi

    2012-01-01

    The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dose-dependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.

  10. REPLICATION TOOL AND METHOD OF PROVIDING A REPLICATION TOOL

    DEFF Research Database (Denmark)

    2016-01-01

    The invention relates to a replication tool (1, 1a, 1b) for producing a part (4) with a microscale textured replica surface (5a, 5b, 5c, 5d). The replication tool (1, 1a, 1b) comprises a tool surface (2a, 2b) defining a general shape of the item. The tool surface (2a, 2b) comprises a microscale...... energy directors on flange portions thereof uses the replication tool (1, 1a, 1b) to form an item (4) with a general shape as defined by the tool surface (2a, 2b). The formed item (4) comprises a microscale textured replica surface (5a, 5b, 5c, 5d) with a lateral arrangement of polydisperse microscale...

  11. Interaction of the Sliding Clamp β-Subunit and Hda, a DnaA-Related Protein

    OpenAIRE

    Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

    2004-01-01

    In Escherichia coli, interactions between the replication initiation protein DnaA, the β subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation...

  12. Physical Interaction between Replication Protein A (RPA) and MRN: Involvement of RPA2 Phosphorylation and the N-terminus of RPA1

    OpenAIRE

    Oakley, Greg; Tillison, Kristin; Opiyo, Stephen; Glanzer, Jason; Horn, Jeffrey M.; Patrick, Steve M.

    2009-01-01

    Replication protein A (RPA) is a heterotrimeric protein consisting of RPA1, RPA2 and RPA3 subunits that binds to ssDNA with high affinity. The response to replication stress requires the recruitment of RPA and the MRE11/RAD50/NBS1 (MRN) complex. RPA bound to ssDNA stabilizes stalled replication forks by recruiting checkpoint proteins involved in fork stabilization. MRN can bind DNA structures encountered at stalled or collapsed replication forks, such as ssDNA-dsDNA junctions or breaks and pr...

  13. Cyclophilin B facilitates the replication of Orf virus

    OpenAIRE

    Zhao, Kui; Li, Jida; He, Wenqi; Song, Deguang; Zhang, Ximu; Zhang, Di; Zhou, Yanlong; Gao, Feng

    2017-01-01

    Background Viruses interact with host cellular factors to construct a more favourable environment for their efficient replication. Expression of cyclophilin B (CypB), a cellular peptidyl-prolyl cis-trans isomerase (PPIase), was found to be significantly up-regulated. Recently, a number of studies have shown that CypB is important in the replication of several viruses, including Japanese encephalitis virus (JEV), hepatitis C virus (HCV) and human papillomavirus type 16 (HPV 16). However, the f...

  14. Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

    Science.gov (United States)

    Han, Xiangzi; Mayca Pozo, Franklin; Wisotsky, Jacob N; Wang, Benlian; Jacobberger, James W; Zhang, Youwei

    2015-05-08

    Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Biomarkers of replicative senescence revisited

    DEFF Research Database (Denmark)

    Nehlin, Jan

    2016-01-01

    Biomarkers of replicative senescence can be defined as those ultrastructural and physiological variations as well as molecules whose changes in expression, activity or function correlate with aging, as a result of the gradual exhaustion of replicative potential and a state of permanent cell cycle...... arrest. The biomarkers that characterize the path to an irreversible state of cell cycle arrest due to proliferative exhaustion may also be shared by other forms of senescence-inducing mechanisms. Validation of senescence markers is crucial in circumstances where quiescence or temporary growth arrest may...... be triggered or is thought to be induced. Pre-senescence biomarkers are also important to consider as their presence indicate that induction of aging processes is taking place. The bona fide pathway leading to replicative senescence that has been extensively characterized is a consequence of gradual reduction...

  16. Involvement of proteasomal subunits zeta and iota in RNA degradation.

    Science.gov (United States)

    Petit, F; Jarrousse, A S; Dahlmann, B; Sobek, A; Hendil, K B; Buri, J; Briand, Y; Schmid, H P

    1997-01-01

    We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism. PMID:9337855

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

    Science.gov (United States)

    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…

  18. N-terminally truncated POM121C inhibits HIV-1 replication.

    Directory of Open Access Journals (Sweden)

    Hideki Saito

    Full Text Available Recent studies have identified host cell factors that regulate early stages of HIV-1 infection including viral cDNA synthesis and orientation of the HIV-1 capsid (CA core toward the nuclear envelope, but it remains unclear how viral DNA is imported through the nuclear pore and guided to the host chromosomal DNA. Here, we demonstrate that N-terminally truncated POM121C, a component of the nuclear pore complex, blocks HIV-1 infection. This truncated protein is predominantly localized in the cytoplasm, does not bind to CA, does not affect viral cDNA synthesis, reduces the formation of 2-LTR and diminished the amount of integrated proviral DNA. Studies with an HIV-1-murine leukemia virus (MLV chimeric virus carrying the MLV-derived Gag revealed that Gag is a determinant of this inhibition. Intriguingly, mutational studies have revealed that the blockade by N-terminally-truncated POM121C is closely linked to its binding to importin-β/karyopherin subunit beta 1 (KPNB1. These results indicate that N-terminally-truncated POM121C inhibits HIV-1 infection after completion of reverse transcription and before integration, and suggest an important role for KPNB1 in HIV-1 replication.

  19. Distinct functions of human RecQ helicases during DNA replication.

    Science.gov (United States)

    Urban, Vaclav; Dobrovolna, Jana; Janscak, Pavel

    2017-06-01

    DNA replication is the most vulnerable process of DNA metabolism in proliferating cells and therefore it is tightly controlled and coordinated with processes that maintain genomic stability. Human RecQ helicases are among the most important factors involved in the maintenance of replication fork integrity, especially under conditions of replication stress. RecQ helicases promote recovery of replication forks being stalled due to different replication roadblocks of either exogenous or endogenous source. They prevent generation of aberrant replication fork structures and replication fork collapse, and are involved in proper checkpoint signaling. The essential role of human RecQ helicases in the genome maintenance during DNA replication is underlined by association of defects in their function with cancer predisposition. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Structural properties of replication origins in yeast DNA sequences

    International Nuclear Information System (INIS)

    Cao Xiaoqin; Zeng Jia; Yan Hong

    2008-01-01

    Sequence-dependent DNA flexibility is an important structural property originating from the DNA 3D structure. In this paper, we investigate the DNA flexibility of the budding yeast (S. Cerevisiae) replication origins on a genome-wide scale using flexibility parameters from two different models, the trinucleotide and the tetranucleotide models. Based on analyzing average flexibility profiles of 270 replication origins, we find that yeast replication origins are significantly rigid compared with their surrounding genomic regions. To further understand the highly distinctive property of replication origins, we compare the flexibility patterns between yeast replication origins and promoters, and find that they both contain significantly rigid DNAs. Our results suggest that DNA flexibility is an important factor that helps proteins recognize and bind the target sites in order to initiate DNA replication. Inspired by the role of the rigid region in promoters, we speculate that the rigid replication origins may facilitate binding of proteins, including the origin recognition complex (ORC), Cdc6, Cdt1 and the MCM2-7 complex

  1. The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence.

    Science.gov (United States)

    Uzunova, Sonya Dimitrova; Zarkov, Alexander Stefanov; Ivanova, Anna Marianova; Stoynov, Stoyno Stefanov; Nedelcheva-Veleva, Marina Nedelcheva

    2014-01-01

    The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle.

  2. Chameleon Chasing II: A Replication.

    Science.gov (United States)

    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…

  3. Hyperthermia stimulates HIV-1 replication.

    Directory of Open Access Journals (Sweden)

    Ferdinand Roesch

    Full Text Available 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.

  4. Adressing Replication and Model Uncertainty

    DEFF Research Database (Denmark)

    Ebersberger, Bernd; Galia, Fabrice; Laursen, Keld

    innovation survey data for France, Germany and the UK, we conduct a ‘large-scale’ replication using the Bayesian averaging approach of classical estimators. Our method tests a wide range of determinants of innovation suggested in the prior literature, and establishes a robust set of findings on the variables...

  5. Replication of kinetoplast minicircle DNA

    International Nuclear Information System (INIS)

    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

  6. Manual of Cupule Replication Technology

    Directory of Open Access Journals (Sweden)

    Giriraj Kumar

    2015-09-01

    Full Text Available Throughout the world, iconic rock art is preceded by non-iconic rock art. Cupules (manmade, roughly semi-hemispherical depressions on rocks form the major bulk of the early non-iconic rock art globally. The antiquity of cupules extends back to the Lower Paleolithic in Asia and Africa, hundreds of thousand years ago. When one observes these cupules, the inquisitive mind poses so many questions with regard to understanding their technology, reasons for selecting the site, which rocks were used to make the hammer stones used, the skill and cognitive abilities employed to create the different types of cupules, the objective of their creation, their age, and so on. Replication of the cupules can provide satisfactory answers to some of these questions. Comparison of the hammer stones and cupules produced by the replication process with those obtained from excavation can provide support to observations. This paper presents a manual of cupule replication technology based on our experience of cupule replication on hard quartzite rock near Daraki-Chattan in the Chambal Basin, India.

  7. On Scalability and Replicability of Smart Grid Projects—A Case Study

    Directory of Open Access Journals (Sweden)

    Lukas Sigrist

    2016-03-01

    Full Text Available This paper studies the scalability and replicability of smart grid projects. Currently, most smart grid projects are still in the R&D or demonstration phases. The full roll-out of the tested solutions requires a suitable degree of scalability and replicability to prevent project demonstrators from remaining local experimental exercises. Scalability and replicability are the preliminary requisites to perform scaling-up and replication successfully; therefore, scalability and replicability allow for or at least reduce barriers for the growth and reuse of the results of project demonstrators. The paper proposes factors that influence and condition a project’s scalability and replicability. These factors involve technical, economic, regulatory and stakeholder acceptance related aspects, and they describe requirements for scalability and replicability. In order to assess and evaluate the identified scalability and replicability factors, data has been collected from European and national smart grid projects by means of a survey, reflecting the projects’ view and results. The evaluation of the factors allows quantifying the status quo of on-going projects with respect to the scalability and replicability, i.e., they provide a feedback on to what extent projects take into account these factors and on whether the projects’ results and solutions are actually scalable and replicable.

  8. Crinivirus replication and host interactions

    Directory of Open Access Journals (Sweden)

    Zsofia A Kiss

    2013-05-01

    Full Text Available Criniviruses comprise one of the genera within the family Closteroviridae. Members in this family are restricted to the phloem and rely on whitefly vectors of the genera Bemisia and/or Trialeurodes for plant-to-plant transmission. All criniviruses have bipartite, positive-sense ssRNA genomes, although there is an unconfirmed report of one having a tripartite genome. Lettuce infectious yellows virus (LIYV is the type species of the genus, the best studied so far of the criniviruses and the first for which a reverse genetics system was available. LIYV RNA 1 encodes for proteins predicted to be involved in replication, and alone is competent for replication in protoplasts. Replication results in accumulation of cytoplasmic vesiculated membranous structures which are characteristic of most studied members of the Closteroviridae. These membranous structures, often referred to as BYV-type vesicles, are likely sites of RNA replication. LIYV RNA 2 is replicated in trans when co-infecting cells with RNA 1, but is temporally delayed relative to RNA1. Efficient RNA 2 replication also is dependent on the RNA 1-encoded RNA binding protein, P34. No LIYV RNA 2-encoded proteins have been shown to affect RNA replication, but at least four, CP, CPm, Hsp70h, and p59 are virion structural components and CPm is a determinant of whitefly transmissibility. Roles of other LIYV RNA 2-encoded proteins are largely as yet unknown, but P26 is a non-virion protein that accumulates in cells as characteristic plasmalemma deposits which in plants are localized within phloem parenchyma and companion cells over plasmodesmata connections to sieve elements. The two remaining crinivirus-conserved RNA 2-encoded proteins are P5 and P9. P5 is 39 amino acid protein and is encoded at the 5’ end of RNA 2 as ORF1 and is part of the hallmark closterovirus gene array. The orthologous gene in BYV has been shown to play a role in cell-to-cell movement and indicated to be localized to the

  9. Muscular subunits transplantation for facial reanimation

    Directory of Open Access Journals (Sweden)

    Hazan André Salo Buslik

    2006-01-01

    Full Text Available PURPOSE: To present an alternative technique for reconstruction of musculocutaneous damages in the face transferring innervated subsegments(subunits of the latissimus dorsi flap for replacement of various facial mimetic muscles. METHODS: One clinical case of trauma with skin and mimetic muscles damage is described as an example of the technique. The treatment was performed with microsurgical transfer of latissimus dorsi muscle subunits. Each subunit present shape and dimensions of the respective mimetic muscles replaced. The origin, insertions and force vectors for the mimicmuscle lost were considered. Each subsegment has its own arterial and venous supply with a motor nerve component for the muscular unit. RESULTS: Pre and one year postoperative photos registration of static and dynamic mimic aspects, as well as digital electromyography digital data of the patients were compared. The transplanted muscular units presented myoeletric activity, fulfilling both the functional and cosmetic aspect. CONCLUSION: This technique seems to be a promising way to deal with the complex musculocutaneous losses of the face as well as facial palsy.

  10. Influvac, a trivalent inactivated subunit influenza vaccine.

    Science.gov (United States)

    Zuccotti, Gian Vincenzo; Fabiano, Valentina

    2011-01-01

    Influenza represents a major sanitary and socio-economic burden and vaccination is universally considered the most effective strategy for preventing the disease and its complications. Traditional influenza vaccines have been on the market since the late 1940s, with million of doses administered annually worldwide, and demonstrated a substantial efficacy and safety. The trivalent inactivated subunit vaccine has been available for more than 25 years and has been studied in healthy children, adults and the elderly and in people affected by underlying chronic medical conditions. We describe vaccine technology focusing on subunit vaccine production procedures and mode of action and provide updated information on efficacy and safety available data. A review of efficacy and safety data in healthy subjects and in high risk populations from major sponsor- and investigator-driven studies. The vaccine showed a good immunogenicity and a favorable safety profile in all target groups. In the panorama of actually available influenza vaccines, trivalent inactivated subunit vaccine represents a well-established tool for preventing flu and the associated complications.

  11. GEMC1 is a TopBP1-interacting protein required for chromosomal DNA replication.

    Science.gov (United States)

    Balestrini, Alessia; Cosentino, Claudia; Errico, Alessia; Garner, Elizabeth; Costanzo, Vincenzo

    2010-05-01

    Many of the factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication is poorly understood in multicellular organisms. Here, we report the identification of GEMC1 (geminin coiled-coil containing protein 1), a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus laevis egg extract we show that Xenopus GEMC1 (xGEMC1) binds to the checkpoint and replication factor TopBP1, which promotes binding of xGEMC1 to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin. Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.

  12. GEMC1 is a TopBP1 interacting protein required for chromosomal DNA replication

    Science.gov (United States)

    Balestrini, Alessia; Cosentino, Claudia; Errico, Alessia; Garner, Elizabeth; Costanzo, Vincenzo

    2010-01-01

    Many factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication in complex multicellular organisms is poorly understood. Here, we report the identification of GEMC1, a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus egg extract we show that Xenopus GEMC1 (xGEMC1) binds to checkpoint and replication factor TopBP1, which promotes xGEMC1 binding to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 directly interacts with replication factors such as Cdc45 and Cdk2-CyclinE by which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication whereas depletion of xGEMC1 prevents DNA replication onset due to impairment of Cdc45 loading onto chromatin. Likewise, inhibition of GEMC1 expression by morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in higher eukaryotes by mediating TopBP1 and Cdk2 dependent recruitment of Cdc45 onto replication origins. PMID:20383140

  13. DNA replication stress restricts ribosomal DNA copy number

    Science.gov (United States)

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

    2017-01-01

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

  14. DNA replication stress restricts ribosomal DNA copy number.

    Science.gov (United States)

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

    2017-09-01

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

  15. DNA replication stress restricts ribosomal DNA copy number.

    Directory of Open Access Journals (Sweden)

    Devika Salim

    2017-09-01

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

  16. Fungal mediator tail subunits contain classical transcriptional activation domains.

    Science.gov (United States)

    Liu, Zhongle; Myers, Lawrence C

    2015-04-01

    Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. Soybean glycinin subunits: Characterization of physicochemical and adhesion properties.

    Science.gov (United States)

    Mo, Xiaoqun; Zhong, Zhikai; Wang, Donghai; Sun, Xiuzhi

    2006-10-04

    Soybean proteins have shown great potential for applications as renewable and environmentally friendly adhesives. The objective of this work was to study physicochemical and adhesion properties of soy glycinin subunits. Soybean glycinin was extracted from soybean flour and then fractionated into acidic and basic subunits with an estimated purity of 90 and 85%, respectively. Amino acid composition of glycinin subunits was determined. The high hydrophobic amino acid content is a major contributor to the solubility behavior and water resistance of the basic subunits. Acidic subunits and glycinin had similar solubility profiles, showing more than 80% solubility at pH 2.0-4.0 or 6.5-12.0, whereas basic subunits had considerably lower solubility with the minimum at pH 4.5-8.0. Thermal analysis using a differential scanning calorimeter suggested that basic subunits form new oligomeric structures with higher thermal stability than glycinin but no highly ordered structures present in isolated acidic subunits. The wet strength of basic subunits was 160% more than that of acidic subunits prepared at their respective isoelectric points (pI) and cured at 130 degrees C. Both pH and the curing temperature significantly affected adhesive performance. High-adhesion water resistance was usually observed for adhesives from protein prepared at their pI values and cured at elevated temperatures. Basic subunits are responsible for the water resistance of glycinin and are a good starting material for the development of water-resistant adhesives.

  18. epsilon, a New Subunit of RNA Polymerase Found in Gram-Positive Bacteria

    Czech Academy of Sciences Publication Activity Database

    Keller, A. N.; Yang, X.; Wiedermannová, Jana; Delumeau, O.; Krásný, Libor; Lewis, P. J.

    2014-01-01

    Roč. 196, č. 20 (2014), s. 3622-3632 ISSN 0021-9193 R&D Projects: GA ČR(CZ) GBP305/12/G034 Institutional support: RVO:61388971 Keywords : RNA polymerase * subunit * X-ray crystallography Subject RIV: EE - Microbiology, Virology Impact factor: 2.808, year: 2014

  19. The TFIIH Subunit p89 (XPB Localizes to the Centrosome during Mitosis

    Directory of Open Access Journals (Sweden)

    Achim Weber

    2010-01-01

    Full Text Available Background: The general transcription factor II H (TFIIH, comprised of a core complex and an associated CAK-complex, functions in transcription, DNA repair and cell cycle control. Mutations of the two largest subunits, p89 (XPB and p80 (XPD, cause the hereditary cancer-prone syndrome xeroderma pigmentosum.

  20. Molecular Evolution and Functional Diversification of Replication Protein A1 in Plants

    Science.gov (United States)

    Aklilu, Behailu B.; Culligan, Kevin M.

    2016-01-01

    Replication protein A (RPA) is a heterotrimeric, single-stranded DNA binding complex required for eukaryotic DNA replication, repair, and recombination. RPA is composed of three subunits, RPA1, RPA2, and RPA3. In contrast to single RPA subunit genes generally found in animals and yeast, plants encode multiple paralogs of RPA subunits, suggesting subfunctionalization. Genetic analysis demonstrates that five Arabidopsis thaliana RPA1 paralogs (RPA1A to RPA1E) have unique and overlapping functions in DNA replication, repair, and meiosis. We hypothesize here that RPA1 subfunctionalities will be reflected in major structural and sequence differences among the paralogs. To address this, we analyzed amino acid and nucleotide sequences of RPA1 paralogs from 25 complete genomes representing a wide spectrum of plants and unicellular green algae. We find here that the plant RPA1 gene family is divided into three general groups termed RPA1A, RPA1B, and RPA1C, which likely arose from two progenitor groups in unicellular green algae. In the family Brassicaceae the RPA1B and RPA1C groups have further expanded to include two unique sub-functional paralogs RPA1D and RPA1E, respectively. In addition, RPA1 groups have unique domains, motifs, cis-elements, gene expression profiles, and pattern of conservation that are consistent with proposed functions in monocot and dicot species, including a novel C-terminal zinc-finger domain found only in plant RPA1C-like sequences. These results allow for improved prediction of RPA1 subunit functions in newly sequenced plant genomes, and potentially provide a unique molecular tool to improve classification of Brassicaceae species. PMID:26858742

  1. Defects of mtDNA Replication Impaired Mitochondrial Biogenesis During Trypanosoma cruzi Infection in Human Cardiomyocytes and Chagasic Patients: The Role of Nrf1/2 and Antioxidant Response

    Science.gov (United States)

    Wan, Xianxiu; Gupta, Shivali; Zago, Maria P.; Davidson, Mercy M.; Dousset, Pierre; Amoroso, Alejandro; Garg, Nisha Jain

    2012-01-01

    Background Mitochondrial dysfunction is a key determinant in chagasic cardiomyopathy development in mice; however, its relevance in human Chagas disease is not known. We determined if defects in mitochondrial biogenesis and dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1)–regulated transcriptional pathways constitute a mechanism or mechanisms underlying mitochondrial oxidative-phosphorylation (OXPHOS) deficiency in human Chagas disease. Methods and Results We utilized human cardiomyocytes and left-ventricular tissue from chagasic and other cardiomyopathy patients and healthy donors (n>6/group). We noted no change in citrate synthase activity, yet mRNA and/or protein levels of subunits of the respiratory complexes were significantly decreased in Trypanosoma cruzi–infected cardiomyocytes (0 to 24 hours) and chagasic hearts. We observed increased mRNA and decreased nuclear localization of PGC-1-coactivated transcription factors, yet the expression of genes for PPARγ-regulated fatty acid oxidation and nuclear respiratory factor (NRF1/2)–regulated mtDNA replication and transcription machinery was enhanced in infected cardiomyocytes and chagasic hearts. The D-loop formation was normal or higher, but mtDNA replication and mtDNA content were decreased by 83% and 40% to 65%, respectively. Subsequently, we noted that reactive oxygen species (ROS), oxidative stress, and mtDNA oxidation were significantly increased, yet NRF1/2-regulated antioxidant gene expression remained compromised in infected cardiomyocytes and chagasic hearts. Conclusions The replication of mtDNA was severely compromised, resulting in a significant loss of mtDNA and expression of OXPHOS genes in T cruzi–infected cardiomyocytes and chagasic hearts. Our data suggest increased ROS generation and selective functional incapacity of NRF2-mediated antioxidant gene expression played a role in the defects in mtDNA replication and unfitness of mtDNA for

  2. Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering

    Directory of Open Access Journals (Sweden)

    Antoun Ayman

    2004-01-01

    Full Text Available Light scattering and standard stopped-flow techniques were used to monitor rapid association of ribosomal subunits during initiation of eubacterial protein synthesis. The effects of the initiation factors IF1, IF2, IF3 and buffer conditions on subunit association were studied along with the role of GTP in this process. The part of light scattering theory that is essential for kinetic measurements is high-lighted in the main text and a more general treatment of Rayleigh scattering from macromolecules is given in an appendix.

  3. Glycosylation of alpha(2)delta(1) subunit: a sweet talk with Ca(v)1.2 channels

    Czech Academy of Sciences Publication Activity Database

    Lazniewska, Joanna; Weiss, Norbert

    2016-01-01

    Roč. 35, č. 3 (2016), s. 239-242 ISSN 0231-5882 R&D Projects: GA ČR GA15-13556S; GA MŠk 7AMB15FR015 Institutional support: RVO:61388963 Keywords : calcium channel * Ca(v)1.2 channel * ancillary subunit * alpha(2)delta(1) subunit * glycosylation * trafficking Subject RIV: CE - Biochemistry Impact factor: 1.170, year: 2016

  4. Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site.

    Science.gov (United States)

    Fuchs, Gabriele; Petrov, Alexey N; Marceau, Caleb D; Popov, Lauren M; Chen, Jin; O'Leary, Seán E; Wang, Richard; Carette, Jan E; Sarnow, Peter; Puglisi, Joseph D

    2015-01-13

    Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

  5. Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis.

    Science.gov (United States)

    Boehringer, Daniel; O'Farrell, Heather C; Rife, Jason P; Ban, Nenad

    2012-03-23

    The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.

  6. Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis*

    Science.gov (United States)

    Boehringer, Daniel; O'Farrell, Heather C.; Rife, Jason P.; Ban, Nenad

    2012-01-01

    The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3′-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation. PMID:22308031

  7. Evaluation of peptide designing strategy against subunit reassociation in mucin 1: A steered molecular dynamics approach.

    Directory of Open Access Journals (Sweden)

    J Lesitha Jeeva Kumari

    Full Text Available Subunit reassociation in mucin 1, a breast cancer tumor marker, is reported as one of the critical factors for its cytoplasmic activation. Inhibition of its heterodimeric association would therefore result in loss of its function and alter disease progression. The present study aimed at evaluating peptide inhibitor designing strategies that may serve as antagonist against this receptor-ligand alliance. Several peptides and their derivatives were designed based on native residues, subunit interface, hydrogen bonding and secondary structure. Docking studies with the peptides were carried on the receptor subunit and their binding affinities were evaluated using steered molecular dynamics simulation and umbrella sampling. Our results showed that among all the different classes of peptides evaluated, the receptor based peptide showed the highest binding affinity. This result was concurrent with the experimental observation that the receptor-ligand alliance in mucin 1 is highly specific. Our results also show that peptide ligand against this subunit association is only stabilized through native residue inter-protein interaction irrespective of the peptide structure, peptide length and number of hydrogen bonds. Consistency in binding affinity, pull force and free energy barrier was observed with only the receptor derived peptides which resulted in favorable interprotein interactions at the interface. Several observations were made and discussed which will eventually lead to designing efficient peptide inhibitors against mucin 1 heterodimeric subunit reassociation.

  8. The yeast replicative aging model.

    Science.gov (United States)

    He, Chong; Zhou, Chuankai; Kennedy, Brian K

    2018-03-08

    It has been nearly three decades since the budding yeast Saccharomyces cerevisiae became a significant model organism for aging research and it has emerged as both simple and powerful. The replicative aging assay, which interrogates the number of times a "mother" cell can divide and produce "daughters", has been a stalwart in these studies, and genetic approaches have led to the identification of hundreds of genes impacting lifespan. More recently, cell biological and biochemical approaches have been developed to determine how cellular processes become altered with age. Together, the tools are in place to develop a holistic view of aging in this single-celled organism. Here, we summarize the current state of understanding of yeast replicative aging with a focus on the recent studies that shed new light on how aging pathways interact to modulate lifespan in yeast. Copyright © 2018. Published by Elsevier B.V.

  9. Replicator dynamics in value chains

    DEFF Research Database (Denmark)

    Cantner, Uwe; Savin, Ivan; Vannuccini, Simone

    2016-01-01

    The pure model of replicator dynamics though providing important insights in the evolution of markets has not found much of empirical support. This paper extends the model to the case of firms vertically integrated in value chains. We show that i) by taking value chains into account, the replicator...... dynamics may revert its effect. In these regressive developments of market selection, firms with low fitness expand because of being integrated with highly fit partners, and the other way around; ii) allowing partner's switching within a value chain illustrates that periods of instability in the early...... stage of industry life-cycle may be the result of an 'optimization' of partners within a value chain providing a novel and simple explanation to the evidence discussed by Mazzucato (1998); iii) there are distinct differences in the contribution to market selection between the layers of a value chain...

  10. Replication confers β cell immaturity.

    Science.gov (United States)

    Puri, Sapna; Roy, Nilotpal; Russ, Holger A; Leonhardt, Laura; French, Esra K; Roy, Ritu; Bengtsson, Henrik; Scott, Donald K; Stewart, Andrew F; Hebrok, Matthias

    2018-02-02

    Pancreatic β cells are highly specialized to regulate systemic glucose levels by secreting insulin. In adults, increase in β-cell mass is limited due to brakes on cell replication. In contrast, proliferation is robust in neonatal β cells that are functionally immature as defined by a lower set point for glucose-stimulated insulin secretion. Here we show that β-cell proliferation and immaturity are linked by tuning expression of physiologically relevant, non-oncogenic levels of c-Myc. Adult β cells induced to replicate adopt gene expression and metabolic profiles resembling those of immature neonatal β that proliferate readily. We directly demonstrate that priming insulin-producing cells to enter the cell cycle promotes a functionally immature phenotype. We suggest that there exists a balance between mature functionality and the ability to expand, as the phenotypic state of the β cell reverts to a less functional one in response to proliferative cues.

  11. Chromatin replication and histone dynamics

    DEFF Research Database (Denmark)

    Alabert, Constance; Jasencakova, Zuzana; Groth, Anja

    2017-01-01

    Inheritance of the DNA sequence and its proper organization into chromatin is fundamental for genome stability and function. Therefore, how specific chromatin structures are restored on newly synthesized DNA and transmitted through cell division remains a central question to understand cell fate...... choices and self-renewal. Propagation of genetic information and chromatin-based information in cycling cells entails genome-wide disruption and restoration of chromatin, coupled with faithful replication of DNA. In this chapter, we describe how cells duplicate the genome while maintaining its proper...... organization into chromatin. We reveal how specialized replication-coupled mechanisms rapidly assemble newly synthesized DNA into nucleosomes, while the complete restoration of chromatin organization including histone marks is a continuous process taking place throughout the cell cycle. Because failure...

  12. Live Replication of Paravirtual Machines

    OpenAIRE

    Stodden, Daniel

    2009-01-01

    Virtual machines offer a fair degree of system state encapsulation, which promotes practical advances in fault tolerance, system debugging, profiling and security applications. This work investigates deterministic replay and semi-active replication for system paravirtualization, a software discipline trading guest kernel binar compatibility for reduced dependency on costly trap-and-emulate techniques. A primary contribution is evidence that trace capturing under a piecewise deterministic exec...

  13. In vitro replication of poliovirus

    International Nuclear Information System (INIS)

    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' 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

  14. Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography

    OpenAIRE

    Sauguet , Ludovic; Raia , Pierre; Henneke , Ghislaine; Delarue , Marc

    2016-01-01

    International audience; Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has ...

  15. The δ subunit of RNA polymerase guides promoter selectivity and virulence in Staphylococcus aureus.

    Science.gov (United States)

    Weiss, Andy; Ibarra, J Antonio; Paoletti, Jessica; Carroll, Ronan K; Shaw, Lindsey N

    2014-04-01

    In Gram-positive bacteria, and particularly the Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the δ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogen Staphylococcus aureus. We showed conservation of important structural regions of RpoE in S. aureus and other species and demonstrated binding to core RNAP that is mediated by the β and/or β' subunits. To identify the impact of the δ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in the rpoE mutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage SA3usa) were strongly upregulated. Phenotypically, the rpoE mutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the δ subunit of RNAP is a critical component of the S. aureus transcription machinery and plays an important role during infection.

  16. Cyclophilin B facilitates the replication of Orf virus.

    Science.gov (United States)

    Zhao, Kui; Li, Jida; He, Wenqi; Song, Deguang; Zhang, Ximu; Zhang, Di; Zhou, Yanlong; Gao, Feng

    2017-06-15

    Viruses interact with host cellular factors to construct a more favourable environment for their efficient replication. Expression of cyclophilin B (CypB), a cellular peptidyl-prolyl cis-trans isomerase (PPIase), was found to be significantly up-regulated. Recently, a number of studies have shown that CypB is important in the replication of several viruses, including Japanese encephalitis virus (JEV), hepatitis C virus (HCV) and human papillomavirus type 16 (HPV 16). However, the function of cellular CypB in ORFV replication has not yet been explored. Suppression subtractive hybridization (SSH) technique was applied to identify genes differentially expressed in the ORFV-infected MDBK cells at an early phase of infection. Cellular CypB was confirmed to be significantly up-regulated by quantitative reverse transcription-PCR (qRT-PCR) analysis and Western blotting. The role of CypB in ORFV infection was further determined using Cyclosporin A (CsA) and RNA interference (RNAi). Effect of CypB gene silencing on ORFV replication by 50% tissue culture infectious dose (TCID 50 ) assay and qRT-PCR detection. In the present study, CypB was found to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection. Cyclosporin A (CsA) exhibited suppressive effects on ORFV replication through the inhibition of CypB. Silencing of CypB gene inhibited the replication of ORFV in MDBK cells. In conclusion, these data suggest that CypB is critical for the efficient replication of the ORFV genome. Cellular CypB was confirmed to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection, which could effectively facilitate the replication of ORFV.

  17. Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ε

    Science.gov (United States)

    Zahurancik, Walter J.; Baranovskiy, Andrey G.; Tahirov, Tahir H.; Suo, Zucai

    2015-01-01

    Numerous genetic studies have provided compelling evidence to establish DNA polymerase ε (Polε) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polε is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3′ → 5′ exonuclease domain common to many replicative polymerases. In addition, Polε possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Polε heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polε in vitro. However, similar studies of the human Polε heterote-tramer (hPolε) have been limited by the difficulty of obtaining hPolε in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolε from insect host cells has allowed for isolation of greater amounts of active hPolε, thus enabling a more detailed kinetic comparison between hPolε and an active N-terminal fragment of the hPolε catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolε. We observe that the small subunits increase DNA binding by hPolε relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3′ → 5′ exonuclease activity of hPolε is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPolε and sway hPolε toward DNA synthesis rather than proofreading. PMID:25684708

  18. DNA replication stress: from molecular mechanisms to human disease.

    Science.gov (United States)

    Muñoz, Sergio; Méndez, Juan

    2017-02-01

    The genome of proliferating cells must be precisely duplicated in each cell division cycle. Chromosomal replication entails risks such as the possibility of introducing breaks and/or mutations in the genome. Hence, DNA replication requires the coordinated action of multiple proteins and regulatory factors, whose deregulation causes severe developmental diseases and predisposes to cancer. In recent years, the concept of "replicative stress" (RS) has attracted much attention as it impinges directly on genomic stability and offers a promising new avenue to design anticancer therapies. In this review, we summarize recent progress in three areas: (1) endogenous and exogenous factors that contribute to RS, (2) molecular mechanisms that mediate the cellular responses to RS, and (3) the large list of diseases that are directly or indirectly linked to RS.

  19. Replication of micro and nano surface geometries

    DEFF Research Database (Denmark)

    Hansen, Hans Nørgaard; Hocken, R.J.; Tosello, Guido

    2011-01-01

    The paper describes the state-of-the-art in replication of surface texture and topography at micro and nano scale. The description includes replication of surfaces in polymers, metals and glass. Three different main technological areas enabled by surface replication processes are presented......: manufacture of net-shape micro/nano surfaces, tooling (i.e. master making), and surface quality control (metrology, inspection). Replication processes and methods as well as the metrology of surfaces to determine the degree of replication are presented and classified. Examples from various application areas...... are given including replication for surface texture measurements, surface roughness standards, manufacture of micro and nano structured functional surfaces, replicated surfaces for optical applications (e.g. optical gratings), and process chains based on combinations of repeated surface replication steps....

  20. Adenovirus sequences required for replication in vivo.

    OpenAIRE

    Wang, K; Pearson, G D

    1985-01-01

    We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occup...

  1. Parametrised Constants and Replication for Spatial Mobility

    DEFF Research Database (Denmark)

    Hüttel, Hans; Haagensen, Bjørn

    2009-01-01

    Parametrised replication and replication are common ways of expressing infinite computation in process calculi. While parametrised constants can be encoded using replication in the π-calculus, this changes in the presence of spatial mobility as found in e.g. the distributed π- calculus...... of the distributed π-calculus with parametrised constants and replication are incomparable. On the other hand, we shall see that there exists a simple encoding of recursion in mobile ambients....

  2. Human aldolase B subunit-specific radioimmunoassay

    International Nuclear Information System (INIS)

    Asaka, M.; Alpert, E.

    1983-01-01

    A radioimmunoassay was developed for the direct quantification of aldolase B in human serum and tissues. The method is a double-antibody radioimmunoassay technique using radioiodinated aldolase B homopolymer as ligand, chicken antibodies to aldolase B and rabbit antibodies to chicken IgG. This radioimmunoassay was shown to be specific for the aldolase B subunit, with no cross-reactivity with either human aldolase A subunit or homopolymeric human aldolase C (C 4 ). The lowest measurable amount by this method was 2 ng/ml. Aldolase B is predominantly found in normal liver tissue, with relatively-high aldolase B levels also observed in kidney. Aldolase B levels in the serum obtained from 11 normal subjects ranged from 23 to 38 ng/ml, with a mean of 28.5 +- 9.2 (S.D.) ng/ml. Almost all of patients with hepatitis had serum aldolase B levels greater than 30 ng/ml. In cancer patients, serum aldolase B was slightly elevated in patients with metastatic liver cancer and primary lever cell carcinoma, whereas no elevation of serum aldolase B was shown in patients without liver metastasis. (Auth.)

  3. 36 CFR 910.64 - Replication.

    Science.gov (United States)

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Replication. 910.64 Section 910.64 Parks, Forests, and Public Property PENNSYLVANIA AVENUE DEVELOPMENT CORPORATION GENERAL... DEVELOPMENT AREA Glossary of Terms § 910.64 Replication. Replication means the process of using modern methods...

  4. Exploiting replicative stress to treat cancer

    DEFF Research Database (Denmark)

    Dobbelstein, Matthias; Sørensen, Claus Storgaard

    2015-01-01

    DNA replication in cancer cells is accompanied by stalling and collapse of the replication fork and signalling in response to DNA damage and/or premature mitosis; these processes are collectively known as 'replicative stress'. Progress is being made to increase our understanding of the mechanisms...

  5. Variance Swap Replication: Discrete or Continuous?

    Directory of Open Access Journals (Sweden)

    Fabien Le Floc’h

    2018-02-01

    Full Text Available The popular replication formula to price variance swaps assumes continuity of traded option strikes. In practice, however, there is only a discrete set of option strikes traded on the market. We present here different discrete replication strategies and explain why the continuous replication price is more relevant.

  6. Subunit stoichiometry of the chloroplast photosystem I complex

    International Nuclear Information System (INIS)

    Bruce, B.D.; Malkin, R.

    1988-01-01

    A native photosystem I (PS I) complex and a PS I core complex depleted of antenna subunits has been isolated from the uniformly 14 C-labeled aquatic higher plant, Lemna. These complexes have been analyzed for their subunit stoichiometry by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods. The results for both preparations indicate that one copy of each high molecular mass subunit is present per PS I complex and that a single copy of most low molecular mass subunits is also present. These results suggest that iron-sulfur center X, an early PS I electron acceptor proposed to bind to the high molecular mass subunits, contains a single [4Fe-4S] cluster which is bound to a dimeric structure of high molecular mass subunits, each providing 2 cysteine residues to coordinate this cluster

  7. Human acid-labile subunit deficiency: clinical, endocrine and metabolic consequences

    NARCIS (Netherlands)

    Domené, Horacio M.; Hwa, Vivian; Argente, Jesús; Wit, Jan M.; Wit, Jaan M.; Camacho-Hübner, Cecilia; Jasper, Héctor G.; Pozo, Jesús; van Duyvenvoorde, Hermine A.; Yakar, Shoshana; Fofanova-Gambetti, Olga V.; Rosenfeld, Ron G.; Scaglia, Paula A.; Bengolea, Sonia V.; Lteif, Aida; Kirmani, Salman; Mahmud, Farid H.; Frystyk, Jan; Hermus, Ad; Twickler, T. B.; Kempers, Marlies J. E.; Barrios, Vicente; Martos-Moreno, Gabriel A.; David, Alessia; Rose, Stephen

    2009-01-01

    The majority of insulin-like growth factor (IGF)-I and IGF-II circulate in the serum as a complex with the insulin-like growth factor binding protein (IGFBP)-3 or IGFBP-5, and an acid-labile subunit (ALS). The function of ALS is to prolong the half-life of the IGF-I-IGFBP-3/IGFBP-5 binary complexes.

  8. Transcriptional regulators of Na, K-ATPase subunits

    OpenAIRE

    Zhiqin eLi; Sigrid A Langhans

    2015-01-01

    The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during developme...

  9. DNA replication machinery is required for development in Drosophila.

    Science.gov (United States)

    Kohzaki, Hidetsugu; Asano, Maki; Murakami, Yota

    2018-01-01

     In Drosophila , some factors involved in chromosome replication seem to be involved in gene amplification and endoreplication, which are actively utilized in particular tissue development, but direct evidence has not been shown. Therefore, we examined the effect of depletion of replication factors on these processes. First, we confirmed RNAi knockdown can be used for the depletion of replication factors by comparing the phenotypes of RNAi knockdown and deletion or point mutants of the components of DNA licensing factor, MCM2, MCM4 and Cdt1. Next, we found that tissue-specific RNAi knockdown of replication factors caused tissue-specific defects, probably due to defects in DNA replication. In particular, we found that depletion inhibited gene amplification of the chorion gene in follicle cells and endoreplication in salivary glands, showing that chromosomal DNA replication factors are required for these processes. Finally, using RNAi, we screened the genes for chromosomal DNA replication that affected tissue development. Interestingly, wing specific knockdown of Mcm10 induced wing formation defects. These results suggest that some components of chromosomal replication machinery are directly involved in tissue development.

  10. Potential biomarkers of DNA replication stress in cancer

    DEFF Research Database (Denmark)

    Ren, Liqun; Chen, Long; Wu, Wei

    2017-01-01

    Oncogene activation is an established driver of tumorigenesis. An apparently inevitable consequence of oncogene activation is the generation of DNA replication stress (RS), a feature common to most cancer cells. RS, in turn, is a causal factor in the development of chromosome instability (CIN...

  11. Replication dynamics of the yeast genome.

    Science.gov (United States)

    Raghuraman, M K; Winzeler, E A; Collingwood, D; Hunt, S; Wodicka, L; Conway, A; Lockhart, D J; Davis, R W; Brewer, B J; Fangman, W L

    2001-10-05

    Oligonucleotide microarrays were used to map the detailed topography of chromosome replication in the budding yeast Saccharomyces cerevisiae. The times of replication of thousands of sites across the genome were determined by hybridizing replicated and unreplicated DNAs, isolated at different times in S phase, to the microarrays. Origin activations take place continuously throughout S phase but with most firings near mid-S phase. Rates of replication fork movement vary greatly from region to region in the genome. The two ends of each of the 16 chromosomes are highly correlated in their times of replication. This microarray approach is readily applicable to other organisms, including humans.

  12. Chromosomal DNA replication of Vicia faba cells

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1976-01-01

    The chromosomal DNA replication of higher plant cells has been investigated by DNA fiber autoradiography. The nuclear DNA fibers of Vicia root meristematic cells are organized into many tandem arrays of replication units or replicons which exist as clusters with respect to replication. DNA is replicated bidirectionally from the initiation points at the average rate of 0.15 μm/min at 20 0 C, and the average interinitiation interval is about 16 μm. The manner of chromosomal DNA replication in this higher plant is similar to that found in other eukaryotic cells at a subchromosomal level. (auth.)

  13. The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating assembly of the viral replicase complex.

    Science.gov (United States)

    Prasanth, K Reddisiva; Barajas, Daniel; Nagy, Peter D

    2015-03-01

    RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role

  14. Inferential misconceptions and replication crisis

    Directory of Open Access Journals (Sweden)

    Norbert Hirschauer

    2016-12-01

    Full Text Available Misinterpretations of the p value and the introduction of bias through arbitrary analytical choices have been discussed in the literature for decades. Nonetheless, they seem to have persisted in empirical research, and criticisms of p value misuses have increased in the recent past due to the non-replicability of many studies. Unfortunately, the critical concerns that have been raised in the literature are scattered over many disciplines, often linguistically confusing, and differing in their main reasons for criticisms. Misuses and misinterpretations of the p value are currently being discussed intensely under the label “replication crisis” in many academic disciplines and journals, ranging from specialized scientific journals to Nature and Science. In a drastic response to the crisis, the editors of the journal Basic and Applied Social Psychology even decided to ban the use of p values from future publications at the beginning of 2015, a fact that has certainly added fuel to the discussions in the relevant scientific forums. Finally, in early March, the American Statistical Association released a brief formal statement on p values that explicitly addresses misuses and misinterpretations. In this context, we systematize the most serious flaws related to the p value and discuss suggestions of how to prevent them and reduce the rate of false discoveries in the future.

  15. Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks

    DEFF Research Database (Denmark)

    Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia

    2016-01-01

    Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose...... RAD52 facilitates repair of collapsed DNA replication forks in cancer cells....

  16. Repair replication in replicating and nonreplicating DNA after irradiation with uv light

    Energy Technology Data Exchange (ETDEWEB)

    Slor, H.; Cleaver, J.E.

    1978-06-01

    Ultraviolet light induces more pyrimidine dimers and more repair replication in DNA that replicates within 2 to 3 h of irradiation than in DNA that does not replicate during this period. This difference may be due to special conformational changes in DNA and chromatin that might be associated with semiconservative DNA replication.

  17. Escherichia coli DnaE Polymerase Couples Pyrophosphatase Activity to DNA Replication.

    Directory of Open Access Journals (Sweden)

    Fabio Lapenta

    Full Text Available DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics.

  18. The Interstellar Ethics of Self-Replicating Probes

    Science.gov (United States)

    Cooper, K.

    Robotic spacecraft have been our primary means of exploring the Universe for over 50 years. Should interstellar travel become reality it seems unlikely that humankind will stop using robotic probes. These probes will be able to replicate themselves ad infinitum by extracting raw materials from the space resources around them and reconfiguring them into replicas of themselves, using technology such as 3D printing. This will create a colonising wave of probes across the Galaxy. However, such probes could have negative as well as positive consequences and it is incumbent upon us to factor self-replicating probes into our interstellar philosophies and to take responsibility for their actions.

  19. Histone acetylation regulates the time of replication origin firing.

    Science.gov (United States)

    Vogelauer, Maria; Rubbi, Liudmilla; Lucas, Isabelle; Brewer, Bonita J; Grunstein, Michael

    2002-11-01

    The temporal firing of replication origins throughout S phase in yeast depends on unknown determinants within the adjacent chromosomal environment. We demonstrate here that the state of histone acetylation of surrounding chromatin is an important regulator of temporal firing. Deletion of RPD3 histone deacetylase causes earlier origin firing and concurrent binding of the replication factor Cdc45p to origins. In addition, increased acetylation of histones in the vicinity of the late origin ARS1412 by recruitment of the histone acetyltransferase Gcn5p causes ARS1412 alone to fire earlier. These data indicate that histone acetylation is a direct determinant of the timing of origin firing.

  20. Inhibition of Zika Virus Replication by Silvestrol

    Directory of Open Access Journals (Sweden)

    Fabian Elgner

    2018-03-01

    Full Text Available The Zika virus (ZIKV outbreak in 2016 in South America with specific pathogenic outcomes highlighted the need for new antiviral substances with broad-spectrum activities to react quickly to unexpected outbreaks of emerging viral pathogens. Very recently, the natural compound silvestrol isolated from the plant Aglaia foveolata was found to have very potent antiviral effects against the (−-strand RNA-virus Ebola virus as well as against Corona- and Picornaviruses with a (+-strand RNA-genome. This antiviral activity is based on the impaired translation of viral RNA by the inhibition of the DEAD-box RNA helicase eukaryotic initiation factor-4A (eIF4A which is required to unwind structured 5´-untranslated regions (5′-UTRs of several proto-oncogenes and thereby facilitate their translation. Zika virus is a flavivirus with a positive-stranded RNA-genome harboring a 5′-capped UTR with distinct secondary structure elements. Therefore, we investigated the effects of silvestrol on ZIKV replication in A549 cells and primary human hepatocytes. Two different ZIKV strains were used. In both infected A549 cells and primary human hepatocytes, silvestrol has the potential to exert a significant inhibition of ZIKV replication for both analyzed strains, even though the ancestor strain from Uganda is less sensitive to silvestrol. Our data might contribute to identify host factors involved in the control of ZIKV infection and help to develop antiviral concepts that can be used to treat a variety of viral infections without the risk of resistances because a host protein is targeted.

  1. Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication

    Science.gov (United States)

    On, Kin Fan; Beuron, Fabienne; Frith, David; Snijders, Ambrosius P; Morris, Edward P; Diffley, John F X

    2014-01-01

    Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of “firing factors.” Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins. PMID:24566989

  2. Singlet Oxygen-Mediated Oxidation during UVA Radiation Alters the Dynamic of Genomic DNA Replication.

    Directory of Open Access Journals (Sweden)

    Dany Graindorge

    Full Text Available UVA radiation (320-400 nm is a major environmental agent that can exert its deleterious action on living organisms through absorption of the UVA photons by endogenous or exogenous photosensitizers. This leads to the production of reactive oxygen species (ROS, such as singlet oxygen (1O2 and hydrogen peroxide (H2O2, which in turn can modify reversibly or irreversibly biomolecules, such as lipids, proteins and nucleic acids. We have previously reported that UVA-induced ROS strongly inhibit DNA replication in a dose-dependent manner, but independently of the cell cycle checkpoints activation. Here, we report that the production of 1O2 by UVA radiation leads to a transient inhibition of replication fork velocity, a transient decrease in the dNTP pool, a quickly reversible GSH-dependent oxidation of the RRM1 subunit of ribonucleotide reductase and sustained inhibition of origin firing. The time of recovery post irradiation for each of these events can last from few minutes (reduction of oxidized RRM1 to several hours (replication fork velocity and origin firing. The quenching of 1O2 by sodium azide prevents the delay of DNA replication, the decrease in the dNTP pool and the oxidation of RRM1, while inhibition of Chk1 does not prevent the inhibition of origin firing. Although the molecular mechanism remains elusive, our data demonstrate that the dynamic of replication is altered by UVA photosensitization of vitamins via the production of singlet oxygen.

  3. Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli.

    Science.gov (United States)

    Kato , J; Katayama, T

    2001-08-01

    The bacterial DnaA protein binds to the chromosomal origin of replication to trigger a series of initiation reactions, which leads to the loading of DNA polymerase III. In Escherichia coli, once this polymerase initiates DNA synthesis, ATP bound to DnaA is efficiently hydrolyzed to yield the ADP-bound inactivated form. This negative regulation of DnaA, which occurs through interaction with the beta-subunit sliding clamp configuration of the polymerase, functions in the temporal blocking of re-initiation. Here we show that the novel DnaA-related protein, Hda, from E.coli is essential for this regulatory inactivation of DnaA in vitro and in vivo. Our results indicate that the hda gene is required to prevent over-initiation of chromosomal replication and for cell viability. Hda belongs to the chaperone-like ATPase family, AAA(+), as do DnaA and certain eukaryotic proteins essential for the initiation of DNA replication. We propose that the once-per-cell-cycle rule of replication depends on the timely interaction of AAA(+) proteins that comprise the apparatus regulating the activity of the initiator of replication.

  4. Singlet Oxygen-Mediated Oxidation during UVA Radiation Alters the Dynamic of Genomic DNA Replication

    Science.gov (United States)

    Graindorge, Dany; Martineau, Sylvain; Machon, Christelle; Arnoux, Philippe; Guitton, Jérôme; Francesconi, Stefania; Frochot, Céline; Sage, Evelyne; Girard, Pierre-Marie

    2015-01-01

    UVA radiation (320–400 nm) is a major environmental agent that can exert its deleterious action on living organisms through absorption of the UVA photons by endogenous or exogenous photosensitizers. This leads to the production of reactive oxygen species (ROS), such as singlet oxygen (1O2) and hydrogen peroxide (H2O2), which in turn can modify reversibly or irreversibly biomolecules, such as lipids, proteins and nucleic acids. We have previously reported that UVA-induced ROS strongly inhibit DNA replication in a dose-dependent manner, but independently of the cell cycle checkpoints activation. Here, we report that the production of 1O2 by UVA radiation leads to a transient inhibition of replication fork velocity, a transient decrease in the dNTP pool, a quickly reversible GSH-dependent oxidation of the RRM1 subunit of ribonucleotide reductase and sustained inhibition of origin firing. The time of recovery post irradiation for each of these events can last from few minutes (reduction of oxidized RRM1) to several hours (replication fork velocity and origin firing). The quenching of 1O2 by sodium azide prevents the delay of DNA replication, the decrease in the dNTP pool and the oxidation of RRM1, while inhibition of Chk1 does not prevent the inhibition of origin firing. Although the molecular mechanism remains elusive, our data demonstrate that the dynamic of replication is altered by UVA photosensitization of vitamins via the production of singlet oxygen. PMID:26485711

  5. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    DEFF Research Database (Denmark)

    Schmidt, Signe Tandrup; Foged, Camilla; Korsholm, Karen Smith

    2016-01-01

    be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode......The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens...... of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR expression profile of the target APCs. Here, we review state-of-the-art formulation approaches employed for the inclusion of immunostimulators and subunit...

  6. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    Directory of Open Access Journals (Sweden)

    Signe Tandrup Schmidt

    2016-03-01

    Full Text Available The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI. Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs, which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the

  7. Overcoming natural replication barriers: differential helicase requirements.

    Science.gov (United States)

    Anand, Ranjith P; Shah, Kartik A; Niu, Hengyao; Sung, Patrick; Mirkin, Sergei M; Freudenreich, Catherine H

    2012-02-01

    DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.

  8. Myristoylated α subunits of guanine nucleotide-binding regulatory proteins

    International Nuclear Information System (INIS)

    Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

    1987-01-01

    Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the α subunits of G/sub s/ (stimulatory) (α 45 and α 52 ), a 41-kDa subunit of G/sub i/ (inhibitory) (α 41 ), a 40-kDa protein (α 40 ), and the 36-kDa β subunit. No protein that comigrated with the α subunit of G 0 (unknown function) (α 39 ) was detected. In cells grown in the presence of [ 3 H]myristic acid, α 41 and α 40 contained 3 H label, while the β subunit did not. Chemical analysis of lipids attached covalently to purified α 41 and α 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein β and γ subunits and of G/sub t/ (Transducin) subunits (α, β, and γ) failed to reveal fatty acids. The fatty acid associated with α 41 , α 40 , and α 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins

  9. Development of a Subunit Vaccine for Contagious Bovine ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Their work has set the stage for commercial development of a sub-unit vaccine. ... The sub-unit vaccine will be cost-effective, easy to produce, and safe. How it will make a ... IDRC invites applications for the IDRC Doctoral Research Awards.

  10. SMC1-Mediated Intra-S-Phase Arrest Facilitates Bocavirus DNA Replication

    Science.gov (United States)

    Luo, Yong; Deng, Xuefeng; Cheng, Fang; Li, Yi

    2013-01-01

    Activation of a host DNA damage response (DDR) is essential for DNA replication of minute virus of canines (MVC), a member of the genus Bocavirus of the Parvoviridae family; however, the mechanism by which DDR contributes to viral DNA replication is unknown. In the current study, we demonstrate that MVC infection triggers the intra-S-phase arrest to slow down host cellular DNA replication and to recruit cellular DNA replication factors for viral DNA replication. The intra-S-phase arrest is regulated by ATM (ataxia telangiectasia-mutated kinase) signaling in a p53-independent manner. Moreover, we demonstrate that SMC1 (structural maintenance of chromosomes 1) is the key regulator of the intra-S-phase arrest induced during infection. Either knockdown of SMC1 or complementation with a dominant negative SMC1 mutant blocks both the intra-S-phase arrest and viral DNA replication. Finally, we show that the intra-S-phase arrest induced during MVC infection was caused neither by damaged host cellular DNA nor by viral proteins but by replicating viral genomes physically associated with the DNA damage sensor, the Mre11-Rad50-Nbs1 (MRN) complex. In conclusion, the feedback loop between MVC DNA replication and the intra-S-phase arrest is mediated by ATM-SMC1 signaling and plays a critical role in MVC DNA replication. Thus, our findings unravel the mechanism underlying DDR signaling-facilitated MVC DNA replication and demonstrate a novel strategy of DNA virus-host interaction. PMID:23365434

  11. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    International Nuclear Information System (INIS)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

    2014-01-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication

  12. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Gagnon, David [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Gjoerup, Ole [Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111 (United States); Archambault, Jacques [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Bullock, Peter A., E-mail: Peter.Bullock@tufts.edu [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States)

    2014-11-15

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.

  13. Replication of Merkel cell polyomavirus induces reorganization of promyelocytic leukemia nuclear bodies.

    Science.gov (United States)

    Neumann, Friederike; Czech-Sioli, Manja; Dobner, Thomas; Grundhoff, Adam; Schreiner, Sabrina; Fischer, Nicole

    2016-11-01

    Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma (MCC), a rare but aggressive skin cancer. The virus is highly prevalent: 60-80 % of adults are seropositive; however, cells permissive for MCPyV infection are unknown. Consequently, very little information about the MCPyV life cycle is available. Until recently, MCPyV replication could only be studied using a semi-permissive in vitro replication system (Neumann et al., 2011; Feng et al., 2011, Schowalter et al., 2011). MCPyV replication most likely depends on subnuclear structures such as promyelocytic leukemia protein nuclear bodies (PML-NBs), which are known to play regulatory roles in the infection of many DNA viruses. Here, we investigated PML-NB components as candidate host factors to control MCPyV DNA replication. We showed that PML-NBs change in number and size in cells actively replicating MCPyV proviral DNA. We observed a significant increase in PML-NBs in cells positive for MCPyV viral DNA replication. Interestingly, a significant amount of cells actively replicating MCPyV did not show any Sp100 expression. While PML and Daxx had no effect on MCPyV DNA replication, MCPyV replication was increased in cells depleted for Sp100, strongly suggesting that Sp100 is a negative regulator of MCPyV DNA replication.

  14. Studies on the subunits of human glycoprotein hormones in relation to reproduction

    International Nuclear Information System (INIS)

    Hagen, C.

    1977-01-01

    In this review summarising present knowledge of the biological and immunological activity of the subunits of human glycoprotein hormones, the specificity of the α-subunit and β-subunit radioimmunoassays are discussed. The crossreaction studies performed with the α-subunit radioimmunoassays are aummarised in one table while those with the β-subunit radioimmunoassays are presented in a second table. (JIW)

  15. Amplified Self-replication of DNA Origami Nanostructures through Multi-cycle Fast-annealing Process

    Science.gov (United States)

    Zhou, Feng; Zhuo, Rebecca; He, Xiaojin; Sha, Ruojie; Seeman, Nadrian; Chaikin, Paul

    We have developed a non-biological self-replication process using templated reversible association of components and irreversible linking with annealing and UV cycles. The current method requires a long annealing time, up to several days, to achieve the specific self-assembly of DNA nanostructures. In this work, we accomplished the self-replication with a shorter time and smaller replication rate per cycle. By decreasing the ramping time, we obtained the comparable replication yield within 90 min. Systematic studies show that the temperature and annealing time play essential roles in the self-replication process. In this manner, we can amplify the self-replication process to a factor of 20 by increasing the number of cycles within the same amount of time.

  16. BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication.

    Science.gov (United States)

    Morosky, Stefanie; Lennemann, Nicholas J; Coyne, Carolyn B

    2016-05-15

    Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of BPIFB6 expression

  17. BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication

    Science.gov (United States)

    Morosky, Stefanie; Lennemann, Nicholas J.

    2016-01-01

    ABSTRACT Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. IMPORTANCE Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of

  18. Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography.

    Science.gov (United States)

    Sauguet, Ludovic; Raia, Pierre; Henneke, Ghislaine; Delarue, Marc

    2016-08-22

    Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has the same 'double-psi β-barrel' architecture seen in the RNA polymerase (RNAP) superfamily, which includes multi-subunit transcriptases of all domains of life, homodimeric RNA-silencing pathway RNAPs and atypical viral RNAPs. This finding bridges together, in non-viral world, DNA transcription and DNA replication within the same protein superfamily. This study documents further the complex evolutionary history of the DNA replication apparatus in different domains of life and proposes a classification of all extant DNAPs.

  19. Replicating chromatin: a tale of histones

    DEFF Research Database (Denmark)

    Groth, Anja

    2009-01-01

    Chromatin serves structural and functional roles crucial for genome stability and correct gene expression. This organization must be reproduced on daughter strands during replication to maintain proper overlay of epigenetic fabric onto genetic sequence. Nucleosomes constitute the structural...... framework of chromatin and carry information to specify higher-order organization and gene expression. When replication forks traverse the chromosomes, nucleosomes are transiently disrupted, allowing the replication machinery to gain access to DNA. Histone recycling, together with new deposition, ensures...

  20. Comparative genomic analysis of multi-subunit tethering complexes demonstrates an ancient pan-eukaryotic complement and sculpting in Apicomplexa.

    Directory of Open Access Journals (Sweden)

    Christen M Klinger

    Full Text Available Apicomplexa are obligate intracellular parasites that cause tremendous disease burden world-wide. They utilize a set of specialized secretory organelles in their invasive process that require delivery of components for their biogenesis and function, yet the precise mechanisms underpinning such processes remain unclear. One set of potentially important components is the multi-subunit tethering complexes (MTCs, factors increasingly implicated in all aspects of vesicle-target interactions. Prompted by the results of previous studies indicating a loss of membrane trafficking factors in Apicomplexa, we undertook a bioinformatic analysis of MTC conservation. Building on knowledge of the ancient presence of most MTC proteins, we demonstrate the near complete retention of MTCs in the newly available genomes for Guillardiatheta and Bigelowiellanatans. The latter is a key taxonomic sampling point as a basal sister taxa to the group including Apicomplexa. We also demonstrate an ancient origin of the CORVET complex subunits Vps8 and Vps3, as well as the TRAPPII subunit Tca17. Having established that the lineage leading to Apicomplexa did at one point possess the complete eukaryotic complement of MTC components, we undertook a deeper taxonomic investigation in twelve apicomplexan genomes. We observed excellent conservation of the VpsC core of the HOPS and CORVET complexes, as well as the core TRAPP subunits, but sparse conservation of TRAPPII, COG, Dsl1, and HOPS/CORVET-specific subunits. However, those subunits that we did identify appear to be expressed with similar patterns to the fully conserved MTC proteins, suggesting that they may function as minimal complexes or with analogous partners. Strikingly, we failed to identify any subunits of the exocyst complex in all twelve apicomplexan genomes, as well as the dinoflagellate Perkinsus marinus. Overall, we demonstrate reduction of MTCs in Apicomplexa and their ancestors, consistent with modification during

  1. Enzymatic recognition of DNA replication origins

    International Nuclear Information System (INIS)

    Stayton, M.M.; Bertsch, L.; Biswas, S.

    1983-01-01

    In this paper we discuss the process of recognition of the complementary-strand origin with emphasis on RNA polymerase action in priming M13 DNA replication, the role of primase in G4 DNA replication, and the function of protein n, a priming protein, during primosome assembly. These phage systems do not require several of the bacterial DNA replication enzymes, particularly those involved in the regulation of chromosome copy number of the initiatiion of replication of duplex DNA. 51 references, 13 figures, 1 table

  2. The Regulation of NF-κB Subunits by Phosphorylation

    Directory of Open Access Journals (Sweden)

    Frank Christian

    2016-03-01

    Full Text Available The NF-κB transcription factor is the master regulator of the inflammatory response and is essential for the homeostasis of the immune system. NF-κB regulates the transcription of genes that control inflammation, immune cell development, cell cycle, proliferation, and cell death. The fundamental role that NF-κB plays in key physiological processes makes it an important factor in determining health and disease. The importance of NF-κB in tissue homeostasis and immunity has frustrated therapeutic approaches aimed at inhibiting NF-κB activation. However, significant research efforts have revealed the crucial contribution of NF-κB phosphorylation to controlling NF-κB directed transactivation. Importantly, NF-κB phosphorylation controls transcription in a gene-specific manner, offering new opportunities to selectively target NF-κB for therapeutic benefit. This review will focus on the phosphorylation of the NF-κB subunits and the impact on NF-κB function.

  3. Potential of Cationic Liposomes as Adjuvants/Delivery Systems for Tuberculosis Subunit Vaccines.

    Science.gov (United States)

    Khademi, Farzad; Taheri, Ramezan Ali; Momtazi-Borojeni, Amir Abbas; Farnoosh, Gholamreza; Johnston, Thomas P; Sahebkar, Amirhossein

    2018-04-27

    The weakness of the BCG vaccine and its highly variable protective efficacy in controlling tuberculosis (TB) in different age groups as well as in different geographic areas has led to intense efforts towards the development and design of novel vaccines. Currently, there are several strategies to develop novel TB vaccines. Each strategy has its advantages and disadvantages. However, the most important of these strategies is the development of subunit vaccines. In recent years, the use of cationic liposome-based vaccines has been considered due to their capacity to elicit strong humoral and cellular immune responses against TB infections. In this review, we aim to evaluate the potential for cationic liposomes to be used as adjuvants/delivery systems for eliciting immune responses against TB subunit vaccines. The present review shows that cationic liposomes have extensive applications either as adjuvants or delivery systems, to promote immune responses against Mycobacterium tuberculosis (Mtb) subunit vaccines. To overcome several limitations of these particles, they were used in combination with other immunostimulatory factors such as TDB, MPL, TDM, and Poly I:C. Cationic liposomes can provide long-term storage of subunit TB vaccines at the injection site, confer strong electrostatic interactions with APCs, potentiate both humoral and cellular (CD4 and CD8) immune responses, and induce a strong memory response by the immune system. Therefore, cationic liposomes can increase the potential of different TB subunit vaccines by serving as adjuvants/delivery systems. These properties suggest the use of cationic liposomes to produce an efficient vaccine against TB infections.

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

    Science.gov (United States)

    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.

  5. Activation of human herpesvirus replication by apoptosis.

    Science.gov (United States)

    Prasad, Alka; Remick, Jill; Zeichner, Steven L

    2013-10-01

    A central feature of herpesvirus biology is the ability of herpesviruses to remain latent within host cells. Classically, exposure to inducing agents, like activating cytokines or phorbol esters that stimulate host cell signal transduction events, and epigenetic agents (e.g., butyrate) was thought to end latency. We recently showed that Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus-8 [HHV-8]) has another, alternative emergency escape replication pathway that is triggered when KSHV's host cell undergoes apoptosis, characterized by the lack of a requirement for the replication and transcription activator (RTA) protein, accelerated late gene kinetics, and production of virus with decreased infectivity. Caspase-3 is necessary and sufficient to initiate the alternative replication program. HSV-1 was also recently shown to initiate replication in response to host cell apoptosis. These observations suggested that an alternative apoptosis-triggered replication program might be a general feature of herpesvirus biology and that apoptosis-initiated herpesvirus replication may have clinical implications, particularly for herpesviruses that almost universally infect humans. To explore whether an alternative apoptosis-initiated replication program is a common feature of herpesvirus biology, we studied cell lines latently infected with Epstein-Barr virus/HHV-4, HHV-6A, HHV-6B, HHV-7, and KSHV. We found that apoptosis triggers replication for each HHV studied, with caspase-3 being necessary and sufficient for HHV replication. An alternative apoptosis-initiated replication program appears to be a common feature of HHV biology. We also found that commonly used cytotoxic chemotherapeutic agents activate HHV replication, which suggests that treatments that promote apoptosis may lead to activation of latent herpesviruses, with potential clinical significance.

  6. Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

    Directory of Open Access Journals (Sweden)

    Stephanie Munk

    2017-10-01

    Full Text Available The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites. We find co-regulation of central DNA damage and replication stress responders, of which the ATR-activating factor TOPBP1 is the most highly regulated. Using pharmacological inhibition of the DNA damage response kinases ATR and ATM, we find that these factors regulate global protein SUMOylation in the protein networks that protect DNA upon replication stress and fork breakage, pointing to integration between phosphorylation and SUMOylation in the cellular systems that protect DNA integrity.

  7. DNA replication and cancer: From dysfunctional replication origin activities to therapeutic opportunities.

    Science.gov (United States)

    Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

    2016-06-01

    A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways to promote genome integrity during DNA replication. This includes suppressing new replication origin firing, stabilization of replicating forks, and the safe restart of forks to prevent any loss of genetic information. Here, we describe mechanisms by which oncogenes can interfere with DNA replication thereby causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. The light subunit of system bo,+ is fully functional in the absence of the heavy subunit

    OpenAIRE

    Reig, Núria; Chillarón, Josep; Bartoccioni, Paola; Fernández, Esperanza; Bendahan, Annie; Zorzano, Antonio; Kanner, Baruch; Palacín, Manuel; Bertran, Joan

    2002-01-01

    The heteromeric amino acid transporters are composed of a type II glycoprotein and a non-glycosylated polytopic membrane protein. System bo,+ exchanges dibasic for neutral amino acids. It is composed of rBAT and bo,+AT, the latter being the polytopic membrane subunit. Mutations in either of them cause malfunction of the system, leading to cystinuria. bo,+AT-reconstituted systems from HeLa or MDCK cells catalysed transport of arginine that was totally dependent on the presence of one of the bo...

  9. Replication and Robustness in Developmental Research

    Science.gov (United States)

    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…

  10. Three Conceptual Replication Studies in Group Theory

    Science.gov (United States)

    Melhuish, Kathleen

    2018-01-01

    Many studies in mathematics education research occur with a nonrepresentative sample and are never replicated. To challenge this paradigm, I designed a large-scale study evaluating student conceptions in group theory that surveyed a national, representative sample of students. By replicating questions previously used to build theory around student…

  11. Using Replication Projects in Teaching Research Methods

    Science.gov (United States)

    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…

  12. Dynamic behavior of DNA replication domains

    NARCIS (Netherlands)

    Manders, E. M.; Stap, J.; Strackee, J.; van Driel, R.; Aten, J. A.

    1996-01-01

    Like many nuclear processes, DNA replication takes place in distinct domains that are scattered throughout the S-phase nucleus. Recently we have developed a fluorescent double-labeling procedure that allows us to visualize nascent DNA simultaneously with "newborn" DNA that had replicated earlier in

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

    Science.gov (United States)

    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…

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

    Science.gov (United States)

    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…

  15. Surface Microstructure Replication in Injection Moulding

    DEFF Research Database (Denmark)

    Hansen, Hans Nørgaard; Arlø, Uffe Rolf

    2005-01-01

    topography is transcribed onto the plastic part through complex mechanisms. This replication however, is not perfect, and the replication quality depends on the plastic material properties, the topography itself, and the process conditions. This paper describes and discusses an investigation of injection...... moulding of surface microstructures. Emphasis is put on the ability to replicate surface microstructures under normal injection moulding conditions, notably with low cost materials at low mould temperatures. The replication of surface microstructures in injection moulding has been explored...... for Polypropylene at low mould temperatures. The process conditions were varied over the recommended process window for the material. The geometry of the obtained structures was analyzed. Evidence suggests that step height replication quality depends linearly on structure width in a certain range. Further...

  16. Surface microstructure replication in injection molding

    DEFF Research Database (Denmark)

    Theilade, Uffe Arlø; Hansen, Hans Nørgaard

    2006-01-01

    topography is transcribed onto the plastic part through complex mechanisms. This replication, however, is not perfect, and the replication quality depends on the plastic material properties, the topography itself, and the process conditions. This paper describes and discusses an investigation of injection...... molding of surface microstructures. The fundamental problem of surface microstructure replication has been studied. The research is based on specific microstructures as found in lab-on-a-chip products and on rough surfaces generated from EDM (electro discharge machining) mold cavities. Emphasis is put...... on the ability to replicate surface microstructures under normal injection-molding conditions, i.e., with commodity materials within typical process windows. It was found that within typical process windows the replication quality depends significantly on several process parameters, and especially the mold...

  17. Suppression of Poxvirus Replication by Resveratrol.

    Science.gov (United States)

    Cao, Shuai; Realegeno, Susan; Pant, Anil; Satheshkumar, Panayampalli S; Yang, Zhilong

    2017-01-01

    Poxviruses continue to cause serious diseases even after eradication of the historically deadly infectious human disease, smallpox. Poxviruses are currently being developed as vaccine vectors and cancer therapeutic agents. Resveratrol is a natural polyphenol stilbenoid found in plants that has been shown to inhibit or enhance replication of a number of viruses, but the effect of resveratrol on poxvirus replication is unknown. In the present study, we found that resveratrol dramatically suppressed the replication of vaccinia virus (VACV), the prototypic member of poxviruses, in various cell types. Resveratrol also significantly reduced the replication of monkeypox virus, a zoonotic virus that is endemic in Western and Central Africa and causes human mortality. The inhibitory effect of resveratrol on poxviruses is independent of VACV N1 protein, a potential resveratrol binding target. Further experiments demonstrated that resveratrol had little effect on VACV early gene expression, while it suppressed VACV DNA synthesis, and subsequently post-replicative gene expression.

  18. Suppression of Poxvirus Replication by Resveratrol

    Directory of Open Access Journals (Sweden)

    Shuai Cao

    2017-11-01

    Full Text Available Poxviruses continue to cause serious diseases even after eradication of the historically deadly infectious human disease, smallpox. Poxviruses are currently being developed as vaccine vectors and cancer therapeutic agents. Resveratrol is a natural polyphenol stilbenoid found in plants that has been shown to inhibit or enhance replication of a number of viruses, but the effect of resveratrol on poxvirus replication is unknown. In the present study, we found that resveratrol dramatically suppressed the replication of vaccinia virus (VACV, the prototypic member of poxviruses, in various cell types. Resveratrol also significantly reduced the replication of monkeypox virus, a zoonotic virus that is endemic in Western and Central Africa and causes human mortality. The inhibitory effect of resveratrol on poxviruses is independent of VACV N1 protein, a potential resveratrol binding target. Further experiments demonstrated that resveratrol had little effect on VACV early gene expression, while it suppressed VACV DNA synthesis, and subsequently post-replicative gene expression.

  19. Host ESCRT proteins are required for bromovirus RNA replication compartment assembly and function.

    Directory of Open Access Journals (Sweden)

    Arturo Diaz

    2015-03-01

    Full Text Available Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV RNA replication occurs on perinuclear endoplasmic reticulum (ER membranes in ~70 nm vesicular invaginations (spherules. BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.

  20. Interaction of the regulatory subunit of the cAMP-dependent protein kinase with PATZ1 (ZNF278)

    International Nuclear Information System (INIS)

    Yang, Weng-Lang; Ravatn, Roald; Kudoh, Kazuya; Alabanza, Leah; Chin, Khew-Voon

    2010-01-01

    The effects of cAMP in cell are predominantly mediated by the cAMP-dependent protein kinase (PKA), which is composed of two genetically distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzyme R 2 C 2 . The only known function for the R subunit is that of inhibiting the activity of the C subunit kinase. It has been shown that overexpression of RIα, but not the C subunit kinase, is associated with neoplastic transformation. In addition, it has also been demonstrated that mutation in the RIα, but not the C subunit is associated with increased resistance to the DNA-damaging anticancer drug cisplatin, thus suggesting that the RIα subunit of PKA may have functions independent of the kinase. We show here that the RIα subunit interacts with a BTB/POZ domain zinc-finger transcription factor, PATZ1 (ZNF278), and co-expression with RIα results in its sequestration in the cytoplasm. The cytoplasmic/nuclear translocation is inducible by cAMP. C-terminus deletion abolishes PATZ1 interaction with RIα and results in its localization in the nucleus. PATZ1 transactivates the cMyc promoter and the presence of cAMP and co-expression with RIα modulates its transactivation. Moreover, PATZ1 is aberrantly expressed in cancer. Taken together, our results showed a potentially novel mechanism of cAMP signaling mediated through the interaction of RIα with PATZ1 that is independent of the kinase activity of PKA, and the aberrant expression of PATZ1 in cancer point to its role in cell growth regulation.

  1. Interaction of the regulatory subunit of the cAMP-dependent protein kinase with PATZ1 (ZNF278)

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Weng-Lang [Long Island Jewish Medical Center, North Shore University Hospital, Manhasset, NY 11030 (United States); Ravatn, Roald [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Kudoh, Kazuya [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Department of Obstetrics and Gynecology, National Defense Medical College, Tokorozawa, Saitama (Japan); Alabanza, Leah [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Chin, Khew-Voon, E-mail: khew-voon.chin@utoledo.edu [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States)

    2010-01-15

    The effects of cAMP in cell are predominantly mediated by the cAMP-dependent protein kinase (PKA), which is composed of two genetically distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzyme R{sub 2}C{sub 2}. The only known function for the R subunit is that of inhibiting the activity of the C subunit kinase. It has been shown that overexpression of RI{alpha}, but not the C subunit kinase, is associated with neoplastic transformation. In addition, it has also been demonstrated that mutation in the RI{alpha}, but not the C subunit is associated with increased resistance to the DNA-damaging anticancer drug cisplatin, thus suggesting that the RI{alpha} subunit of PKA may have functions independent of the kinase. We show here that the RI{alpha} subunit interacts with a BTB/POZ domain zinc-finger transcription factor, PATZ1 (ZNF278), and co-expression with RI{alpha} results in its sequestration in the cytoplasm. The cytoplasmic/nuclear translocation is inducible by cAMP. C-terminus deletion abolishes PATZ1 interaction with RI{alpha} and results in its localization in the nucleus. PATZ1 transactivates the cMyc promoter and the presence of cAMP and co-expression with RI{alpha} modulates its transactivation. Moreover, PATZ1 is aberrantly expressed in cancer. Taken together, our results showed a potentially novel mechanism of cAMP signaling mediated through the interaction of RI{alpha} with PATZ1 that is independent of the kinase activity of PKA, and the aberrant expression of PATZ1 in cancer point to its role in cell growth regulation.

  2. Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle.

    Science.gov (United States)

    Pasion, S G; Brown, G W; Brown, L M; Ray, D S

    1994-12-01

    In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.

  3. A New Replication Norm for Psychology

    Directory of Open Access Journals (Sweden)

    Etienne P LeBel

    2015-10-01

    Full Text Available In recent years, there has been a growing concern regarding the replicability of findings in psychology, including a mounting number of prominent findings that have failed to replicate via high-powered independent replication attempts. In the face of this replicability “crisis of confidence”, several initiatives have been implemented to increase the reliability of empirical findings. In the current article, I propose a new replication norm that aims to further boost the dependability of findings in psychology. Paralleling the extant social norm that researchers should peer review about three times as many articles that they themselves publish per year, the new replication norm states that researchers should aim to independently replicate important findings in their own research areas in proportion to the number of original studies they themselves publish per year (e.g., a 4:1 original-to-replication studies ratio. I argue this simple approach could significantly advance our science by increasing the reliability and cumulative nature of our empirical knowledge base, accelerating our theoretical understanding of psychological phenomena, instilling a focus on quality rather than quantity, and by facilitating our transformation toward a research culture where executing and reporting independent direct replications is viewed as an ordinary part of the research process. To help promote the new norm, I delineate (1 how each of the major constituencies of the research process (i.e., funders, journals, professional societies, departments, and individual researchers can incentivize replications and promote the new norm and (2 any obstacles each constituency faces in supporting the new norm.

  4. Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

    Science.gov (United States)

    Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

    2017-05-01

    Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

  5. Telomeres and replicative senescence: Is it only length that counts?

    Science.gov (United States)

    von Zglinicki, T

    2001-07-26

    Telomeres are well established as a major 'replicometer', counting the population doublings in primary human cell cultures and ultimately triggering replicative senescence. However, neither is the pace of this biological clock inert, nor is there a fixed threshold telomere length acting as the universal trigger of replicative senescence. The available data suggest that opening of the telomeric loop and unscheduled exposure of the single-stranded G-rich telomeric overhang might act like a semaphore to signal senescent cell cycle arrest. Short telomere length, telomeric single-strand breaks, low levels of loop-stabilizing proteins, or other factors may trigger this opening of the loop. Thus, both telomere shortening and the ultimate signalling into senescence are able to integrate different environmental and genetic factors, especially oxidative stress-mediated damage, which might otherwise become a thread to genomic stability.

  6. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.

    Directory of Open Access Journals (Sweden)

    Kèvin Knoops

    2008-09-01

    Full Text Available Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV, replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200-300 nm, and "vesicle packets" apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this "replication network" will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions.

  7. Specificity and Function of Archaeal DNA Replication Initiator Proteins

    Directory of Open Access Journals (Sweden)

    Rachel Y. Samson

    2013-02-01

    Full Text Available Chromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC that recruits the replicative helicase MCM(2-7 via Cdc6 and Cdt1. We find that the three origins in the single chromosome of the archaeon Sulfolobus islandicus are specified by distinct initiation factors. While two origins are dependent on archaeal homologs of eukaryal Orc1 and Cdc6, the third origin is instead reliant on an archaeal Cdt1 homolog. We exploit the nonessential nature of the orc1-1 gene to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels the protein’s structure rather than that of the DNA template.

  8. Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism.

    Science.gov (United States)

    Reynolds, John J; Bicknell, Louise S; Carroll, Paula; Higgs, Martin R; Shaheen, Ranad; Murray, Jennie E; Papadopoulos, Dimitrios K; Leitch, Andrea; Murina, Olga; Tarnauskaitė, Žygimantė; Wessel, Sarah R; Zlatanou, Anastasia; Vernet, Audrey; von Kriegsheim, Alex; Mottram, Rachel M A; Logan, Clare V; Bye, Hannah; Li, Yun; Brean, Alexander; Maddirevula, Sateesh; Challis, Rachel C; Skouloudaki, Kassiani; Almoisheer, Agaadir; Alsaif, Hessa S; Amar, Ariella; Prescott, Natalie J; Bober, Michael B; Duker, Angela; Faqeih, Eissa; Seidahmed, Mohammed Zain; Al Tala, Saeed; Alswaid, Abdulrahman; Ahmed, Saleem; Al-Aama, Jumana Yousuf; Altmüller, Janine; Al Balwi, Mohammed; Brady, Angela F; Chessa, Luciana; Cox, Helen; Fischetto, Rita; Heller, Raoul; Henderson, Bertram D; Hobson, Emma; Nürnberg, Peter; Percin, E Ferda; Peron, Angela; Spaccini, Luigina; Quigley, Alan J; Thakur, Seema; Wise, Carol A; Yoon, Grace; Alnemer, Maha; Tomancak, Pavel; Yigit, Gökhan; Taylor, A Malcolm R; Reijns, Martin A M; Simpson, Michael A; Cortez, David; Alkuraya, Fowzan S; Mathew, Christopher G; Jackson, Andrew P; Stewart, Grant S

    2017-04-01

    To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

  9. Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis

    Science.gov (United States)

    Hua, Hui; Namdar, Mandana; Ganier, Olivier; Gregan, Juraj; Méchali, Marcel; Kearsey, Stephen E.

    2013-01-01

    Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I–II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry. PMID:23303250

  10. Genetic analysis of the cytoplasmic dynein subunit families.

    Science.gov (United States)

    Pfister, K Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M C

    2006-01-01

    Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

  11. Genetic analysis of the cytoplasmic dynein subunit families.

    Directory of Open Access Journals (Sweden)

    K Kevin Pfister

    2006-01-01

    Full Text Available Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

  12. Rif1 acts through Protein Phosphatase 1 but independent of replication timing to suppress telomere extension in budding yeast.

    Science.gov (United States)

    Kedziora, Sylwia; Gali, Vamsi K; Wilson, Rosemary H C; Clark, Kate R M; Nieduszynski, Conrad A; Hiraga, Shin-Ichiro; Donaldson, Anne D

    2018-05-04

    The Rif1 protein negatively regulates telomeric TG repeat length in the budding yeast Saccharomyces cerevisiae, but how it prevents telomere over-extension is unknown. Rif1 was recently shown to control DNA replication by acting as a Protein Phosphatase 1 (PP1)-targeting subunit. Therefore, we investigated whether Rif1 controls telomere length by targeting PP1 activity. We find that a Rif1 mutant defective for PP1 interaction causes a long-telomere phenotype, similar to that of rif1Δ cells. Tethering PP1 at a specific telomere partially substitutes for Rif1 in limiting TG repeat length, confirming the importance of PP1 in telomere length control. Ablating Rif1-PP1 interaction is known to cause precocious activation of telomere-proximal replication origins and aberrantly early telomere replication. However, we find that Rif1 still limits telomere length even if late replication is forced through deletion of nearby replication origins, indicating that Rif1 can control telomere length independent of replication timing. Moreover we find that, even at a de novo telomere created after DNA synthesis during a mitotic block, Rif1-PP1 interaction is required to suppress telomere lengthening and prevent inappropriate recruitment of Tel1 kinase. Overall, our results show that Rif1 controls telomere length by recruiting PP1 to directly suppress telomerase-mediated TG repeat lengthening.

  13. Development of replication-deficient adenovirus malaria vaccines.

    Science.gov (United States)

    Hollingdale, Michael R; Sedegah, Martha; Limbach, Keith

    2017-03-01

    Malaria remains a major threat to endemic populations and travelers, including military personnel to these areas. A malaria vaccine is feasible, as radiation attenuated sporozoites induce nearly 100% efficacy. Areas covered: This review covers current malaria clinical trials using adenoviruses and pre-clinical research. Heterologous prime-boost regimens, including replication-deficient human adenovirus 5 (HuAd5) carrying malaria antigens, are efficacious. However, efficacy appears to be adversely affected by pre-existing anti-HuAd5 antibodies. Current strategies focus on replacing HuAd5 with rarer human adenoviruses or adenoviruses isolated from non-human primates (NHPs). The chimpanzee adenovirus ChAd63 is undergoing evaluation in clinical trials including infants in malaria-endemic areas. Key antigens have been identified and are being used alone, in combination, or with protein subunit vaccines. Gorilla adenoviruses carrying malaria antigens are also currently being evaluated in preclinical models. These replacement adenovirus vectors will be successfully used to develop vaccines against malaria, as well as other infectious diseases. Expert commentary: Simplified prime-boost single shot regimens, dry-coated live vector vaccines or silicon microneedle arrays could be developed for malaria or other vaccines. Replacement vectors with similar or superior immunogenicity have rapidly advanced, and several are now in extensive Phase 2 and beyond in malaria as well as other diseases, notably Ebola.

  14. Data from Investigating Variation in Replicability: A “Many Labs” Replication Project

    Directory of Open Access Journals (Sweden)

    Richard A. Klein

    2014-04-01

    Full Text Available This dataset is from the Many Labs Replication Project in which 13 effects were replicated across 36 samples and over 6,000 participants. Data from the replications are included, along with demographic variables about the participants and contextual information about the environment in which the replication was conducted. Data were collected in-lab and online through a standardized procedure administered via an online link. The dataset is stored on the Open Science Framework website. These data could be used to further investigate the results of the included 13 effects or to study replication and generalizability more broadly.

  15. NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62.

    Science.gov (United States)

    Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

    2011-07-01

    Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus.

  16. Structure of the Escherichia coli RNA polymerase α subunit C-terminal domain

    International Nuclear Information System (INIS)

    Lara-González, Samuel; Birktoft, Jens J.; Lawson, Catherine L.

    2010-01-01

    The crystal structure of the dimethyllysine derivative of the E. coli RNA polymerase α subunit C-terminal domain is reported at 2.0 Å resolution. The α subunit C-terminal domain (αCTD) of RNA polymerase (RNAP) is a key element in transcription activation in Escherichia coli, possessing determinants responsible for the interaction of RNAP with DNA and with transcription factors. Here, the crystal structure of E. coli αCTD (α subunit residues 245–329) determined to 2.0 Å resolution is reported. Crystals were obtained after reductive methylation of the recombinantly expressed domain. The crystals belonged to space group P2 1 and possessed both pseudo-translational symmetry and pseudo-merohedral twinning. The refined coordinate model (R factor = 0.193, R free = 0.236) has improved geometry compared with prior lower resolution determinations of the αCTD structure [Jeon et al. (1995 ▶), Science, 270, 1495–1497; Benoff et al. (2002 ▶), Science, 297, 1562–1566]. An extensive dimerization interface formed primarily by N- and C-terminal residues is also observed. The new coordinates will facilitate the improved modeling of αCTD-containing multi-component complexes visualized at lower resolution using X-ray crystallography and electron-microscopy reconstruction

  17. The AMP-activated protein kinase beta 1 subunit modulates erythrocyte integrity.

    Science.gov (United States)

    Cambridge, Emma L; McIntyre, Zoe; Clare, Simon; Arends, Mark J; Goulding, David; Isherwood, Christopher; Caetano, Susana S; Reviriego, Carmen Ballesteros; Swiatkowska, Agnieszka; Kane, Leanne; Harcourt, Katherine; Adams, David J; White, Jacqueline K; Speak, Anneliese O

    2017-01-01

    Failure to maintain a normal in vivo erythrocyte half-life results in the development of hemolytic anemia. Half-life is affected by numerous factors, including energy balance, electrolyte gradients, reactive oxygen species, and membrane plasticity. The heterotrimeric AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that acts as a critical regulator of cellular energy balance. Previous roles for the alpha 1 and gamma 1 subunits in the control of erythrocyte survival have been reported. In the work described here, we studied the role of the beta 1 subunit in erythrocytes and observed microcytic anemia with compensatory extramedullary hematopoiesis together with splenomegaly and increased osmotic resistance. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  18. In silico predictions of LH2 ring sizes from the crystal structure of a single subunit using molecular dynamics simulations.

    Science.gov (United States)

    Janosi, Lorant; Keer, Harindar; Cogdell, Richard J; Ritz, Thorsten; Kosztin, Ioan

    2011-07-01

    Most of the currently known light-harvesting complexes 2 (LH2) rings are formed by 8 or 9 subunits. As of now, questions like "what factors govern the LH2 ring size?" and "are there other ring sizes possible?" remain largely unanswered. Here, we investigate by means of molecular dynamics (MD) simulations and stochastic modeling the possibility of predicting the size of an LH2 ring from the sole knowledge of the high resolution crystal structure of a single subunit. Starting with single subunits of two LH2 rings with known size, that is, an 8-ring from Rs. moliscianum (MOLI) and a 9-ring from Rps. acidophila (ACI), and one with unknown size (referred to as X), we build atomic models of subunit dimers corresponding to assumed 8-, 9-, and 10-ring geometries. After inserting each of the dimers into a lipid-water environment, we determine the preferred angle between the corresponding subunits by three methods: (1) energy minimization, (2) free MD simulations, and (3) potential of mean force calculations. We find that the results from all three methods are consistent with each other, and when taken together, it allows one to predict with reasonable level of confidence the sizes of the corresponding ring structures. One finds that X and ACI very likely form a 9-ring, while MOLI is more likely to form an 8-ring than a 9-ring. Finally, we discuss both the merits and limitations of all three prediction methods. Copyright © 2011 Wiley-Liss, Inc.

  19. The Inherent Asymmetry of DNA Replication.

    Science.gov (United States)

    Snedeker, Jonathan; Wooten, Matthew; Chen, Xin

    2017-10-06

    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 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 in DNA replication 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 decisions related to patterning and development.

  20. Ultrastructural Characterization of Zika Virus Replication Factories

    Directory of Open Access Journals (Sweden)

    Mirko Cortese

    2017-02-01

    Full Text Available Summary: 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. : Cortese et al. show that ZIKV infection in both human hepatoma and neuronal progenitor cells induces drastic structural modification of the cellular architecture. Microtubules and intermediate filaments surround the viral replication factory composed of vesicles corresponding to ER membrane invagination toward the ER lumen. Importantly, alteration of microtubule flexibility impairs ZIKV replication. Keywords: Zika virus, flavivirus, human neural progenitor cells, replication factories, replication organelles, microtubules, intermediate filaments, electron microscopy, electron tomography, live-cell imaging

  1. Cellular Aspects of Prion Replication In Vitro

    Science.gov (United States)

    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

  2. Human C4orf14 interacts with the mitochondrial nucleoid and is involved in the biogenesis of the small mitochondrial ribosomal subunit.

    Science.gov (United States)

    He, J; Cooper, H M; Reyes, A; Di Re, M; Kazak, L; Wood, S R; Mao, C C; Fearnley, I M; Walker, J E; Holt, I J

    2012-07-01

    The bacterial homologue of C4orf14, YqeH, has been linked to assembly of the small ribosomal subunit. Here, recombinant C4orf14 isolated from human cells, co-purified with the small, 28S subunit of the mitochondrial ribosome and the endogenous protein co-fractionated with the 28S subunit in sucrose gradients. Gene silencing of C4orf14 specifically affected components of the small subunit, leading to decreased protein synthesis in the organelle. The GTPase of C4orf14 was critical to its interaction with the 28S subunit, as was GTP. Therefore, we propose that C4orf14, with bound GTP, binds to components of the 28S subunit facilitating its assembly, and GTP hydrolysis acts as the release mechanism. C4orf14 was also found to be associated with human mitochondrial nucleoids, and C4orf14 gene silencing caused mitochondrial DNA depletion. In vitro C4orf14 is capable of binding to DNA. The association of C4orf14 with mitochondrial translation factors and the mitochondrial nucleoid suggests that the 28S subunit is assembled at the mitochondrial nucleoid, enabling the direct transfer of messenger RNA from the nucleoid to the ribosome in the organelle.

  3. MYC and the Control of DNA Replication

    Science.gov (United States)

    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. Replicated Data Management for Mobile Computing

    CERN Document Server

    Douglas, Terry

    2008-01-01

    Managing data in a mobile computing environment invariably involves caching or replication. In many cases, a mobile device has access only to data that is stored locally, and much of that data arrives via replication from other devices, PCs, and services. Given portable devices with limited resources, weak or intermittent connectivity, and security vulnerabilities, data replication serves to increase availability, reduce communication costs, foster sharing, and enhance survivability of critical information. Mobile systems have employed a variety of distributed architectures from client-server

  5. Interaction of the Sliding Clamp β-Subunit and Hda, a DnaA-Related Protein

    Science.gov (United States)

    Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

    2004-01-01

    In Escherichia coli, interactions between the replication initiation protein DnaA, the β subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and β proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with β in vitro. A new β-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified β-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind β. A 10-amino-acid peptide containing the E. coli Hda β-binding motif was shown to compete with Hda for binding to β in an Hda-β interaction assay. These results establish that the interaction of Hda with β is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication. PMID:15150238

  6. Interaction of the sliding clamp beta-subunit and Hda, a DnaA-related protein.

    Science.gov (United States)

    Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

    2004-06-01

    In Escherichia coli, interactions between the replication initiation protein DnaA, the beta subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and beta proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with beta in vitro. A new beta-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified beta-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind beta. A 10-amino-acid peptide containing the E. coli Hda beta-binding motif was shown to compete with Hda for binding to beta in an Hda-beta interaction assay. These results establish that the interaction of Hda with beta is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.

  7. NF-κB p50 subunit knockout impairs late LTP and alters long term memory in the mouse hippocampus

    Directory of Open Access Journals (Sweden)

    Oikawa Kensuke

    2012-07-01

    Full Text Available Abstract Background Nuclear factor kappa B (NF-κB is a transcription factor typically expressed with two specific subunits (p50, p65. Investigators have reported that NF-κB is activated during the induction of in vitro long term potentiation (LTP, a paradigm of synaptic plasticity and correlate of memory, suggesting that NF-κB may be necessary for some aspects of memory encoding. Furthermore, NF-κB has been implicated as a potential requirement in behavioral tests of memory. Unfortunately, very little work has been done to explore the effects of deleting specific NF-κB subunits on memory. Studies have shown that NF-κB p50 subunit deletion (p50−/− leads to memory deficits, however some recent studies suggest the contrary where p50−/− mice show enhanced memory in the Morris water maze (MWM. To more critically explore the role of the NF-κB p50 subunit in synaptic plasticity and memory, we assessed long term spatial memory in vivo using the MWM, and synaptic plasticity in vitro utilizing high frequency stimuli capable of eliciting LTP in slices from the hippocampus of NF-κB p50−/− versus their controls (p50+/+. Results We found that the lack of the NF-κB p50 subunit led to significant decreases in late LTP and in selective but significant alterations in MWM tests (i.e., some improvements during acquisition, but deficits during retention. Conclusions These results support the hypothesis that the NF-κ p50 subunit is required in long term spatial memory in the hippocampus.

  8. Molecular cloning and expression of heteromeric ACCase subunit genes from Jatropha curcas.

    Science.gov (United States)

    Gu, Keyu; Chiam, Huihui; Tian, Dongsheng; Yin, Zhongchao

    2011-04-01

    Acetyl-CoA carboxylase (ACCase) catalyzes the biotin-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, which is the essential first step in the biosynthesis of long-chain fatty acids. ACCase exists as a multi-subunit enzyme in most prokaryotes and the chloroplasts of most plants and algae, while it is present as a multi-domain enzyme in the endoplasmic reticulum of most eukaryotes. The heteromeric ACCase of higher plants consists of four subunits: an α-subunit of carboxyltransferase (α-CT, encoded by accA gene), a biotin carboxyl carrier protein (BCCP, encoded by accB gene), a biotin carboxylase (BC, encoded by accC gene) and a β-subunit of carboxyltransferase (β-CT, encoded by accD gene). In this study, we cloned and characterized the genes accA, accB1, accC and accD that encode the subunits of heteromeric ACCase in Jatropha (Jatropha curcas), a potential biofuel plant. The full-length cDNAs of the four subunit genes were isolated from a Jatropha cDNA library and by using 5' RACE, whereas the genomic clones were obtained from a Jatropha BAC library. They encode a 771 amino acid (aa) α-CT, a 286-aa BCCP1, a 537-aa BC and a 494-aa β-CT, respectively. The single-copy accA, accB1 and accC genes are nuclear genes, while the accD gene is located in chloroplast genome. Jatropha α-CT, BCCP1, BC and β-CT show high identity to their homologues in other higher plants at amino acid level and contain all conserved domains for ACCase activity. The accA, accB1, accC and accD genes are temporally and spatially expressed in the leaves and endosperm of Jatropha plants, which are regulated by plant development and environmental factors. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  9. What Should Researchers Expect When They Replicate Studies? A Statistical View of Replicability in Psychological Science.

    Science.gov (United States)

    Patil, Prasad; Peng, Roger D; Leek, Jeffrey T

    2016-07-01

    A recent study of the replicability of key psychological findings is a major contribution toward understanding the human side of the scientific process. Despite the careful and nuanced analysis reported, the simple narrative disseminated by the mass, social, and scientific media was that in only 36% of the studies were the original results replicated. In the current study, however, we showed that 77% of the replication effect sizes reported were within a 95% prediction interval calculated using the original effect size. Our analysis suggests two critical issues in understanding replication of psychological studies. First, researchers' intuitive expectations for what a replication should show do not always match with statistical estimates of replication. Second, when the results of original studies are very imprecise, they create wide prediction intervals-and a broad range of replication effects that are consistent with the original estimates. This may lead to effects that replicate successfully, in that replication results are consistent with statistical expectations, but do not provide much information about the size (or existence) of the true effect. In this light, the results of the Reproducibility Project: Psychology can be viewed as statistically consistent with what one might expect when performing a large-scale replication experiment. © The Author(s) 2016.

  10. Mapping replication origins in yeast chromosomes.

    Science.gov (United States)

    Brewer, B J; Fangman, W L

    1991-07-01

    The replicon hypothesis, first proposed in 1963 by Jacob and Brenner, states that DNA replication is controlled at sites called origins. Replication origins have been well studied in prokaryotes. However, the study of eukaryotic chromosomal origins has lagged behind, because until recently there has been no method for reliably determining the identity and location of origins from eukaryotic chromosomes. Here, we review a technique we developed with the yeast Saccharomyces cerevisiae that allows both the mapping of replication origins and an assessment of their activity. Two-dimensional agarose gel electrophoresis and Southern hybridization with total genomic DNA are used to determine whether a particular restriction fragment acquires the branched structure diagnostic of replication initiation. The technique has been used to localize origins in yeast chromosomes and assess their initiation efficiency. In some cases, origin activation is dependent upon the surrounding context. The technique is also being applied to a variety of eukaryotic organisms.

  11. Advancing Polymerase Ribozymes Towards Self-Replication

    Science.gov (United States)

    Tjhung, K. F.; Joyce, G. F.

    2017-07-01

    Autocatalytic replication and evolution in vitro by (i) a cross-chiral RNA polymerase catalyzing polymerization of mononucleotides of the opposite handedness; (ii) non-covalent assembly of component fragments of an existing RNA polymerase ribozyme.

  12. Initiation of Replication in Escherichia coli

    DEFF Research Database (Denmark)

    Frimodt-Møller, Jakob

    The circular chromosome of Escherichia coli is replicated by two replisomes assembled at the unique origin and moving in the opposite direction until they meet in the less well defined terminus. The key protein in initiation of replication, DnaA, facilitates the unwinding of double-stranded DNA...... to single-stranded DNA in oriC. Although DnaA is able to bind both ADP and ATP, DnaA is only active in initiation when bound to ATP. Although initiation of replication, and the regulation of this, is thoroughly investigated it is still not fully understood. The overall aim of the thesis was to investigate...... the regulation of initiation, the effect on the cell when regulation fails, and if regulation was interlinked to chromosomal organization. This thesis uncovers that there exists a subtle balance between chromosome replication and reactive oxygen species (ROS) inflicted DNA damage. Thus, failure in regulation...

  13. LHCb Data Replication During SC3

    CERN Multimedia

    Smith, A

    2006-01-01

    LHCb's participation in LCG's Service Challenge 3 involves testing the bulk data transfer infrastructure developed to allow high bandwidth distribution of data across the grid in accordance with the computing model. To enable reliable bulk replication of data, LHCb's DIRAC system has been integrated with gLite's File Transfer Service middleware component to make use of dedicated network links between LHCb computing centres. DIRAC's Data Management tools previously allowed the replication, registration and deletion of files on the grid. For SC3 supplementary functionality has been added to allow bulk replication of data (using FTS) and efficient mass registration to the LFC replica catalog.Provisional performance results have shown that the system developed can meet the expected data replication rate required by the computing model in 2007. This paper details the experience and results of integration and utilisation of DIRAC with the SC3 transfer machinery.

  14. Molecular Mechanisms of DNA Replication Checkpoint Activation

    Directory of Open Access Journals (Sweden)

    Bénédicte Recolin

    2014-03-01

    Full Text Available 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.

  15. Locating Nearby Copies of Replicated Internet Servers

    National Research Council Canada - National Science Library

    Guyton, James D; Schwartz, Michael F

    1995-01-01

    In this paper we consider the problem of choosing among a collection of replicated servers focusing on the question of how to make choices that segregate client/server traffic according to network topology...

  16. The Legal Road To Replicating Silicon Valley

    OpenAIRE

    John Armour; Douglas Cumming

    2004-01-01

    Must policymakers seeking to replicate the success of Silicon Valley’s venture capital market first replicate other US institutions, such as deep and liquid stock markets? Or can legal reforms alone make a significant difference? In this paper, we compare the economic and legal determinants of venture capital investment, fundraising and exits. We introduce a cross-sectional and time series empirical analysis across 15 countries and 13 years of data spanning an entire business cycle. We show t...

  17. Evolution of Database Replication Technologies for WLCG

    OpenAIRE

    Baranowski, Zbigniew; Pardavila, Lorena Lobato; Blaszczyk, Marcin; Dimitrov, Gancho; Canali, Luca

    2015-01-01

    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 databas...

  18. Pituitary glycoprotein hormone a-subunit secretion by cirrhotic patients

    Directory of Open Access Journals (Sweden)

    Oliveira M.C.

    1999-01-01

    Full Text Available Secretion of the a-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the a-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6% presented hypogonadism (which was central in 45 cases and primary in 2, 7 were eugonadal, and 9 women were in normal menopause. The serum a-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively, with an intra-assay coefficient of variation (CV of less than 5% and an interassay CV of 5%, and sensitivity limit of 4 ng/l. The serum a-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the a-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01 and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02. Among men with central hypogonadism there was a negative correlation between a-subunit levels and total testosterone levels (r = 0.54, P<0.01 as well as free testosterone levels (r = -0.53, P<0.01. In conclusion, although the a-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver.

  19. The scenario on the origin of translation in the RNA world: in principle of replication parsimony

    Directory of Open Access Journals (Sweden)

    Ma Wentao

    2010-11-01

    Full Text Available Abstract Background It is now believed that in the origin of life, proteins should have been "invented" in an RNA world. However, due to the complexity of a possible RNA-based proto-translation system, this evolving process seems quite complicated and the associated scenario remains very blurry. Considering that RNA can bind amino acids with specificity, it has been reasonably supposed that initial peptides might have been synthesized on "RNA templates" containing multiple amino acid binding sites. This "Direct RNA Template (DRT" mechanism is attractive because it should be the simplest mechanism for RNA to synthesize peptides, thus very likely to have been adopted initially in the RNA world. Then, how this mechanism could develop into a proto-translation system mechanism is an interesting problem. Presentation of the hypothesis Here an explanation to this problem is shown considering the principle of "replication parsimony" --- genetic information tends to be utilized in a parsimonious way under selection pressure, due to its replication cost (e.g., in the RNA world, nucleotides and ribozymes for RNA replication. Because a DRT would be quite long even for a short peptide, its replication cost would be great. Thus the diversity and the length of functional peptides synthesized by the DRT mechanism would be seriously limited. Adaptors (proto-tRNAs would arise to allow a DRT's complementary strand (called "C-DRT" here to direct the synthesis of the same peptide synthesized by the DRT itself. Because the C-DRT is a necessary part in the DRT's replication, fewer turns of the DRT's replication would be needed to synthesize definite copies of the functional peptide, thus saving the replication cost. Acting through adaptors, C-DRTs could transform into much shorter templates (called "proto-mRNAs" here and substitute the role of DRTs, thus significantly saving the replication cost. A proto-rRNA corresponding to the small subunit rRNA would then emerge

  20. Suppression of feline coronavirus replication in vitro by cyclosporin A

    Directory of Open Access Journals (Sweden)

    Tanaka Yoshikazu

    2012-04-01

    Full Text Available Abstract The feline infectious peritonitis virus (FIPV is a member of the feline coronavirus family that causes FIP, which is incurable and fatal in cats. Cyclosporin A (CsA, an immunosuppressive agent that targets the nuclear factor pathway of activated T-cells (NF-AT to bind cellular cyclophilins (CyP, dose-dependently inhibited FIPV replication in vitro. FK506 (an immunosuppressor of the pathway that binds cellular FK506-binding protein (FKBP but not CyP did not affect FIPV replication. Neither cell growth nor viability changed in the presence of either CsA or FK506, and these factors did not affect the NF-AT pathway in fcwf-4 cells. Therefore, CsA does not seem to exert inhibitory effects via the NF-AT pathway. In conclusion, CsA inhibited FIPV replication in vitro and further studies are needed to verify the practical value of CsA as an anti-FIPV treatment in vivo.

  1. Promotion of Hendra Virus Replication by MicroRNA 146a

    Science.gov (United States)

    Marsh, Glenn A.; Jenkins, Kristie A.; Gantier, Michael P.; Tizard, Mark L.; Middleton, Deborah; Lowenthal, John W.; Haining, Jessica; Izzard, Leonard; Gough, Tamara J.; Deffrasnes, Celine; Stambas, John; Robinson, Rachel; Heine, Hans G.; Pallister, Jackie A.; Foord, Adam J.; Bean, Andrew G.; Wang, Lin-Fa

    2013-01-01

    Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease. PMID:23345523

  2. ATAD2 is an epigenetic reader of newly synthesized histone marks during DNA replication.

    Science.gov (United States)

    Koo, Seong Joo; Fernández-Montalván, Amaury E; Badock, Volker; Ott, Christopher J; Holton, Simon J; von Ahsen, Oliver; Toedling, Joern; Vittori, Sarah; Bradner, James E; Gorjánácz, Mátyás

    2016-10-25

    ATAD2 (ATPase family AAA domain-containing protein 2) is a chromatin regulator harboring an AAA+ ATPase domain and a bromodomain, previously proposed to function as an oncogenic transcription co-factor. Here we suggest that ATAD2 is also required for DNA replication. ATAD2 is co-expressed with genes involved in DNA replication in various cancer types and predominantly expressed in S phase cells where it localized on nascent chromatin (replication sites). Our extensive biochemical and cellular analyses revealed that ATAD2 is recruited to replication sites through a direct interaction with di-acetylated histone H4 at K5 and K12, indicative of newly synthesized histones during replication-coupled chromatin reassembly. Similar to ATAD2-depletion, ectopic expression of ATAD2 mutants that are deficient in binding to these di-acetylation marks resulted in reduced DNA replication and impaired loading of PCNA onto chromatin, suggesting relevance of ATAD2 in DNA replication. Taken together, our data show a novel function of ATAD2 in cancer and for the first time identify a reader of newly synthesized histone di-acetylation-marks during replication.

  3. Modes of DNA repair and replication

    International Nuclear Information System (INIS)

    Hanawalt, P.; Kondo, S.

    1979-01-01

    Modes of DNA repair and replication require close coordination as well as some overlap of enzyme functions. Some classes of recovery deficient mutants may have defects in replication rather than repair modes. Lesions such as the pyrimidine dimers produced by ultraviolet light irradiation are the blocks to normal DNA replication in vivo and in vitro. The DNA synthesis by the DNA polymerase 1 of E. coli is blocked at one nucleotide away from the dimerized pyrimidines in template strands. Thus, some DNA polymerases seem to be unable to incorporate nucleotides opposite to the non-pairing lesions in template DNA strands. The lesions in template DNA strands may block the sequential addition of nucleotides in the synthesis of daughter strands. Normal replication utilizes a constitutive ''error-free'' mode that copies DNA templates with high fidelity, but which may be totally blocked at a lesion that obscures the appropriate base pairing specificity. It might be expected that modified replication system exhibits generally high error frequency. The error rate of DNA polymerases may be controlled by the degree of phosphorylation of the enzyme. Inducible SOS system is controlled by recA genes that also control the pathways for recombination. It is possible that SOS system involves some process other than the modification of a blocked replication apparatus to permit error-prone transdimer synthesis. (Yamashita, S.)

  4. Replication and robustness in developmental research.

    Science.gov (United States)

    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).

  5. Nonequilibrium Entropic Bounds for Darwinian Replicators

    Directory of Open Access Journals (Sweden)

    Jordi Piñero

    2018-01-01

    Full Text Available Life evolved on our planet by means of a combination of Darwinian selection and innovations leading to higher levels of complexity. The emergence and selection of replicating entities is a central problem in prebiotic evolution. Theoretical models have shown how populations of different types of replicating entities exclude or coexist with other classes of replicators. Models are typically kinetic, based on standard replicator equations. On the other hand, the presence of thermodynamical constraints for these systems remain an open question. This is largely due to the lack of a general theory of statistical methods for systems far from equilibrium. Nonetheless, a first approach to this problem has been put forward in a series of novel developements falling under the rubric of the extended second law of thermodynamics. The work presented here is twofold: firstly, we review this theoretical framework and provide a brief description of the three fundamental replicator types in prebiotic evolution: parabolic, malthusian and hyperbolic. Secondly, we employ these previously mentioned techinques to explore how replicators are constrained by thermodynamics. Finally, we comment and discuss where further research should be focused on.

  6. Mediator Subunit Med28 Is Essential for Mouse Peri-Implantation Development and Pluripotency.

    Directory of Open Access Journals (Sweden)

    Lin Li

    Full Text Available The multi-subunit mammalian Mediator complex acts as an integrator of transcriptional regulation by RNA Polymerase II, and has emerged as a master coordinator of development and cell fate determination. We previously identified the Mediator subunit, MED28, as a cytosolic binding partner of merlin, the Neurofibromatosis 2 (NF2 tumor suppressor, and thus MED28 is distinct in having a cytosolic role as an NF2 interacting protein as well as a nuclear role as a Mediator complex subunit. Although limited in vitro studies have been performed on MED28, its in vivo function remains unknown. Employing a knockout mouse model, we describe for the first time the requirement for Med28 in the developing mouse embryo. Med28-deficiency causes peri-implantation lethality resulting from the loss of pluripotency of the inner cell mass accompanied by reduced expression of key pluripotency transcription factors Oct4 and Nanog. Further, overexpression of Med28 in mouse embryonic fibroblasts enhances the efficiency of their reprogramming to pluripotency. Cre-mediated inactivation of Med28 in induced pluripotent stem cells shows that Med28 is required for their survival. Intriguingly, heterozygous loss of Med28 results in differentiation of induced pluripotent stem cells into extraembryonic trophectoderm and primitive endoderm lineages. Our findings document the essential role of Med28 in the developing embryo as well as in acquisition and maintenance of pluripotency during reprogramming.

  7. Short communication: molecular characterization of dog and cat p65 subunits of NF-kappaB.

    Science.gov (United States)

    Ishikawa, Shingo; Takemitsu, Hiroshi; Li, Gebin; Mori, Nobuko; Yamamoto, Ichiro; Arai, Toshiro

    2015-04-01

    Nuclear factor kappa B (NF-κB) plays an important role in the immune system. The p65 subunit is an important part of NF-κB unit, and studies of dog and cat p65 subunits of NF-κB (dp65 and cp65) are important in understanding their immune function. In this study, we described the molecular characterization of dp65 and cp65. The dp65 and cp65 complementary DNA encoded 542 and 555 amino acids, respectively, showing a high sequence homology with the mammalian p65 subunit (>87.5%). Quantitative polymerase chain reaction revealed that the p65 messenger RNA is highly expressed in the dog stomach and cat heart and adipose tissue. Functional NF-κB promoter-luciferase reporter vectors revealed that our isolated dp65 and cp65 cDNA encodes a functionally active protein. Transiently expressed dp65 and cp65 up-regulated pro-inflammatory cytokine expression levels in dog and cat, respectively. These findings suggest that dp65 and cp65 play important roles in regulating immune function. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

    Science.gov (United States)

    Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

    2012-01-01

    The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

  9. Organization of Replication of Ribosomal DNA in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Linskens, Maarten H.K.; Huberman, Joel A.

    1988-01-01

    Using recently developed replicon mapping techniques, we have analyzed the replication of the ribosomal DNA in Saccharomyces cerevisiae. The results show that (i) the functional origin of replication colocalizes with an autonomously replicating sequence element previously mapped to the

  10. The replicative DNA polymerase of herpes simplex virus 1 exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities

    OpenAIRE

    Bogani, Federica; Boehmer, Paul E.

    2008-01-01

    Base excision repair (BER) is essential for maintaining genome stability both to counter the accumulation of unusual bases and to protect from base loss in the DNA. Herpes simplex virus 1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery, including enzymes involved in nucleotide metabolism. We report on a replicative family B and a herpesvirus-encoded DNA Pol that possesses DNA lyase activity. We have discovered that the catalytic subunit of the HSV-1 DNA polymeras...

  11. The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes.

    Science.gov (United States)

    Hemsley, Piers A; Hurst, Charlotte H; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R; De Cothi, Elizabeth A; Steele, John F; Knight, Heather

    2014-01-01

    The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation-induced freezing tolerance. In addition, these three subunits are required for low temperature-induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced.

  12. How many bootstrap replicates are necessary?

    Science.gov (United States)

    Pattengale, Nicholas D; Alipour, Masoud; Bininda-Emonds, Olaf R P; Moret, Bernard M E; Stamatakis, Alexandros

    2010-03-01

    Phylogenetic bootstrapping (BS) is a standard technique for inferring confidence values on phylogenetic trees that is based on reconstructing many trees from minor variations of the input data, trees called replicates. BS is used with all phylogenetic reconstruction approaches, but we focus here on one of the most popular, maximum likelihood (ML). Because ML inference is so computationally demanding, it has proved too expensive to date to assess the impact of the number of replicates used in BS on the relative accuracy of the support values. For the same reason, a rather small number (typically 100) of BS replicates are computed in real-world studies. Stamatakis et al. recently introduced a BS algorithm that is 1 to 2 orders of magnitude faster than previous techniques, while yielding qualitatively comparable support values, making an experimental study possible. In this article, we propose stopping criteria--that is, thresholds computed at runtime to determine when enough replicates have been generated--and we report on the first large-scale experimental study to assess the effect of the number of replicates on the quality of support values, including the performance of our proposed criteria. We run our tests on 17 diverse real-world DNA--single-gene as well as multi-gene--datasets, which include 125-2,554 taxa. We find that our stopping criteria typically stop computations after 100-500 replicates (although the most conservative criterion may continue for several thousand replicates) while producing support values that correlate at better than 99.5% with the reference values on the best ML trees. Significantly, we also find that the stopping criteria can recommend very different numbers of replicates for different datasets of comparable sizes. Our results are thus twofold: (i) they give the first experimental assessment of the effect of the number of BS replicates on the quality of support values returned through BS, and (ii) they validate our proposals for

  13. Probing the functional subunits of the tonoplast H+-ATPase

    International Nuclear Information System (INIS)

    Randall, S.K.; Lai, S.; Sze, H.

    1986-01-01

    The tonoplast ATPase of oat roots is composed of at least three polypeptides of 72, 60, and 16 kDa. The 16 kDA polypeptide covalently binds N,N'-dicyclohexylcarbodiimide and is postulated to be a component of the proton channel. Initial studies to identify other subunits indicate that both the 72 and 60 kDa subunits covalently bind 14 C]-7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and [ 14 C]N-ethylamleimide, inhibitors of the tonoplast ATPase. ATP prevents binding of these inhibitors suggesting that both the 72 and 60 kDa subunits are involved in substrate binding. Polyclonal antibody has been made to the 72 kDa subunit. Western blot analysis of tonoplast vesicles reveals single reactive polypeptide (72 kDa). The antibody shows no cross-reactivity towards either the mitochondrial F 1 -ATPase or the plasma membrane ATPase. This antibody specifically inhibits ATP hydrolysis and ATP-dependent H + pumping in native tonoplast vesicles. The authors conclude that the 72 kDa subunit is intimately associated with the catalytic (or ATP-binding) site

  14. A rolling circle replication mechanism produces multimeric lariats of mitochondrial DNA in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Samantha C Lewis

    2015-02-01

    Full Text Available Mitochondrial DNA (mtDNA encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans.

  15. MMSET is dynamically regulated during cell-cycle progression and promotes normal DNA replication.

    Science.gov (United States)

    Evans, Debra L; Zhang, Haoxing; Ham, Hyoungjun; Pei, Huadong; Lee, SeungBaek; Kim, JungJin; Billadeau, Daniel D; Lou, Zhenkun

    2016-01-01

    The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin Recognition Complex (ORC) binds to future replication origins, coordinating with multiple factors to load the minichromosome maintenance (MCM) complex onto future replication origins as part of the pre-replication complex (pre-RC). The pre-RC machinery, in turn, remains inactive until the subsequent S phase when it is required for replication fork formation, thereby initiating DNA replication. Multiple myeloma SET domain-containing protein (MMSET, a.k.a. WHSC1, NSD2) is a histone methyltransferase that is frequently overexpressed in aggressive cancers and is essential for normal human development. Several studies have suggested a role for MMSET in cell-cycle regulation; however, whether MMSET is itself regulated during cell-cycle progression has not been examined. In this study, we report that MMSET is degraded during S phase in a cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) and proteasome-dependent manner. Notably, we also report defects in DNA replication and a decreased association of pre-RC factors with chromatin in MMSET-depleted cells. Taken together, our results suggest a dynamic regulation of MMSET levels throughout the cell cycle, and further characterize the role of MMSET in DNA replication and cell-cycle progression.

  16. Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus.

    Directory of Open Access Journals (Sweden)

    Kai Xu

    2016-10-01

    Full Text Available Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs. The VRCs built by Tomato bushy stunt virus (TBSV are enriched with phosphatidylethanolamine (PE through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5-positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment.

  17. MOF Suppresses Replication Stress and Contributes to Resolution of Stalled Replication Forks.

    Science.gov (United States)

    Singh, Dharmendra Kumar; Pandita, Raj K; Singh, Mayank; Chakraborty, Sharmistha; Hambarde, Shashank; Ramnarain, Deepti; Charaka, Vijaya; Ahmed, Kazi Mokim; Hunt, Clayton R; Pandita, Tej K

    2018-03-15

    The human MOF (hMOF) protein belongs to the MYST family of histone acetyltransferases and plays a critical role in transcription and the DNA damage response. MOF is essential for cell proliferation; however, its role during replication and replicative stress is unknown. Here we demonstrate that cells depleted of MOF and under replicative stress induced by cisplatin, hydroxyurea, or camptothecin have reduced survival, a higher frequency of S-phase-specific chromosome damage, and increased R-loop formation. MOF depletion decreased replication fork speed and, when combined with replicative stress, also increased stalled replication forks as well as new origin firing. MOF interacted with PCNA, a key coordinator of replication and repair machinery at replication forks, and affected its ubiquitination and recruitment to the DNA damage site. Depletion of MOF, therefore, compromised the DNA damage repair response as evidenced by decreased Mre11, RPA70, Rad51, and PCNA focus formation, reduced DNA end resection, and decreased CHK1 phosphorylation in cells after exposure to hydroxyurea or cisplatin. These results support the argument that MOF plays an important role in suppressing replication stress induced by genotoxic agents at several stages during the DNA damage response. Copyright © 2018 American Society for Microbiology.

  18. Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants.

    Science.gov (United States)

    Xu, Kai; Nagy, Peter D

    2017-04-01

    Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication have been gaining full attention only recently. Previous work with Tomato bushy stunt tombusvirus (TBSV) in model host yeast has revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we showed that the TBSV p33 and p92 replication proteins could bind to sterols in vitro The sterol binding by p33 is supported by cholesterol recognition/interaction amino acid consensus (CRAC) and CARC-like sequences within the two transmembrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also showed the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis on both the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The data support the notion that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Together, the results obtained in this study and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication. IMPORTANCE One intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly, or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are

  19. X-irradiation affects all DNA replication intermediates when inhibiting replication initiation

    International Nuclear Information System (INIS)

    Loenn, U.; Karolinska Hospital, Stockholm

    1982-01-01

    When a human melanoma line was irradiated with 10 Gy, there was, after 30 to 60 min, a gradual reduction in the DNA replication rate. Ten to twelve hours after the irradiation, the DNA replication had returned to near normal rate. The results showed tht low dose-rate X-irradiation inhibits preferentially the formation of small DNA replication intermediates. There is no difference between the inhibition of these replication intermediates formed only in the irradiated cells and those formed also in untreated cells. (U.K.)

  20. Realistic Vascular Replicator for TAVR Procedures.

    Science.gov (United States)

    Rotman, Oren M; Kovarovic, Brandon; Sadasivan, Chander; Gruberg, Luis; Lieber, Baruch B; Bluestein, Danny

    2018-04-13

    Transcatheter aortic valve replacement (TAVR) is an over-the-wire procedure for treatment of severe aortic stenosis (AS). TAVR valves are conventionally tested using simplified left heart simulators (LHS). While those provide baseline performance reliably, their aortic root geometries are far from the anatomical in situ configuration, often overestimating the valves' performance. We report on a novel benchtop patient-specific arterial replicator designed for testing TAVR and training interventional cardiologists in the procedure. The Replicator is an accurate model of the human upper body vasculature for training physicians in percutaneous interventions. It comprises of fully-automated Windkessel mechanism to recreate physiological flow conditions. Calcified aortic valve models were fabricated and incorporated into the Replicator, then tested for performing TAVR procedure by an experienced cardiologist using the Inovare valve. EOA, pressures, and angiograms were monitored pre- and post-TAVR. A St. Jude mechanical valve was tested as a reference that is less affected by the AS anatomy. Results in the Replicator of both valves were compared to the performance in a commercial ISO-compliant LHS. The AS anatomy in the Replicator resulted in a significant decrease of the TAVR valve performance relative to the simplified LHS, with EOA and transvalvular pressures comparable to clinical data. Minor change was seen in the mechanical valve performance. The Replicator showed to be an effective platform for TAVR testing. Unlike a simplified geometric anatomy LHS, it conservatively provides clinically-relevant outcomes and complement it. The Replicator can be most valuable for testing new valves under challenging patient anatomies, physicians training, and procedural planning.

  1. Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

    Science.gov (United States)

    Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

    1999-03-05

    Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

  2. TIA-1 and TIAR interact with 5'-UTR of enterovirus 71 genome and facilitate viral replication.

    Science.gov (United States)

    Wang, Xiaohui; Wang, Huanru; Li, Yixuan; Jin, Yu; Chu, Ying; Su, Airong; Wu, Zhiwei

    2015-10-16

    Enterovirus 71 is one of the major causative pathogens of HFMD in children. Upon infection, the viral RNA is translated in an IRES-dependent manner and requires several host factors for effective replication. Here, we found that T-cell-restricted intracellular antigen 1 (TIA-1), and TIA-1 related protein (TIAR) were translocated from nucleus to cytoplasm after EV71 infection and localized to the sites of viral replication. We found that TIA-1 and TIAR can facilitate EV71 replication by enhancing the viral genome synthesis in host cells. We demonstrated that both proteins bound to the stem-loop I of 5'-UTR of viral genome and improved the stability of viral genomic RNA. Our results suggest that TIA-1 and TIAR are two new host factors that interact with 5-UTR of EV71 genome and positively regulate viral replication. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Novel subunit structure observed for noncooperative hemoglobin from Urechis caupo.

    Science.gov (United States)

    Kolatkar, P R; Meador, W E; Stanfield, R L; Hackert, M L

    1988-03-05

    Tetrameric hemoglobin from the "fat innkeeper" worm Urechis caupo possesses a novel subunit arrangement having an "inside out" quaternary structure in that the G/H helices are located on the outer surface of the tetramer. A 5-A resolution crystal structure reveals that although the individual subunits are beta-like, having a distinct D helix and the general myoglobin fold, the subunit contacts are very different from those previously observed for hemoglobins. Furthermore, the hemoglobin from U. caupo is also quite different from the unusual hemoglobin tetramer from clam which also has its G/H helices on the outer surface but with the hemes in close proximity through E-F helical contacts (Royer, W. E., Jr., Love, W. E., and Fenderson, F. F. (1985) Nature 316, 277-280).

  4. Dynamic properties of motor proteins with two subunits

    International Nuclear Information System (INIS)

    Kolomeisky, Anatoly B; III, Hubert Phillips

    2005-01-01

    The dynamics of motor protein molecules consisting of two subunits is investigated using simple discrete stochastic models. Exact steady-state analytical expressions are obtained for velocities and dispersions for any number of intermediate states and conformations between the corresponding binding states of proteins. These models enable us to provide a detailed description and comparison of two different mechanisms of the motion of motor proteins along the linear tracks: the hand-over-hand mechanism, when the motion of subunits alternate; and the inchworm mechanism, when one subunit is always trailing another one. It is shown that the proteins in the hand-over-hand mechanism move faster and fluctuate more than the molecules in the inchworm mechanism. The effect of external forces on dynamic properties of motor proteins is also discussed. Finally, a quantitative method, based on experimental observations for single motor proteins, is proposed for distinguishing between two mechanisms of motion

  5. CSNAP Is a Stoichiometric Subunit of the COP9 Signalosome

    Directory of Open Access Journals (Sweden)

    Shelly Rozen

    2015-10-01

    Full Text Available The highly conserved COP9 signalosome (CSN complex is a key regulator of all cullin-RING-ubiquitin ligases (CRLs, the largest family of E3 ubiquitin ligases. Until now, it was accepted that the CSN is composed of eight canonical components. Here, we report the discovery of an additional integral and stoichiometric subunit that had thus far evaded detection, and we named it CSNAP (CSN acidic protein. We show that CSNAP binds CSN3, CSN5, and CSN6, and its incorporation into the CSN complex is mediated through the C-terminal region involving conserved aromatic residues. Moreover, depletion of this small protein leads to reduced proliferation and a flattened and enlarged morphology. Finally, on the basis of sequence and structural properties shared by both CSNAP and DSS1, a component of the related 19S lid proteasome complex, we propose that CSNAP, the ninth CSN subunit, is the missing paralogous subunit of DSS1.

  6. Thermostable Subunit Vaccines for Pulmonary Delivery: How Close Are We?

    DEFF Research Database (Denmark)

    Foged, Camilla

    2016-01-01

    , such as influenza, tuberculosis, and Ebola, for which no good universal vaccines exist. At least two pharmaceutical improvements are expected to help filling this gap: i) The development of thermostable vaccine dosage forms, and ii) the full exploitation of the adjuvant technology for subunit vaccines to potentiate...... strong immune responses. This review highlights the status and recent advances in formulation and pulmonary delivery of thermostable human subunit vaccines. Such vaccines are very appealing from compliance, distribution and immunological point of view: Being non-invasive, inhalable vaccines are self...... immunity. Here, I review state of the art and perspectives in formulation design and processing methods for powder-based subunit vaccines intended for pulmonary administration, and present dry powder inhaler technologies suitable for translating these vaccines into clinical trials....

  7. [Single-molecule detection and characterization of DNA replication based on DNA origami].

    Science.gov (United States)

    Wang, Qi; Fan, Youjie; Li, Bin

    2014-08-01

    To investigate single-molecule detection and characterization of DNA replication. Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication. The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis. The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.

  8. Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

    Science.gov (United States)

    Zakian, V A; Brewer, B J; Fangman, W L

    1979-08-01

    Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA

  9. Spacetime replication of continuous variable quantum information

    International Nuclear Information System (INIS)

    Hayden, Patrick; Nezami, Sepehr; Salton, Grant; Sanders, Barry C

    2016-01-01

    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. (paper)

  10. Extremal dynamics in random replicator ecosystems

    Energy Technology Data Exchange (ETDEWEB)

    Kärenlampi, Petri P., E-mail: petri.karenlampi@uef.fi

    2015-10-02

    The seminal numerical experiment by Bak and Sneppen (BS) is repeated, along with computations with replicator models, including a greater amount of features. Both types of models do self-organize, and do obey power-law scaling for the size distribution of activity cycles. However species extinction within the replicator models interferes with the BS self-organized critical (SOC) activity. Speciation–extinction dynamics ruins any stationary state which might contain a steady size distribution of activity cycles. The BS-type activity appears as a dissimilar phenomenon in comparison to speciation–extinction dynamics in the replicator system. No criticality is found from the speciation–extinction dynamics. Neither are speciations and extinctions in real biological macroevolution known to contain any diverging distributions, or self-organization towards any critical state. Consequently, biological macroevolution probably is not a self-organized critical phenomenon. - Highlights: • Extremal Dynamics organizes random replicator ecosystems to two phases in fitness space. • Replicator systems show power-law scaling of activity. • Species extinction interferes with Bak–Sneppen type mutation activity. • Speciation–extinction dynamics does not show any critical phase transition. • Biological macroevolution probably is not a self-organized critical phenomenon.

  11. Replication of cultured lung epithelial cells

    International Nuclear Information System (INIS)

    Guzowski, D.; Bienkowski, R.

    1986-01-01

    The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during the first week showed that the cells contained myofilaments, microvilli and lamellar bodies (markers for type 2 cells). The following changes were observed after the first week: increase in cell size; loss of lamellar bodies and appearance of multivesicular bodies; increase in rough endoplasmic reticulum and golgi; increase in tonafilaments and well-defined junctions. General cell morphology was good for up to 10 wk. Cells cultured on plastic surface degenerated after 1 wk. Cell replication was assayed by autoradiography of cultures exposed to ( 3 H)-thymidine and by direct cell counts. The cells did not replicate during the first week; however, between 2-10 wk the cells incorporated the label and went through approximately 6 population doublings. They have demonstrated that lung alveolar epithelial cells can replicate in culture if they are maintained on an appropriate substrate. The coincidence of ability to replicate and loss of markers for differentiation may reflect the dichotomy between growth and differentiation commonly observed in developing systems

  12. The evolutionary ecology of molecular replicators.

    Science.gov (United States)

    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.

  13. A conserved MCM single-stranded DNA binding element is essential for replication initiation.

    Science.gov (United States)

    Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B; Bell, Stephen P; Enemark, Eric J

    2014-04-01

    The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001.

  14. Distinct functions of human RecQ helicases during DNA replication

    Czech Academy of Sciences Publication Activity Database

    Urban, Václav; Dobrovolná, Jana; Janščák, Pavel

    2017-01-01

    Roč. 225, červen (2017), s. 20-26 ISSN 0301-4622 R&D Projects: GA ČR(CZ) GA14-05743S; GA MŠk LH14037 Institutional support: RVO:68378050 Keywords : DNA replication * Replication stress * RecQ helicases * Genomic instability * Cancer Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 2.402, year: 2016

  15. Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus

    International Nuclear Information System (INIS)

    Zhang Jing; Yamada, Osamu; Sakamoto, Takashi; Yoshida, Hiroshi; Iwai, Takahiro; Matsushita, Yoshihisa; Shimamura, Hideo; Araki, Hiromasa; Shimotohno, Kunitada

    2004-01-01

    Small interfering RNA (siRNA) is currently being evaluated not only as a powerful tool for functional genomics, but also as a potentially promising therapeutic agent for cancer and infectious diseases. Inhibitory effect of siRNA on viral replication has been demonstrated in multiple pathogenic viruses. However, because of the high sequence specificity of siRNA-mediated RNA degradation, antiviral efficacy of siRNA directed to viral genome will be largely limited by emergence of escape variants resistant to siRNA due to high mutation rates of virus, especially RNA viruses such as poliovirus and hepatitis C virus (HCV). To investigate the therapeutic feasibility of siRNAs specific for the putative cellular cofactors for HCV, we constructed adenovirus vectors expressing siRNAs against La, polypyrimidine tract-binding protein (PTB), subunit gamma of human eukaryotic initiation factors 2B (eIF2Bγ), and human VAMP-associated protein of 33 kDa (hVAP-33). Adenoviral-mediated expression of siRNAs markedly diminished expression of the endogenous genes, and silencing of La, PTB, and hVAP-33 by siRNAs substantially blocked HCV replication in Huh-7 cells. Thus, our studies demonstrate the feasibility and potential of adenoviral-delivered siRNAs specific for cellular cofactors in combating HCV infection, which can be used either alone or in combination with siRNA against viral genome to prevent the escape of mutant variants and provide additive or synergistic anti-HCV effects

  16. The Hepatitis B Virus (HBV) HBx Protein Activates AKT To Simultaneously Regulate HBV Replication and Hepatocyte Survival

    Science.gov (United States)

    Rawat, Siddhartha

    2014-01-01

    ABSTRACT Chronic infection with hepatitis B virus (HBV) is a risk factor for developing liver diseases such as hepatocellular carcinoma (HCC). HBx is a multifunctional protein encoded by the HBV genome; HBx stimulates HBV replication and is thought to play an important role in the development of HBV-associated HCC. HBx can activate the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway in some cell lines; however, whether HBx regulates PI3K/AKT signaling in normal hepatocytes has not been evaluated. In studies described here, we assessed HBx activation of PI3K/AKT signaling in an ex vivo model of cultured primary hepatocytes and determined how this HBx activity affects HBV replication. We report that HBx activates AKT in primary hepatocytes and that the activation of AKT decreases HBV replication and HBV mRNA and core protein levels. We show that the transcription factor hepatocyte nuclear factor 4α (HNF4α) is a target of HBx-regulated AKT, and we link HNF4α to HBx-regulated AKT modulation of HBV transcription and replication. Although we and others have shown that HBx stimulates and is likely required for HBV replication, we now report that HBx also activates signals that can diminish the overall level of HBV replication. While this may seem counterintuitive, we show that an important effect of HBx activation of AKT is inhibition of apoptosis. Consequently, our studies suggest that HBx balances HBV replication and cell survival by stimulating signaling pathways that enhance hepatocyte survival at the expense of higher levels of HBV replication. IMPORTANCE Chronic hepatitis B virus (HBV) infection is a common cause of the development of liver cancer. Regulation of cell signaling pathways by the HBV HBx protein is thought to influence the development of HBV-associated liver cancer. HBx stimulates, and may be essential for, HBV replication. We show that HBx activates AKT in hepatocytes to reduce HBV replication. While this seems contradictory to an

  17. Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells

    International Nuclear Information System (INIS)

    Shigeno, Yuta; Uchiumi, Toshio; Nomura, Takaomi

    2016-01-01

    Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential