Promoting interdomain analogical transfer: When creating a problem helps to solve a problem.

PubMed

Minervino, Ricardo A; Olguín, Valeria; Trench, Máximo

2017-02-01

Research on analogical thinking has devised several ways of promoting an abstract encoding of base analogs, thus rendering them more retrievable during later encounters with similar situations lacking surface similarities. Recent studies have begun to explore ways of facilitating transfer at retrieval time, which could facilitate the retrieval of distant analogs learned within contexts that were not specially directed to emphasize their abstract structure. Such studies demonstrate that comparing a target problem to an analogous problem helps students retrieve base analogs that lack surface similarities. To devise more portable ways of enhancing analogical transfer, Experiment 1 replicated Kurtz and Loewenstein's (Memory & Cognition, 35, 334-341, 2007) target-comparison procedure with an additional condition in which participants compared the target to a nonanalogous problem before attempting to reach its solution. Although comparing two analogous targets outperformed the standard transfer condition in promoting analogical transfer, comparing nonanalogous problems did not yield a transfer advantage. Based on prior studies that showed that the activity of creating analogous problems during their initial encoding elicits a more abstract representation of base analogs, in Experiment 2 we assessed whether constructing a second analogous target problem at retrieval time helps participants retrieve superficially dissimilar base analogs. As predicted, target invention increased the retrieval of distant sources. In both experiments we found an association between the quality of the generated schemas and the probability of retrieving a distant base analog from memory.

Selenizing astragalus polysaccharide attenuates PCV2 replication promotion caused by oxidative stress through autophagy inhibition via PI3K/AKT activation.

PubMed

Liu, Dandan; Xu, Jing; Qian, Gang; Hamid, Mohammed; Gan, Fang; Chen, Xingxiang; Huang, Kehe

2018-03-01

Our previous studies have shown that oxidative stress could promote the porcine circovirus type 2 (PCV2) replication, and astragalus polysaccharide (APS)/selenium could suppress PCV2 replication. However, whether selenizing astragalus polysaccharide (sAPS) provides protection against oxidative stress-induced PCV2 replication promotion and the mechanism involved remain unclear. The present study aimed to explore the mechanism of the PCV2 replication promotion induced by oxidative stress and a novel pharmacotherapeutic approach involving the regulation of autophagy of sAPS. Our results showed that H 2 O 2 promoted PCV2 replication via enhancing autophagy by using 3-methyladenine (3-MA) and autophagy-related gene 5 (ATG5) knockdown. Sodium selenite, APS, the mixture of sodium selenite and APS, and sAPS significantly inhibited H 2 O 2 -induced PCV2 replication promotion, respectively. Among these, sAPS exerted maximal inhibitory effect. sAPS could also significantly inhibit autophagy activated by H 2 O 2 and increase the Akt and mTOR phosphorylation. Moreover, LY294002, the specific phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) inhibitor, significantly alleviated the effects of sAPS on autophagy and PCV2 replication. Taken together, we conclude that H 2 O 2 promotes PCV2 replication by inducing autophagy and sAPS attenuates the PCV2 replication promotion through autophagy inhibition via PI3K/AKT activation. Copyright © 2017 Elsevier B.V. All rights reserved.

Checkpoint-dependent RNR induction promotes fork restart after replicative stress.

PubMed

Morafraile, Esther C; Diffley, John F X; Tercero, José Antonio; Segurado, Mónica

2015-01-20

The checkpoint kinase Rad53 is crucial to regulate DNA replication in the presence of replicative stress. Under conditions that interfere with the progression of replication forks, Rad53 prevents Exo1-dependent fork degradation. However, although EXO1 deletion avoids fork degradation in rad53 mutants, it does not suppress their sensitivity to the ribonucleotide reductase (RNR) inhibitor hydroxyurea (HU). In this case, the inability to restart stalled forks is likely to account for the lethality of rad53 mutant cells after replication blocks. Here we show that Rad53 regulates replication restart through the checkpoint-dependent transcriptional response, and more specifically, through RNR induction. Thus, in addition to preventing fork degradation, Rad53 prevents cell death in the presence of HU by regulating RNR-expression and localization. When RNR is induced in the absence of Exo1 and RNR negative regulators, cell viability of rad53 mutants treated with HU is increased and the ability of replication forks to restart after replicative stress is restored.

Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter.

PubMed

Wang, Xiaohong; Liu, Haibin; Ge, Hui; Ajiro, Masahiko; Sharma, Nishi R; Meyers, Craig; Morozov, Pavel; Tuschl, Thomas; Klar, Amar; Court, Donald; Zheng, Zhi-Ming

2017-05-30

The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P 811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis -acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors. IMPORTANCE It has been known for decades that the activity of viral late promoters is associated with viral DNA replication among almost all DNA viruses. However, the mechanism of how DNA replication activates the viral late promoter and what components of the replication machinery are involved remain largely unknown. In this study, we characterized the P 811 promoter region of HPV18 and demonstrated that its activation depends on the orientation of DNA replication. Using single-stranded oligonucleotides targeting the replication fork on either leading or lagging strands, we showed that viral lagging-strand replication activates the promoter. We also identified a transcriptional repressor element located upstream of the promoter transcription start site which interacts with cellular proteins hnRNP D0B and hnRNP A/B and modulates the late promoter activity. This is the first report on how DNA replication activates a viral late promoter. Copyright © 2017 Wang et al.

A Tumor-stroma Targeted Oncolytic Adenovirus Replicated in Human Ovary Cancer Samples and Inhibited Growth of Disseminated Solid Tumors in Mice

PubMed Central

Lopez, M Veronica; Rivera, Angel A; Viale, Diego L; Benedetti, Lorena; Cuneo, Nicasio; Kimball, Kristopher J; Wang, Minghui; Douglas, Joanne T; Zhu, Zeng B; Bravo, Alicia I; Gidekel, Manuel; Alvarez, Ronald D; Curiel, David T; Podhajcer, Osvaldo L

2012-01-01

Targeting the tumor stroma in addition to the malignant cell compartment is of paramount importance to achieve complete tumor regression. In this work, we modified a previously designed tumor stroma-targeted conditionally replicative adenovirus (CRAd) based on the SPARC promoter by introducing a mutated E1A unable to bind pRB and pseudotyped with a chimeric Ad5/3 fiber (Ad F512v1), and assessed its replication/lytic capacity in ovary cancer in vitro and in vivo. AdF512v1 was able to replicate in fresh samples obtained from patients: (i) with primary human ovary cancer; (ii) that underwent neoadjuvant treatment; (iii) with metastatic disease. In addition, we show that four intraperitoneal (i.p.) injections of 5 × 1010 v.p. eliminated 50% of xenografted human ovary tumors disseminated in nude mice. Moreover, AdF512v1 replication in tumor models was enhanced 15–40-fold when the tumor contained a mix of malignant and SPARC-expressing stromal cells (fibroblasts and endothelial cells). Contrary to the wild-type virus, AdF512v1 was unable to replicate in normal human ovary samples while the wild-type virus can replicate. This study provides evidence on the lytic capacity of this CRAd and highlights the importance of targeting the stromal tissue in addition to the malignant cell compartment to achieve tumor regression. PMID:22948673

Glutamine starvation enhances PCV2 replication via the phosphorylation of p38 MAPK, as promoted by reducing glutathione levels.

PubMed

Chen, Xingxiang; Shi, Xiuli; Gan, Fang; Huang, Da; Huang, Kehe

2015-03-18

Glutamine has a positive effect on ameliorating reproductive failure caused by porcine circovirus type 2 (PCV2). However, the mechanism by which glutamine affects PCV2 replication remains unclear. This study was conducted to investigate the effects of glutamine on PCV2 replication and its underlying mechanisms in vitro. The results show that glutamine promoted PK-15 cell viability. Surprisingly, glutamine starvation significantly increased PCV2 replication. The promotion of PCV2 replication by glutamine starvation disappeared after fresh media with 4 mM glutamine was added. Likewise, promotion of PCV2 was observed after adding buthionine sulfoximine (BSO). Glutamine starvation or BSO treatment increased the level of p38 MAPK phosphorylation and PCV2 replication in PK-15 cells. Meanwhile, p38 MAPK phosphorylation and PCV2 replication significantly decreased in p38-knockdown PK-15 cells. Promotion of PCV2 replication caused by glutamine starvation could be blocked in p38-knockdown PK-15 cells. Therefore, glutamine starvation increased PCV2 replication by promoting p38 MAPK activation, which was associated with the down regulation of intracellular glutathione levels. Our findings may contribute toward interpreting the possible pathogenic mechanism of PCV2 and provide a theoretical reference for application of glutamine in controlling porcine circovirus-associated diseases.

Efficient data replication for the delivery of high-quality video content over P2P VoD advertising networks

NASA Astrophysics Data System (ADS)

Ho, Chien-Peng; Yu, Jen-Yu; Lee, Suh-Yin

2011-12-01

Recent advances in modern television systems have had profound consequences for the scalability, stability, and quality of transmitted digital data signals. This is of particular significance for peer-to-peer (P2P) video-on-demand (VoD) related platforms, faced with an immediate and growing demand for reliable service delivery. In response to demands for high-quality video, the key objectives in the construction of the proposed framework were user satisfaction with perceived video quality and the effective utilization of available resources on P2P VoD networks. This study developed a peer-based promoter to support online advertising in P2P VoD networks based on an estimation of video distortion prior to the replication of data stream chunks. The proposed technology enables the recovery of lost video using replicated stream chunks in real time. Load balance is achieved by adjusting the replication level of each candidate group according to the degree-of-distortion, thereby enabling a significant reduction in server load and increased scalability in the P2P VoD system. This approach also promotes the use of advertising as an efficient tool for commercial promotion. Results indicate that the proposed system efficiently satisfies the given fault tolerances.

RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.

PubMed

Elia, Andrew E H; Wang, David C; Willis, Nicholas A; Boardman, Alexander P; Hajdu, Ildiko; Adeyemi, Richard O; Lowry, Elizabeth; Gygi, Steven P; Scully, Ralph; Elledge, Stephen J

2015-10-15

We have used quantitative proteomics to profile ubiquitination in the DNA damage response (DDR). We demonstrate that RPA, which functions as a protein scaffold in the replication stress response, is multiply ubiquitinated upon replication fork stalling. Ubiquitination of RPA occurs on chromatin, involves sites outside its DNA binding channel, does not cause proteasomal degradation, and increases under conditions of fork collapse, suggesting a role in repair at stalled forks. We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination (HR) at stalled replication forks. Mutational analysis suggests that multisite ubiquitination of the entire RPA complex is responsible for repair at stalled forks. Multisite protein group sumoylation is known to promote HR in yeast. Our findings reveal a similar requirement for multisite protein group ubiquitination during HR at stalled forks in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Complete regression of human malignant mesothelioma xenografts following local injection of midkine promoter-driven oncolytic adenovirus

PubMed Central

Kubo, Shuji; Kawasaki, Yoshiko; Yamaoka, Norie; Tagawa, Masatoshi; Kasahara, Noriyuki; Terada, Nobuyuki; Okamura, Haruki

2010-01-01

Background Malignant mesothelioma is a highly aggressive tumor with poor prognosis. Conventional therapies for mesothelioma are generally non-curative, and new treatment paradigms are urgently needed. We hypothesized that the tumor-specific midkine (Mdk) promoter could confer transcriptional targeting to oncolytic adenoviruses for effective treatment of malignant mesothelioma. Methods We analyzed Mdk expression by quantitative RT-PCR in six human mesothelioma cell lines, and tested Mdk promoter activity by luciferase reporter assay. Based on these data, we constructed a replication-selective oncolytic adenovirus, designated AdMdk-E1-iresTK, which contains an Mdk promoter-driven adenoviral E1 gene and HSV-thymidine kinase (TK) suicide gene, and CMV promoter-driven green fluorescent protein (GFP) marker gene. Selectivity of viral replication and cytolysis were characterized in normal vs. mesothelioma cells in vitro, and intratumoral spread and antitumor efficacy were investigated in vivo. Results Mdk promoter activity was restricted in normal cells, but highly activated in mesothelioma cell lines. AdMdk-E1-iresTK was seen to efficiently replicate, produce viral progeny, and spread in multiple mesothelioma cell lines. Lytic spread of AdMdk-E1-iresTK mediated efficient killing of these mesothelioma cells, and its in vitro cytocidal effect was significantly enhanced by treatment with the prodrug, ganciclovir. Intratumoral injection of AdMdk-E1-iresTK caused complete regression of MESO4 and MSTO human mesothelioma xenografts in athymic mice. In vivo fluorescence imaging demonstrated intratumoral spread of AdMdk-E1-iresTK-derived signals, which vanished after tumor eradication. Conclusions These data indicate that transcriptional targeting of viral replication by the Mdk promoter represents a promising general strategy for oncolytic virotherapy of cancers with upregulated Mdk expression, including malignant mesothelioma. PMID:20635326

Detection of neuroendocrine tumors using promoter-specific secreted Gaussia luciferase.

PubMed

Tseng, Alan Wei-Shun; Akerstrom, Victoria; Chen, Chiachen; Breslin, Mary B; Lan, Michael S

2016-01-01

Accurate detection of neuroendocrine (NE) tumors is critically important for better prognosis and treatment outcomes in patients. To demonstrate the efficacy of using an adenoviral vector for the detection of NE tumors, we have constructed a pair of adenoviral vectors which, in combination, can conditionally replicate and release Gaussia luciferase into the circulation after infecting the NE tumors. The expression of these two vectors is regulated upstream by an INSM1-promoter (insulinoma-associated-1) that is specifically active in NE tumors and developing NE tissues, but silenced in normal adult tissues. In order to retain the tumor-specificity of the INSM1 promoter, we have modified the promoter using the core insulator sequence from the chicken β-globin HS4 insulator and the neuronal restrictive silencing element (NRSE). This modified INSM1-promoter can retain NE tumor specificity in an adenoviral construct while driving a mutated adenovirus E1A gene (∆24E1A), the Metridia, or Gaussia luciferase gene. The in vitro cell line and mouse xenograft human tumor studies revealed the NE specificity of the INSM1-promoter in NE lung cancer, neuroblastoma, medulloblastoma, retinoblastoma, and insulinoma. When we combined the INSM1-promoter driven Gaussia luciferase with ∆24E1A, the co-infected NE tumor secreted higher levels of Gaussia luciferase as compared to the INSM1p-Gaussia virus alone. In a mouse subcutaneous xenograft tumor model, the combination viruses secreted detectable level of Gaussia luciferase after infecting an INSM1-positive NE lung tumor for ≥12 days. Therefore, the INSM1-promoter specific conditional replicating adenovirus represents a sensitive diagnostic tool to aid clinicians in the detection of NE tumors.

Effects of the adenovirus 2 late promoter on simian virus 40 transcription and replication.

PubMed Central

Grass, D S; Manley, J L

1986-01-01

A 100-base-pair fragment of adenovirus 2 (Ad2) DNA encompassing the major late transcriptional promoter was inserted into the simian virus 40 (SV40) late promoter region at SV40 nucleotide 294 to study the effects of a strong TATA box-containing promoter on SV40 late transcription. pSVAdE contains the insert in an orientation such that it would promote transcription towards the origin and early region of SV40, while the insert is in the opposite orientation in pSVAdL. Nuclease S1 analysis with 5'-end-labeled probes showed that in cells transfected with pSVAdE, the late mRNA initiation sites are essentially the same as in wild type, demonstrating that an insert of 100 base pairs can have no effect on utilization of the SV40 late start sites. In pSVAdL-transfected cells, however, the major late viral initiation site is now in the insert at +1 with respect to the Ad2 major late cap site. However, all of the SV40 initiation sites are still utilized and with the same efficiency relative to each other as in wild type. Thus, it appears that the Ad2 late promoter and the SV40 late promoter can function independently on the same DNA molecule, even when one promoter is embedded within the other. By using cytosine arabinoside to block DNA replication and thereby inhibit the onset of late expression, it has been shown that both the Ad2 late promoter and the SV40 late promoter have similar requirement for DNA replication in this context. In addition, pSVAdL showed dramatically diminished virus viability and VPI expression compared with both wildtype and pSVAdE. Possible explanations for this unexpected finding are discussed. Images PMID:3001338

SeMet attenuates OTA-induced PCV2 replication promotion by inhibiting autophagy by activating the AKT/mTOR signaling pathway.

PubMed

Qian, Gang; Liu, Dandan; Hu, Junfa; Gan, Fang; Hou, Lili; Zhai, Nianhui; Chen, Xingxiang; Huang, Kehe

2018-02-13

Porcine circovirus type 2 (PCV2) is recognized as the causative agent of porcine circovirus-associated diseases. PCV2 replication could be promoted by low doses of ochratoxin A (OTA) as in our previous study and selenium has been shown to attenuate PCV2 replication. However, the underlying mechanism remains unclear. The aim of the study was to investigate the effects of selenomethionine (SeMet), the major component of organic selenium, on OTA-induced PCV2 replication promotion and its potential mechanism. The present study demonstrates that OTA could promote PCV2 replication as measured by cap protein expression, viral titer, viral DNA copies and the number of infected cells. In addition, OTA could activate autophagy as indicated by up-regulated light chain 3 (LC3)-II and autophagy-related protein 5 expressions and autophagosome formation. Further, OTA could down-regulate p-AKT and p-mTOR expressions and OTA-induced autophagy was inhibited when insulin was applied. SeMet at 2, 4 and 6 μM had significant inhibiting effects against OTA-induced PCV2 replication promotion. Furthermore, SeMet could attenuate OTA-induced autophagy and up-regulate OTA-induced p-AKT and p-mTOR expression inhibition. Rapamycin, an inhibitor of AKT/mTOR, could reverse the effects of SeMet on OTA-induced autophagy and the PCV2 replication promotion. In conclusion, SeMet could block OTA-induced PCV2 replication promotion by inhibiting autophagy by activating the AKT/mTOR pathway. Therefore, SeMet supplementation could be an effective prophylactic strategy against PCV2 infections and autophagy may be a potential marker to develop novel anti-PCV2 drugs.

Strong minor groove base conservation in sequence logos implies DNA distortion or base flipping during replication and transcription initiation.

PubMed

Schneider, T D

2001-12-01

The sequence logo for DNA binding sites of the bacteriophage P1 replication protein RepA shows unusually high sequence conservation ( approximately 2 bits) at a minor groove that faces RepA. However, B-form DNA can support only 1 bit of sequence conservation via contacts into the minor groove. The high conservation in RepA sites therefore implies a distorted DNA helix with direct or indirect contacts to the protein. Here I show that a high minor groove conservation signature also appears in sequence logos of sites for other replication origin binding proteins (Rts1, DnaA, P4 alpha, EBNA1, ORC) and promoter binding proteins (sigma(70), sigma(D) factors). This finding implies that DNA binding proteins generally use non-B-form DNA distortion such as base flipping to initiate replication and transcription.

Regulated Eukaryotic DNA Replication Origin Firing with Purified Proteins

PubMed Central

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

2016-01-01

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 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. PMID:25739503

Regulated eukaryotic DNA replication origin firing with purified proteins.

PubMed

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.

Analysis of re-replication from deregulated origin licensing by DNA fiber spreading

PubMed Central

Dorn, Elizabeth S.; Chastain, Paul D.; Hall, Jonathan R.; Cook, Jeanette Gowen

2009-01-01

A major challenge each human cell-division cycle is to ensure that DNA replication origins do not initiate more than once, a phenomenon known as re-replication. Acute deregulation of replication control ultimately causes extensive DNA damage, cell-cycle checkpoint activation and cell death whereas moderate deregulation promotes genome instability and tumorigenesis. In the absence of detectable increases in cellular DNA content however, it has been difficult to directly demonstrate re-replication or to determine if the ability to re-replicate is restricted to a particular cell-cycle phase. Using an adaptation of DNA fiber spreading we report the direct detection of re-replication on single DNA molecules from human chromosomes. Using this method we demonstrate substantial re-replication within 1 h of S phase entry in cells overproducing the replication factor, Cdt1. Moreover, a comparison of the HeLa cancer cell line to untransformed fibroblasts suggests that HeLa cells produce replication signals consistent with low-level re-replication in otherwise unperturbed cell cycles. Re-replication after depletion of the Cdt1 inhibitor, geminin, in an untransformed fibroblast cell line is undetectable by standard assays but readily quantifiable by DNA fiber spreading analysis. Direct evaluation of re-replicated DNA molecules will promote increased understanding of events that promote or perturb genome stability. PMID:19010964

DNA Replication Is Required for Circadian Clock Function by Regulating Rhythmic Nucleosome Composition.

PubMed

Liu, Xiao; Dang, Yunkun; Matsu-Ura, Toru; He, Yubo; He, Qun; Hong, Christian I; Liu, Yi

2017-07-20

Although the coupling between circadian and cell cycles allows circadian clocks to gate cell division and DNA replication in many organisms, circadian clocks were thought to function independently of cell cycle. Here, we show that DNA replication is required for circadian clock function in Neurospora. Genetic and pharmacological inhibition of DNA replication abolished both overt and molecular rhythmicities by repressing frequency (frq) gene transcription. DNA replication is essential for the rhythmic changes of nucleosome composition at the frq promoter. The FACT complex, known to be involved in histone disassembly/reassembly, is required for clock function and is recruited to the frq promoter in a replication-dependent manner to promote replacement of histone H2A.Z by H2A. Finally, deletion of H2A.Z uncoupled the dependence of the circadian clock on DNA replication. Together, these results establish circadian clock and cell cycle as interdependent coupled oscillators and identify DNA replication as a critical process in the circadian mechanism. Published by Elsevier Inc.

Altered Subcellular Localization of a Tobacco Membrane Raft-Associated Remorin Protein by Tobamovirus Infection and Transient Expression of Viral Replication and Movement Proteins

PubMed Central

Sasaki, Nobumitsu; Takashima, Eita; Nyunoya, Hiroshi

2018-01-01

Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexvirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by tomato mosaic virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamovirus movement. PMID:29868075

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

PubMed Central

Natsume, Toyoaki; Nishimura, Kohei; Minocherhomji, Sheroy; Bhowmick, Rahul; Hickson, Ian D.; Kanemaki, Masato T.

2017-01-01

DNA replication fork progression can be disrupted at difficult to replicate loci in the human genome, which has the potential to challenge chromosome integrity. This replication fork disruption can lead to the dissociation of the replisome and the formation of DNA damage. To model the events 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 (DSBs). Remarkably, these cells maintain some DNA synthesis in the absence of MCM2, and this requires the MCM8–9 complex, a paralog of the MCM2–7 replicative helicase. We show that MCM8–9 functions in a homologous recombination-based pathway downstream from RAD51, which is promoted by DSB induction. This RAD51/MCM8–9 axis is distinct from the recently described RAD52-dependent DNA synthesis pathway that operates in early mitosis at common fragile sites. We propose that stalled replication forks can be restarted in S phase via homologous recombination using MCM8–9 as an alternative replicative helicase. PMID:28487407

Achievement goals, self-handicapping, and performance: a 2 x 2 achievement goal perspective.

PubMed

Ntoumanis, Nikos; Thøgersen-Ntoumani, Cecilie; Smith, Alison L

2009-11-01

Elliot and colleagues (2006) examined the effects of experimentally induced achievement goals, proposed by the trichotomous model, on self-handicapping and performance in physical education. Our study replicated and extended the work of Elliot et al. by experimentally promoting all four goals proposed by the 2 x 2 model (Elliot & McGregor, 2001), measuring the participants' own situational achievement goals, using a relatively novel task, and testing the participants in a group setting. We used a randomized experimental design with four conditions that aimed to induce one of the four goals advanced by the 2 x 2 model. The participants (n = 138) were undergraduates who engaged in a dart-throwing task. The results pertaining to self-handicapping partly replicated Elliot and colleagues' findings by showing that experimentally promoted performance-avoidance goals resulted in less practice. In contrast, the promotion of mastery-avoidance goals did not result in less practice compared with either of the approach goals. Dart-throwing performance did not differ among the four goal conditions. Personal achievement goals did not moderate the effects of experimentally induced goals on self-handicapping and performance. The extent to which mastery-avoidance goals are maladaptive is discussed, as well as the interplay between personal and experimentally induced goals.

Evidence-based fitness promotion in an afterschool setting: implementation fidelity and its policy implications.

PubMed

Thaw, Jean M; Villa, Manuela; Reitman, David; DeLucia, Christian; Gonzalez, Vanessa; Hanson, K Lori

2014-01-01

Little is known about how the adoption of evidence-based physical activity (PA) curricula by out-of-school time (OST) programs affects children's physical fitness, and there are no clear guidelines of what constitutes reasonable gains given the types of PA instruction currently offered in these programs. Using a three-wave, quasi-experimental, naturalistic observation design, this study evaluated the implementation of an evidence-based PA instruction curriculum (Sports, Play, and Active Recreation for Kids [SPARK]) and examined whether the potential health benefits of evidence-based PA instruction can be replicated in this context when compared to OST programs that do not use evidence-based PA curricula. Quality of PA instruction and SPARK implementation fidelity were also assessed. Results indicated that children in the non-evidence-based/standard PA instruction programs engaged in higher levels of moderate-to-vigorous PA (MVPA) and showed greater improvements in fitness levels over time. The findings from this chapter suggest that while it is generally accepted that evidence-based approaches yield higher levels of PA when implemented by researchers under controlled conditions, findings are inconsistent when evidence-based PA instruction is implemented in the field, under presumably less controlled conditions. It appears that when it comes to PA instruction in afterschool, either less structured activities or well-implemented evidence-based practices could be the key to promoting higher PA levels and greater health and fitness for school-aged children. © 2014 WILEY PERIODICALS, INC.

Structural rearrangements in the mitochondrial genome of Drosophila melanogaster induced by elevated levels of the replicative DNA helicase

PubMed Central

Ciesielski, Grzegorz L; Nadalutti, Cristina A; Oliveira, Marcos T; Griffith, Jack D; Kaguni, Laurie S

2018-01-01

Abstract Pathological conditions impairing functions of mitochondria often lead to compensatory upregulation of the mitochondrial DNA (mtDNA) replisome machinery, and the replicative DNA helicase appears to be a key factor in regulating mtDNA copy number. Moreover, mtDNA helicase mutations have been associated with structural rearrangements of the mitochondrial genome. To evaluate the effects of elevated levels of the mtDNA helicase on the integrity and replication of the mitochondrial genome, we overexpressed the helicase in Drosophila melanogaster Schneider cells and analyzed the mtDNA by two-dimensional neutral agarose gel electrophoresis and electron microscopy. We found that elevation of mtDNA helicase levels increases the quantity of replication intermediates and alleviates pausing at the replication slow zones. Though we did not observe a concomitant alteration in mtDNA copy number, we observed deletions specific to the segment of repeated elements in the immediate vicinity of the origin of replication, and an accumulation of species characteristic of replication fork stalling. We also found elevated levels of RNA that are retained in the replication intermediates. Together, our results suggest that upregulation of mtDNA helicase promotes the process of mtDNA replication but also results in genome destabilization. PMID:29432582

Sleeping Beauty transposon-based system for rapid generation of HBV-replicating stable cell lines.

PubMed

Wu, Yong; Zhang, Tian-Ying; Fang, Lin-Lin; Chen, Zi-Xuan; Song, Liu-Wei; Cao, Jia-Li; Yang, Lin; Yuan, Quan; Xia, Ning-Shao

2016-08-01

The stable HBV-replicating cell lines, which carry replication-competent HBV genome stably integrated into the genome of host cell, are widely used to evaluate the effects of antiviral agents. However, current methods to generate HBV-replicating cell lines, which are mostly dependent on random integration of foreign DNA via plasmid transfection, are less-efficient and time-consuming. To address this issue, we constructed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable cell lines carrying replication-competent HBV genome of different genotype. This vector contains a Sleeping Beauty transposon containing HBV 1.3-copy genome with an expression cassette of the SV40 promoter driving red fluorescent protein (mCherry) and self-cleaving P2A peptide linked puromycin resistance gene (PuroR). In addition, a PGK promoter-driven SB100X hyperactive transposase cassette is placed in the outside of the transposon in the same plasmid.The HBV-replicating stable cells could be obtained from pTSMP-HBV transfected HepG2 cells by red fluorescence-activated cell sorting and puromycin resistant cell selection within 4-week. Using this system, we successfully constructed four cell lines carrying replication-competent HBV genome of genotypes A-D. The replication and viral protein expression profiles of these cells were systematically characterized. In conclusion, our study provides a high-efficiency strategy to generate HBV-replicating stable cell lines, which may facilitate HBV-related virological study. Copyright © 2016. Published by Elsevier B.V.

Role of the human cytomegalovirus major immediate-early promoter's 19-base-pair-repeat cyclic AMP-response element in acutely infected cells.

PubMed

Keller, M J; Wheeler, D G; Cooper, E; Meier, J L

2003-06-01

Prior studies have suggested a role of the five copies of the 19-bp-repeat cyclic AMP (cAMP)-response element (CRE) in major immediate-early (MIE) promoter activation, the rate-limiting step in human cytomegalovirus (HCMV) replication. We used two different HCMV genome modification strategies to test this hypothesis in acutely infected cells. We report the following: (i) the CREs do not govern basal levels of MIE promoter activity at a high or low multiplicity of infection (MOI) in human foreskin fibroblast (HFF)- or NTera2-derived neuronal cells; (ii) serum and virion components markedly increase MIE promoter-dependent transcription at a low multiplicity of infection (MOI), but this increase is not mediated by the CREs; (iii) forskolin stimulation of the cAMP signaling pathway induces a two- to threefold increase in MIE RNA levels in a CRE-specific manner at a low MOI in both HFF- and NTera2-derived neuronal cells; and (iv) the CREs do not regulate basal levels of HCMV DNA replication at a high or low MOI in HFF. Their presence does impart a forskolin-induced increase in viral DNA replication at a low MOI but only when basal levels of MIE promoter activity are experimentally diminished. In conclusion, the 19-bp-repeat CREs add to the robust MIE promoter activity that occurs in the acutely infected stimulated cells, although the CREs' greater role may be in other settings.

DNA replication and cancer: From dysfunctional replication origin activities to therapeutic opportunities.

PubMed

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.

Promoting Literacy with Self-Created Grocery Lists on Mobile Devices

ERIC Educational Resources Information Center

Douglas, Karen H.; Uphold, Nicole M.; Steffen, Shannon; Kroesch, Allison M.

2018-01-01

Four middle school students with a developmental disability participated in a multiple probe design across students and replicated across conditions study to evaluate the effectiveness of constant time delay to teach them to create a grocery list on an iPad or iPod touch. The classroom teacher collected data on the percentage of items…

Tumor Necrosis Factor Receptor-Associated Factor 5 Interacts with the NS3 Protein and Promotes Classical Swine Fever Virus Replication.

PubMed

Lv, Huifang; Dong, Wang; Guo, Kangkang; Jin, Mingxing; Li, Xiaomeng; Li, Cunfa; Zhang, Yanming

2018-06-05

Classical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious and high-mortality viral disease, causing huge economic losses in the swine industry worldwide. CSFV non-structural protein 3 (NS3), a multifunctional protein, plays crucial roles in viral replication. However, how NS3 exactly exerts these functions is currently unknown. Here, we identified tumor necrosis factor receptor-associated factor 5 (TRAF5) as a novel binding partner of the NS3 protein via yeast two-hybrid, co-immunoprecipitation and glutathione S -transferase pull-down assays. Furthermore, we observed that TRAF5 promoted CSFV replication in porcine alveolar macrophages (PAMs). Additionally, CSFV infection or NS3 expression upregulated TRAF5 expression, implying that CSFV may exploit TRAF5 via NS3 for better growth. Moreover, CSFV infection and TRAF5 expression activated p38 mitogen activated protein kinase (MAPK) activity, and inhibition of p38 MAPK activation by the SB203580 inhibitor suppressed CSFV replication. Notably, TRAF5 overexpression did not promote CSFV replication following inhibition of p38 MAPK activation. Our findings reveal that TRAF5 promotes CSFV replication via p38 MAPK activation. This work provides a novel insight into the role of TRAF5 in CSFV replication capacity.

Providing Teachers with Research- and Cognitive Learning Theory-Based Instructional Materials for Promoting Students' Metacognition: A Replication Study of a Community College Mathematics Teacher and Curriculum Reformation Program

ERIC Educational Resources Information Center

Nall, Katherine Ligon

2011-01-01

The purpose of this study was to assess the effect of a 3-stage community college mathematics teacher and curriculum conceptual change program on student achievement. The study, which was a replication and extension of Lake (2008), was conducted during Fall 2009 and Spring 2010 terms and focused on teachers' instructional practices relative to…

The development of the conditionally replication-competent adenovirus: replacement of E4 orf1-4 region by exogenous gene.

PubMed

Nam, Jae-Kook; Lee, Mi-Hyang; Seo, Hae-Hyun; Kim, Seok-Ki; Lee, Kang-Huyn; Kim, In-Hoo; Lee, Sang-Jin

2010-05-01

Tumor or tissue specific replicative adenovirus armed with a therapeutic gene has shown a promising anti-cancer therapeutic modality. However, because the genomic packaging capacity is constrained, only a few places inside it are available for transgene insertion. In the present study, we introduce a novel strategy utilizing the early E4 region for the insertion of therapeutic gene(s). We constructed the conditionally replication-competent adenovirus (CRAd), Ad5E4(mRFP) by: (i) replacing the E4/E1a promoter by the prostate-specific enhancer element; (ii) inserting mRFP inside the E4orf1-4 deletion region; and (iii) sub-cloning enhanced green fluorescent protein controlled by cytomegalovirus promoter in the left end of the viral genome. Subsequently, we evaluated its replication abilities and killing activities in vitro, as well as its in vivo anti-tumor efficacy in CWR22rv xenografts. When infected with Ad5E4(mRFP), the number and intensity of the mRFP gene products increased in a prostate cancer cell-specific manner as designed, suggesting that the mRFP gene and E4orfs other than E4orf1-4 were well synthesized from one transcript via alternative splicing as the recombinant adenovirus replicated. As expected from the confirmed virus replication capability, Ad5E4(mRFP) induced cell lysis as potent as the wild-type adenovirus and effectively suppressed tumor growth when tested in the CWR22rv xenografts in nude mice. Furthermore, Ad5E4(endo/angio) harboring an endostatin-angiostatin gene in E4orf1-4 was able to enhance CRAd by replacing mRFP with a therapeutic gene. The approach employed in the present study for the insertion of a therapeutic transgene in CRAd should facilitate the construction of CRAd containing multiple therapeutic genes in the viral genome that may have the potential to serve as highly potent cancer therapeutic reagents. Copyright (c) 2010 John Wiley & Sons, Ltd.

Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Imura, Yoshiyuki, E-mail: imura@brs.nihon-u.ac.jp; Molho, Melissa; Chuang, Chingkai

Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement bothmore » defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase.« less

iAID: an improved auxin-inducible degron system for the construction of a 'tight' conditional mutant in the budding yeast Saccharomyces cerevisiae.

PubMed

Tanaka, Seiji; Miyazawa-Onami, Mayumi; Iida, Tetsushi; Araki, Hiroyuki

2015-08-01

Isolation of a 'tight' conditional mutant of a gene of interest is an effective way of studying the functions of essential genes. Strategies that use ubiquitin-mediated protein degradation to eliminate the product of a gene of interest, such as heat-inducible degron (td) and auxin-inducible degron (AID), are powerful methods for constructing conditional mutants. However, these methods do not work with some genes. Here, we describe an improved AID system (iAID) for isolating tight conditional mutants in the budding yeast Saccharomyces cerevisiae. In this method, transcriptional repression by the 'Tet-OFF' promoter is combined with proteolytic elimination of the target protein by the AID system. To provide examples, we describe the construction of tight mutants of the replication factors Dpb11 and Mcm10, dpb11-iAID, and mcm10-iAID. Because Dpb11 and Mcm10 are required for the initiation of DNA replication, their tight mutants are unable to enter S phase. This is the case for dpb11-iAID and mcm10-iAID cells after the addition of tetracycline and auxin. Both the 'Tet-OFF' promoter and the AID system have been shown to work in model eukaryotes other than budding yeast. Therefore, the iAID system is not only useful in budding yeast, but also can be applied to other model systems to isolate tight conditional mutants. Copyright © 2015 John Wiley & Sons, Ltd.

Inhibition of TGF-β Signaling Promotes Human Pancreatic β-Cell Replication

PubMed Central

Dhawan, Sangeeta; Dirice, Ercument; Kulkarni, Rohit N.

2016-01-01

Diabetes is associated with loss of functional pancreatic β-cells, and restoration of β-cells is a major goal for regenerative therapies. Endogenous regeneration of β-cells via β-cell replication has the potential to restore cellular mass; however, pharmacological agents that promote regeneration or expansion of endogenous β-cells have been elusive. The regenerative capacity of β-cells declines rapidly with age, due to accumulation of p16INK4a, resulting in limited capacity for adult endocrine pancreas regeneration. Here, we show that transforming growth factor-β (TGF-β) signaling via Smad3 integrates with the trithorax complex to activate and maintain Ink4a expression to prevent β-cell replication. Importantly, inhibition of TGF-β signaling can result in repression of the Ink4a/Arf locus, resulting in increased β-cell replication in adult mice. Furthermore, small molecule inhibitors of the TGF-β pathway promote β-cell replication in human islets transplanted into NOD-scid IL-2Rgnull mice. These data reveal a novel role for TGF-β signaling in the regulation of the Ink4a/Arf locus and highlight the potential of using small molecule inhibitors of TGF-β signaling to promote human β-cell replication. PMID:26936960

Synaptogyrin-2 Promotes Replication of a Novel Tick-borne Bunyavirus through Interacting with Viral Nonstructural Protein NSs*

PubMed Central

Sun, Qiyu; Qi, Xian; Zhang, Yan; Wu, Xiaodong; Liang, Mifang; Li, Chuan; Li, Dexin; Cardona, Carol J.; Xing, Zheng

2016-01-01

Synaptogyrin-2 is a non-neuronal member of the synaptogyrin family involved in synaptic vesicle biogenesis and trafficking. Little is known about the function of synaptogyrin-2. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, and leukocytopenia with high mortality, caused by a novel tick-borne phlebovirus in the family Bunyaviridae. Our previous studies have shown that the viral nonstructural protein NSs forms inclusion bodies (IBs) that are involved in viral immune evasion, as well as viral RNA replication. In this study, we sought to elucidate the mechanism by which NSs formed the IBs, a lipid droplet-based structure confirmed by NSs co-localization with perilipin A and adipose differentiation-related protein (ADRP). Through a high throughput screening, we identified synaptogyrin-2 to be highly up-regulated in response to SFTS bunyavirus (SFTSV) infection and to be a promoter of viral replication. We demonstrated that synaptogyrin-2 interacted with NSs and was translocated into the IBs, which were reconstructed from lipid droplets into large structures in infection. Viral RNA replication decreased, and infectious virus titers were lowered significantly when synaptogyrin-2 was silenced in specific shRNA-expressing cells, which correlated with the reduced number of the large IBs restructured from regular lipid droplets. We hypothesize that synaptogyrin-2 is essential to promoting the formation of the IBs to become virus factories for viral RNA replication through its interaction with NSs. These findings unveil the function of synaptogyrin-2 as an enhancer in viral infection. PMID:27226560

Requirement of multiple cis-acting elements in the human cytomegalovirus major immediate-early distal enhancer for viral gene expression and replication.

PubMed

Meier, Jeffery L; Keller, Michael J; McCoy, James J

2002-01-01

We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer's orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at -300 or -345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication.

Chk1 promotes replication fork progression by controlling replication initiation

PubMed Central

Petermann, Eva; Woodcock, Mick; Helleday, Thomas

2010-01-01

DNA replication starts at initiation sites termed replication origins. Metazoan cells contain many more potential origins than are activated (fired) during each S phase. Origin activation is controlled by the ATR checkpoint kinase and its downstream effector kinase Chk1, which suppresses origin firing in response to replication blocks and during normal S phase by inhibiting the cyclin-dependent kinase Cdk2. In addition to increased origin activation, cells deficient in Chk1 activity display reduced rates of replication fork progression. Here we investigate the causal relationship between increased origin firing and reduced replication fork progression. We use the Cdk inhibitor roscovitine or RNAi depletion of Cdc7 to inhibit origin firing in Chk1-inhibited or RNAi-depleted cells. We report that Cdk inhibition and depletion of Cdc7 can alleviate the slow replication fork speeds in Chk1-deficient cells. Our data suggest that increased replication initiation leads to slow replication fork progression and that Chk1 promotes replication fork progression during normal S phase by controlling replication origin activity. PMID:20805465

Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription.

PubMed

Pai, Chen-Chun; Kishkevich, Anastasiya; Deegan, Rachel S; Keszthelyi, Andrea; Folkes, Lisa; Kearsey, Stephen E; De León, Nagore; Soriano, Ignacio; de Bruin, Robertus Antonius Maria; Carr, Antony M; Humphrey, Timothy C

2017-09-12

Chromatin modification through histone H3 lysine 36 methylation by the SETD2 tumor suppressor plays a key role in maintaining genome stability. Here, we describe a role for Set2-dependent H3K36 methylation in facilitating DNA replication and the transcriptional responses to both replication stress and DNA damage through promoting MluI cell-cycle box (MCB) binding factor (MBF)-complex-dependent transcription in fission yeast. Set2 loss leads to reduced MBF-dependent ribonucleotide reductase (RNR) expression, reduced deoxyribonucleoside triphosphate (dNTP) synthesis, altered replication origin firing, and a checkpoint-dependent S-phase delay. Accordingly, prolonged S phase in the absence of Set2 is suppressed by increasing dNTP synthesis. Furthermore, H3K36 is di- and tri-methylated at these MBF gene promoters, and Set2 loss leads to reduced MBF binding and transcription in response to genotoxic stress. Together, these findings provide new insights into how H3K36 methylation facilitates DNA replication and promotes genotoxic stress responses in fission yeast. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

RNAi-Based Suppressor Screens Reveal Genetic Interactions Between the CRL2LRR-1 E3-Ligase and the DNA Replication Machinery in Caenorhabditis elegans

PubMed Central

Ossareh-Nazari, Batool; Katsiarimpa, Anthi; Merlet, Jorge; Pintard, Lionel

2016-01-01

Cullin-RING E3-Ligases (CRLs), the largest family of E3 ubiquitin-Ligases, regulate diverse cellular processes by promoting ubiquitination of target proteins. The evolutionarily conserved Leucine Rich Repeat protein 1 (LRR-1) is a substrate-recognition subunit of a CRL2LRR-1 E3-ligase. Here we provide genetic evidence supporting a role of this E3-enzyme in the maintenance of DNA replication integrity in Caenorhabditis elegans. Through RNAi-based suppressor screens of lrr-1(0) and cul-2(or209ts) mutants, we identified two genes encoding components of the GINS complex, which is part of the Cdc45-MCM-GINS (CMG) replicative helicase, as well as CDC-7 and MUS-101, which drives the assembly of the CMG helicase during DNA replication. In addition, we identified the core components of the ATR/ATL-1 DNA replication checkpoint pathway (MUS-101, ATL-1, CLSP-1, CHK-1). These results suggest that the CRL2LRR-1 E3-ligase acts to modify or degrade factor(s) that would otherwise misregulate the replisome, eventually leading to the activation of the DNA replication checkpoint. PMID:27543292

WV Walks: replication with expanded reach.

PubMed

Reger-Nash, Bill; Bauman, Adrian; Cooper, Linda; Chey, Tien; Simon, Kenneth J; Brann, Maria; Leyden, Kevin M

2008-01-01

WV Walks replicated the Wheeling Walks community-wide campaign methodology to promote physical activity. A social marketing intervention promoted walking among insufficiently active 40- to 65-year-olds throughout the television media market in north-central West Virginia. The intervention included participatory planning, an 8-week mass media-based campaign, and policy and environmental activities. Pre and post random-digit-dial cohort telephone surveys were conducted at baseline and immediately postcampaign in intervention and comparison regions. The campaign resulted in maximal message awareness in north-central WV and demonstrated a significant increase in walking behavior represented by an absolute shift of 12% of the target population from insufficiently active to active (> or = 30 minutes, 5 days per week), versus the comparison community (adjusted odds ratio 1.82, CI: 1.05-3.17). Policy and environmental changes were also evident. This replication study increases our confidence that the initial effects observed in the Wheeling Walks intervention are generalizable to other similar rural communities.

Emotion-based preventive intervention: Effectively promoting emotion knowledge and adaptive behavior among at-risk preschoolers.

PubMed

Finlon, Kristy J; Izard, Carroll E; Seidenfeld, Adina; Johnson, Stacy R; Cavadel, Elizabeth Woodburn; Ewing, E Stephanie Krauthamer; Morgan, Judith K

2015-11-01

Effectiveness studies of preschool social-emotional programs are needed in low-income, diverse populations to help promote the well-being of at-risk children. Following an initial program efficacy study 2 years prior, 248 culturally diverse Head Start preschool children participated in the current effectiveness trial and received either the Emotion-Based Prevention Program (EBP) or the I Can Problem Solve (ICPS) intervention. Pre- and postdata collection included direct child assessment, teacher report, parent interview, and independent observations. Teachers implementing the EBP intervention demonstrated good and consistent fidelity to the program. Overall, children in EBP classrooms gained more emotion knowledge and displayed greater decreases in negative emotion expressions and internalizing behaviors across the implementation period as compared to children in ICPS classrooms. In addition, cumulative risk, parental depressive symptoms, and classroom climate significantly moderated treatment effects. For children experiencing more stress or less support, EBP produced more successful outcomes than did ICPS. These results provide evidence of EBP sustainability and program effectiveness, as did previous findings that demonstrated EBP improvements in emotion knowledge, regulation skills, and behavior problems replicated under unsupervised program conditions.

Replication of urban innovations - prioritization of strategies for the replication of Dhaka's community-based decentralized composting model.

PubMed

Yedla, Sudhakar

2012-01-01

Dhaka's community-based decentralized composting (DCDC) is a successful demonstration of solid waste management by adopting low-cost technology, local resources community participation and partnerships among the various actors involved. This paper attempts to understand the model, necessary conditions, strategies and their priorities to replicate DCDC in the other developing cities of Asia. Thirteen strategies required for its replication are identified and assessed based on various criteria, namely transferability, longevity, economic viability, adaptation and also overall replication. Priority setting by multi-criteria analysis by applying analytic hierarchy process revealed that immediate transferability without long-term and economic viability consideration is not advisable as this would result in unsustainable replication of DCDC. Based on the analysis, measures to ensure the product quality control; partnership among stakeholders (public-private-community); strategies to achieve better involvement of the private sector in solid waste management (entrepreneurship in approach); simple and low-cost technology; and strategies to provide an effective interface among the complementing sectors are identified as important strategies for its replication.

Requirement of Multiple cis-Acting Elements in the Human Cytomegalovirus Major Immediate-Early Distal Enhancer for Viral Gene Expression and Replication

PubMed Central

Meier, Jeffery L.; Keller, Michael J.; McCoy, James J.

2002-01-01

We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer’s orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at −300 or −345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication. PMID:11739696

Tissue-specific, tumor-selective, replication-competent adenovirus vector for cancer gene therapy.

PubMed

Doronin, K; Kuppuswamy, M; Toth, K; Tollefson, A E; Krajcsi, P; Krougliak, V; Wold, W S

2001-04-01

We have previously described two replication-competent adenovirus vectors, named KD1 and KD3, for potential use in cancer gene therapy. KD1 and KD3 have two small deletions in the E1A gene that restrict efficient replication of these vectors to human cancer cell lines. These vectors also have increased capacity to lyse cells and spread from cell to cell because they overexpress the adenovirus death protein, an adenovirus protein required for efficient cell lysis and release of adenovirus from the cell. We now describe a new vector, named KD1-SPB, which is the KD1 vector with the E4 promoter replaced by the promoter for surfactant protein B (SPB). SPB promoter activity is restricted in the adult to type II alveolar epithelial cells and bronchial epithelial cells. Because KD1-SPB has the E1A mutations, it should replicate within and destroy only alveolar and bronchial cancer cells. We show that KD1-SPB replicates, lyses cells, and spreads from cell to cell as well as does KD1 in H441 cells, a human cancer cell line where the SPB promoter is active. KD1-SPB replicates, lyses cells, and spreads only poorly in Hep3B liver cancer cells. Replication was determined by expression of the E4ORF3 protein, viral DNA accumulation, fiber synthesis, and virus yield. Cell lysis and vector spread were measured by lactate dehydrogenase release and a "vector spread" assay. In addition to Hep3B cells, KD1-SPB also did not express E4ORF3 in HT29.14S (colon), HeLa (cervix), KB (nasopharynx), or LNCaP (prostate) cancer cell lines, in which the SPB promoter is not expected to be active. Following injection into H441 or Hep3B tumors growing in nude mice, KD1-SPB caused a three- to fourfold suppression of growth of H441 tumors, similar to that seen with KD1. KD1-SPB had only a minimal effect on the growth of Hep3B tumors, whereas KD1 again caused a three- to fourfold suppression. These results establish that the adenovirus E4 promoter can be replaced by a tissue-specific promoter in a replication-competent vector. The vector has three engineered safety features: the tissue-specific promoter, the mutations in E1A that preclude efficient replication in nondividing cells, and a deletion of the E3 genes which shield the virus from attack by the immune system. KD1-SPB may have use in treating human lung cancers in which the SPB promoter is active.

Cyclooxygenase-2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents.

PubMed

Lin, Chun-Kuang; Tseng, Chin-Kai; Wu, Yu-Hsuan; Liaw, Chih-Chuang; Lin, Chun-Yu; Huang, Chung-Hao; Chen, Yen-Hsu; Lee, Jin-Ching

2017-03-20

Cyclooxygenase-2 (COX-2) is one of the important mediators of inflammation in response to viral infection, and it contributes to viral replication, for example, cytomegalovirus or hepatitis C virus replication. The role of COX-2 in dengue virus (DENV) replication remains unclear. In the present study, we observed an increased level of COX-2 in patients with dengue fever compared with healthy donors. Consistent with the clinical data, an elevated level of COX-2 expression was also observed in DENV-infected ICR suckling mice. Using cell-based experiments, we revealed that DENV-2 infection significantly induced COX-2 expression and prostaglandin E 2 (PGE 2 ) production in human hepatoma Huh-7 cells. The exogenous expression of COX-2 or PGE 2 treatment dose-dependently enhanced DENV-2 replication. In contrast, COX-2 gene silencing and catalytic inhibition sufficiently suppressed DENV-2 replication. In an ICR suckling mouse model, we identified that the COX-2 inhibitor NS398 protected mice from succumbing to life-threatening DENV-2 infection. By using COX-2 promoter-based analysis and specific inhibitors against signaling molecules, we identified that NF-κB and MAPK/JNK are critical factors for DENV-2-induced COX-2 expression and viral replication. Altogether, our results reveal that COX-2 is an important factor for DENV replication and can serve as a potential target for developing therapeutic agents against DENV infection.

The microRNA-99 family modulates hepatitis B virus replication by promoting IGF-1R/PI3K/Akt/mTOR/ULK1 signaling-induced autophagy.

PubMed

Lin, Yong; Deng, Wanyu; Pang, Jinke; Kemper, Thekla; Hu, Jing; Yin, Jian; Zhang, Jiming; Lu, Mengji

2017-05-01

MicroRNAs are small highly conserved noncoding RNAs that are widely expressed in multicellular organisms and participate in the regulation of various cellular processes including autophagy and viral replication. Evidently, microRNAs are able to modulate host gene expression and thereby inhibit or enhance hepatitis B virus (HBV) replication. The miR-99 family members are highly expressed in the liver. Interestingly, the plasma levels of miR-99 family in the peripheral blood correspond with HBV DNA loads. Thus, we asked whether the miR-99 family regulated HBV replication and analyzed the underlying molecular mechanism. Compared with primary hepatocytes, miR-99 family expression was downregulated in hepatoma cells. Transfection of miR-99a, miR-99b, and miR-100 markedly increased HBV replication, progeny secretion, and antigen expression in hepatoma cells. However, miR-99 family had no effect on HBV transcription and HBV promoter activities, suggesting that they regulate HBV replication at posttranscriptional steps. Consistent with bioinformatic analysis and recent reports, ectopic expression of miR-99 family attenuated IGF-1R/Akt/mTOR pathway signaling and repressed insulin-stimulated activation in hepatoma cells. Moreover, the experimental data demonstrated that the miR-99 family promoted autophagy through mTOR/ULK1 signaling and thereby enhanced HBV replication. In conclusion, the miR-99 family promotes HBV replication posttranscriptionally through IGF-1R/PI3K/Akt/mTOR/ULK1 signaling-induced autophagy. © 2016 John Wiley & Sons Ltd.

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

PubMed Central

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

Synaptogyrin-2 Promotes Replication of a Novel Tick-borne Bunyavirus through Interacting with Viral Nonstructural Protein NSs.

PubMed

Sun, Qiyu; Qi, Xian; Zhang, Yan; Wu, Xiaodong; Liang, Mifang; Li, Chuan; Li, Dexin; Cardona, Carol J; Xing, Zheng

2016-07-29

Synaptogyrin-2 is a non-neuronal member of the synaptogyrin family involved in synaptic vesicle biogenesis and trafficking. Little is known about the function of synaptogyrin-2. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, and leukocytopenia with high mortality, caused by a novel tick-borne phlebovirus in the family Bunyaviridae. Our previous studies have shown that the viral nonstructural protein NSs forms inclusion bodies (IBs) that are involved in viral immune evasion, as well as viral RNA replication. In this study, we sought to elucidate the mechanism by which NSs formed the IBs, a lipid droplet-based structure confirmed by NSs co-localization with perilipin A and adipose differentiation-related protein (ADRP). Through a high throughput screening, we identified synaptogyrin-2 to be highly up-regulated in response to SFTS bunyavirus (SFTSV) infection and to be a promoter of viral replication. We demonstrated that synaptogyrin-2 interacted with NSs and was translocated into the IBs, which were reconstructed from lipid droplets into large structures in infection. Viral RNA replication decreased, and infectious virus titers were lowered significantly when synaptogyrin-2 was silenced in specific shRNA-expressing cells, which correlated with the reduced number of the large IBs restructured from regular lipid droplets. We hypothesize that synaptogyrin-2 is essential to promoting the formation of the IBs to become virus factories for viral RNA replication through its interaction with NSs. These findings unveil the function of synaptogyrin-2 as an enhancer in viral infection. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jha, Jyoti K.; Li, Mi; Ghirlando, Rodolfo

Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations inrctBthat reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in amore » dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding. IMPORTANCE The capacity of proteins to undergo remodeling provides opportunities to control their function. However, remodeling remains a poorly understood aspect of the structure-function paradigm due to its dynamic nature. Here we have studied remodeling of the initiator of replication ofVibrio choleraeChr2 by the molecular chaperone, DnaK. We show that DnaK binds to a site on the Chr2 initiator (RctB) that promotes initiation by reducing the initiator’s propensity to dimerize. Dimerization of the initiator of the putative plasmid progenitor of Chr2 is also reduced by DnaK, which promotes initiation. Paradoxically, the DnaK binding also promotes replication inhibition by reducing an autoinhibitory activity of RctB. In the plasmid-to-chromosome transition, it appears that the initiator has acquired an autoinhibitory activity and along with it a new chaperone activity that apparently helps to control replication inhibition independently of replication promotion.« less

Intrinsically bent DNA in replication origins and gene promoters.

PubMed

Gimenes, F; Takeda, K I; Fiorini, A; Gouveia, F S; Fernandez, M A

2008-06-24

Intrinsically bent DNA is an alternative conformation of the DNA molecule caused by the presence of dA/dT tracts, 2 to 6 bp long, in a helical turn phase DNA or with multiple intervals of 10 to 11 bp. Other than flexibility, intrinsic bending sites induce DNA curvature in particular chromosome regions such as replication origins and promoters. Intrinsically bent DNA sites are important in initiating DNA replication, and are sometimes found near to regions associated with the nuclear matrix. Many methods have been developed to localize bent sites, for example, circular permutation, computational analysis, and atomic force microscopy. This review discusses intrinsically bent DNA sites associated with replication origins and gene promoter regions in prokaryote and eukaryote cells. We also describe methods for identifying bent DNA sites for circular permutation and computational analysis.

Lysine residues K66, K109, and K110 in the bovine foamy virus transactivator protein are required for transactivation and viral replication.

PubMed

Zhang, Suzhen; Cui, Xiaoxu; Li, Jing; Liang, Zhibin; Qiao, Wentao; Tan, Juan

2016-04-01

Bovine foamy virus (BFV) is a complex retrovirus that infects cattle. Like all retroviruses, BFV encodes a transactivator Tas protein (BTas) that increases gene transcription from viral promoters. BFV encodes two promoters that can interact with BTas, a conserved promoter in the 5' long terminal repeat (LTR) and a unique internal promoter (IP). Our previous study showed that BTas is acetylated by p300 at residues K66, K109, and K110, which markedly enhanced the ability of BTas to bind to DNA. However, whether these residues are important for BFV replication was not determined. Therefore, in this study we provide direct evidence that BTas is required for BFV replication and demonstrate that residues K66, K109, and K110 are critical for BTas function and BFV replication. Full-length infectious clones were generated, which were BTas deficient or contained lysine to arginine (K→R) mutations at position 66, 109, and/or 110. In vivo data indicated that K→R mutations at positions 66, 109, and 110 in BTas impaired transactivation of both the LTR and IP promoters. In addition, the K→R mutations in full-length infectious clones reduced expression of viral proteins, and the triple mutant and BTas deletion completely abrogated viral replication. Taken together, these results indicate that lysine residues at positions 66, 109, and 110 in the BTas protein are crucial for BFV replication and suggest a potential role for BTas acetylation in regulating the viral life cycle.

Replication intermediates of the linear mitochondrial DNA of Candida parapsilosis suggest a common recombination based mechanism for yeast mitochondria.

PubMed

Gerhold, Joachim M; Sedman, Tiina; Visacka, Katarina; Slezakova, Judita; Tomaska, Lubomir; Nosek, Jozef; Sedman, Juhan

2014-08-15

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria*

PubMed Central

Gerhold, Joachim M.; Sedman, Tiina; Visacka, Katarina; Slezakova, Judita; Tomaska, Lubomir; Nosek, Jozef; Sedman, Juhan

2014-01-01

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations. PMID:24951592

Nonstructural 3 Protein of Hepatitis C Virus Modulates the Tribbles Homolog 3/Akt Signaling Pathway for Persistent Viral Infection

PubMed Central

Tran, Si C.; Pham, Tu M.; Nguyen, Lam N.; Park, Eun-Mee; Lim, Yun-Sook

2016-01-01

ABSTRACT Hepatitis C virus (HCV) infection often causes chronic hepatitis, liver cirrhosis, and ultimately hepatocellular carcinoma. However, the mechanisms underlying HCV-induced liver pathogenesis are still not fully understood. By transcriptome sequencing (RNA-Seq) analysis, we recently identified host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these, tribbles homolog 3 (TRIB3) was selected for further characterization. TRIB3 was initially identified as a binding partner of protein kinase B (also known as Akt). TRIB3 blocks the phosphorylation of Akt and induces apoptosis under endoplasmic reticulum (ER) stress conditions. HCV has been shown to enhance Akt phosphorylation for its own propagation. In the present study, we demonstrated that both mRNA and protein levels of TRIB3 were increased in the context of HCV replication. We further showed that promoter activity of TRIB3 was increased by HCV-induced ER stress. Silencing of TRIB3 resulted in increased RNA and protein levels of HCV, whereas overexpression of TRIB3 decreased HCV replication. By employing an HCV pseudoparticle entry assay, we further showed that TRIB3 was a negative host factor involved in HCV entry. Both in vitro binding and immunoprecipitation assays demonstrated that HCV NS3 specifically interacted with TRIB3. Consequently, the association of TRIB3 and Akt was disrupted by HCV NS3, and thus, TRIB3-Akt signaling was impaired in HCV-infected cells. Moreover, HCV modulated TRIB3 to promote extracellular signal-regulated kinase (ERK) phosphorylation, activator protein 1 (AP-1) activity, and cell migration. Collectively, these data indicate that HCV exploits the TRIB3-Akt signaling pathway to promote persistent viral infection and may contribute to HCV-mediated pathogenesis. IMPORTANCE TRIB3 is a pseudokinase protein that acts as an adaptor in signaling pathways for important cellular processes. So far, the functional involvement of TRIB3 in virus-infected cells has not yet been demonstrated. We showed that both mRNA and protein expression levels of TRIB3 were increased in the context of HCV RNA replication. Gene silencing of TRIB3 increased HCV RNA and protein levels, and thus, overexpression of TRIB3 decreased HCV replication. TRIB3 is known to promote apoptosis by negatively regulating the Akt signaling pathway under ER stress conditions. Most importantly, we demonstrated that the TRIB3-Akt signaling pathway was disrupted by NS3 in HCV-infected cells. These data provide evidence that HCV modulates the TRIB3-Akt signaling pathway to establish persistent viral infection. PMID:27252525

RNAi-Based Suppressor Screens Reveal Genetic Interactions Between the CRL2LRR-1 E3-Ligase and the DNA Replication Machinery in Caenorhabditis elegans.

PubMed

Ossareh-Nazari, Batool; Katsiarimpa, Anthi; Merlet, Jorge; Pintard, Lionel

2016-10-13

Cullin-RING E3-Ligases (CRLs), the largest family of E3 ubiquitin-Ligases, regulate diverse cellular processes by promoting ubiquitination of target proteins. The evolutionarily conserved Leucine Rich Repeat protein 1 (LRR-1) is a substrate-recognition subunit of a CRL2 LRR-1 E3-ligase. Here we provide genetic evidence supporting a role of this E3-enzyme in the maintenance of DNA replication integrity in Caenorhabditis elegans Through RNAi-based suppressor screens of lrr-1(0) and cul-2(or209ts) mutants, we identified two genes encoding components of the GINS complex, which is part of the Cdc45-MCM-GINS (CMG) replicative helicase, as well as CDC-7 and MUS-101, which drives the assembly of the CMG helicase during DNA replication. In addition, we identified the core components of the ATR/ATL-1 DNA replication checkpoint pathway (MUS-101, ATL-1, CLSP-1, CHK-1). These results suggest that the CRL2 LRR-1 E3-ligase acts to modify or degrade factor(s) that would otherwise misregulate the replisome, eventually leading to the activation of the DNA replication checkpoint. Copyright © 2016 Ossareh-Nazari et al.

Cyclooxygenase‐2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents

PubMed Central

Lin, Chun-Kuang; Tseng, Chin-Kai; Wu, Yu-Hsuan; Liaw, Chih-Chuang; Lin, Chun-Yu; Huang, Chung-Hao; Chen, Yen-Hsu; Lee, Jin-Ching

2017-01-01

Cyclooxygenase-2 (COX-2) is one of the important mediators of inflammation in response to viral infection, and it contributes to viral replication, for example, cytomegalovirus or hepatitis C virus replication. The role of COX-2 in dengue virus (DENV) replication remains unclear. In the present study, we observed an increased level of COX-2 in patients with dengue fever compared with healthy donors. Consistent with the clinical data, an elevated level of COX-2 expression was also observed in DENV-infected ICR suckling mice. Using cell-based experiments, we revealed that DENV-2 infection significantly induced COX-2 expression and prostaglandin E2 (PGE2) production in human hepatoma Huh-7 cells. The exogenous expression of COX-2 or PGE2 treatment dose-dependently enhanced DENV-2 replication. In contrast, COX-2 gene silencing and catalytic inhibition sufficiently suppressed DENV-2 replication. In an ICR suckling mouse model, we identified that the COX-2 inhibitor NS398 protected mice from succumbing to life-threatening DENV-2 infection. By using COX-2 promoter-based analysis and specific inhibitors against signaling molecules, we identified that NF-κB and MAPK/JNK are critical factors for DENV-2-induced COX-2 expression and viral replication. Altogether, our results reveal that COX-2 is an important factor for DENV replication and can serve as a potential target for developing therapeutic agents against DENV infection. PMID:28317866

Replication-Competent Controlled Herpes Simplex Virus

PubMed Central

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

2015-01-01

ABSTRACT We present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. IMPORTANCE The alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate, stringent regulation of multiple replication-essential genes. By directing activating heat to the region of virus administration, replication is strictly confined to infected cells within this region. The requirement for antiprogestin provides an additional level of safety, ensuring that virus replication cannot be triggered inadvertently. Replication-competent controlled vectors such as HSV-GS3 may have the potential to be superior to conventional attenuated HSV vaccine and oncolytic vectors without sacrificing safety. PMID:26269179

Biodistribution and Safety Assessment of Bladder Cancer Specific Recombinant Oncolytic Adenovirus in Subcutaneous Xenografts Tumor Model in Nude Mice

PubMed Central

Wang, Fang; Wang, Zhiping; Tian, Hongwei; Qi, Meijiao; Zhai, Zhenxing; Li, Shuwen; Li, Renju; Zhang, Hongjuan; Wang, Wenyun; Fu, Shenjun; Lu, Jianzhong; Rodriguez, Ronald; Guo, Yinglu; Zhou, Liqun

2012-01-01

Background The previous works about safety evaluation for constructed bladder tissue specific adenovirus are poorly documented. Thus, we investigated the biodistribution and body toxicity of bladder specific oncolytic adenovirus Ad-PSCAE-UPII-E1A (APU-E1A) and Ad-PSCAE-UPII-E1A-AR (APU-E1A-AR), providing meaningful information prior to embarking on human clinical trials. Materials and Method Conditionally replicate recombinant adenovirus (CRADs) APU-E1A, APU-EIA-AR were constructed with bladder tissue specific Uroplakin II (UP II) promoter to induce the expression of Ad5E1A gene and E1A-AR fusing gene, and PSCAE was inserted at upstream of promoter to enhance the function of promoter. Based on the cytopathic and anti-tumor effect of bladder cancer, these CRADs were intratumorally injected into subcutaneous xenografts tumor in nude mice. We then determined the toxicity through general health and behavioral assessment, hepatic and hematological toxicity evaluation, macroscopic and microscopic postmortem analyses. The spread of the transgene E1A of adenovirus was detected with RT-PCR and Western blot. Virus replication and distribution were examined with APU-LUC administration and Luciferase Assay. Results General assessment and body weight of the animals did not reveal any alteration in general behavior. The hematological alterations of groups which were injected with 5×108 pfu or higher dose (5×109 pfu) of APU-E1A and APU-E1A-AR showed no difference in comparison with PBS group, and only slight increased transaminases in contrast to PBS group at 5×109 pfu of APU-E1A and APU-E1A-AR were observed. E1A transgene did not disseminate to organs outside of xenograft tumor. Virus replication was not detected in other organs beside tumor according to Luciferase Assay. Conclusions Our study showed that recombinant adenovirus APU-E1A-AR and APU-E1A appear safe with 5×107 pfu and 5×108 pfu intratumorally injection in mice, without any discernable effects on general health and behavior. PMID:22384806

Biodistribution and safety assessment of bladder cancer specific recombinant oncolytic adenovirus in subcutaneous xenografts tumor model in nude mice.

PubMed

Wang, Fang; Wang, Zhiping; Tian, Hongwei; Qi, Meijiao; Zhai, Zhenxing; Li, Shuwen; Li, Renju; Zhang, Hongjuan; Wang, Wenyun; Fu, Shenjun; Lu, Jianzhong; Rodriguez, Ronald; Guo, Yinglu; Zhou, Liqun

2012-04-01

The previous works about safety evaluation for constructed bladder tissue specific adenovirus are poorly documented. Thus, we investigated the biodistribution and body toxicity of bladder specific oncolytic adenovirus Ad-PSCAE-UPII-E1A (APU-E1A) and Ad-PSCAE-UPII-E1A-AR (APU-E1A-AR), providing meaningful information prior to embarking on human clinical trials. Conditionally replicate recombinant adenovirus (CRADs) APU-E1A, APU-EIA-AR were constructed with bladder tissue specific UroplakinII(UPII) promoter to induce the expression of Ad5E1A gene and E1A-AR fusing gene, and PSCAE was inserted at upstream of promoter to enhance the function of promoter. Based on the cytopathic and anti-tumor effect of bladder cancer, these CRADs were intratumorally injected into subcutaneous xenografts tumor in nude mice. We then determined the toxicity through general health and behavioral assessment, hepatic and hematological toxicity evaluation, macroscopic and microscopic postmortem analyses. The spread of the transgene E1A of adenovirus was detected with RT-PCR and Western blot. Virus replication and distribution were examined with APU-LUC administration and Luciferase Assay. General assessment and body weight of the animals did not reveal any alteration in general behavior. The hematological alterations of groups which were injected with 5x10(8) pfu or higher dose (5x10(9) pfu) of APU-E1A and APU-E1A-AR showed no difference in comparison with PBS group, and only slight increased transaminases in contrast to PBS group at 5x10(9) pfu of APU-E1A and APU-E1A-AR were observed. E1A transgene did not disseminate to organs outside of xenograft tumor. Virus replication was not detected in other organs beside tumor according to Luciferase Assay. Our study showed that recombinant adenovirus APU-E1A-AR and APU-E1A appear safe with 5x10(7) pfu and 5x10(8) pfu intratumorally injection in mice, without any discernable effects on general health and behavior.

Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature

DOE Office of Scientific and Technical Information (OSTI.GOV)

Groom, Joseph; Chung, Daehwan; Olson, Daniel G.

2016-01-29

Clostridium thermocellum is a leading candidate for the consolidated bioprocessing of lignocellulosic biomass for the production of fuels and chemicals. A limitation to the engineering of this strain is the availability of stable replicating plasmid vectors for homologous and heterologous expression of genes that provide improved and/or novel pathways for fuel production. Current vectors relay on replicons from mesophilic bacteria and are not stable at the optimum growth temperature of C. thermocellum. To develop more thermostable genetic tools for C. thermocellum, we constructed vectors based on the hyperthermophilic Caldicellulosiruptor bescii replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproduciblymore » transformed C. thermocellum at 60 °C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from C. bescii were functional in C. thermocellum. Phylogenetic analyses of the proteins contained on pBAS2 revealed that the replication initiation protein RepL is unique among thermophiles. Lastly, these results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.« less

Analysis of JC virus DNA replication using a quantitative and high-throughput assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, Jong; Phelan, Paul J.; Chhum, Panharith

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 JCVmore » 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.« less

The MluI cell cycle box (MCB) motifs, but not damage-responsive elements (DREs), are responsible for the transcriptional induction of the rhp51+ gene in response to DNA replication stress.

PubMed

Sartagul, Wugangerile; Zhou, Xin; Yamada, Yuki; Ma, Ning; Tanaka, Katsunori; Furuyashiki, Tomoyuki; Ma, Yan

2014-01-01

DNA replication stress induces the transcriptional activation of rhp51+, a fission yeast recA homolog required for repair of DNA double strand breaks. However, the mechanism by which DNA replication stress activates rhp51+ transcription is not understood. The promoter region of rhp51+ contains two damage-responsive elements (DREs) and two MluI cell cycle box (MCB) motifs. Using luciferase reporter assays, we examined the role of these elements in rhp51+ transcription. The full-length rhp51+ promoter and a promoter fragment containing MCB motifs only, but not a fragment containing DREs, mediated transcriptional activation upon DNA replication stress. Removal of the MCB motifs from the rhp51+ promoter abolished the induction of rhp51+ transcription by DNA replication stress. Consistent with a role for MCB motifs in rhp51+ transcription activation, deletion of the MBF (MCB-binding factor) co-repressors Nrm1 and Yox1 precluded rhp51+ transcriptional induction in response to DNA replication stress. Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51+ transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways. Because MBF is critical for G1/S transcription, we examined how the cell cycle affected rhp51+ transcription. The transcription of rhp51+ and cdc18+, an MBF-dependent G1/S gene, peaked simultaneously in synchronized cdc25-22 cells. Furthermore, DNA replication stress maintained transcription of rhp51+ similarly to cdc18+. Collectively, these results suggest that MBF and its regulators mediate rhp51+ transcription in response to DNA replication stress, and underlie rhp51+ transcription at the G1/S transition.

Origin of life. Primordial genetics: Information transfer in a pre-RNA world based on self-replicating beta-sheet amyloid conformers.

PubMed

Maury, Carl Peter J

2015-10-07

The question of the origin of life on Earth can largely be reduced to the question of what was the first molecular replicator system that was able to replicate and evolve under the presumably very harsh conditions on the early Earth. It is unlikely that a functional RNA could have existed under such conditions and it is generally assumed that some other kind of information system preceded the RNA world. Here, I present an informational molecular system that is stable, self-replicative, environmentally responsive, and evolvable under conditions characterized by high temperatures, ultraviolet and cosmic radiation. This postulated pregenetic system is based on the amyloid fold, a functionally unique polypeptide fold characterized by a cross beta-sheet structure in which the beta strands are arranged perpendicular to the fiber axis. Beside an extraordinary structural robustness, the amyloid fold possesses a unique ability to transmit information by a three-dimensional templating mechanism. In amyloidogenesis short peptide monomers are added one by one to the growing end of the fiber. From the same monomeric subunits several structural variants of amyloid may be formed. Then, in a self-replicative mode, a specific amyloid conformer can act as a template and confer its spatially encoded information to daughter molecular entities in a repetitive way. In this process, the specific conformational information, the spatially changed organization, is transmitted; the coding element is the steric zipper structure, and recognition occurs by amino acid side chain complementarity. The amyloid information system fulfills several basic requirements of a primordial evolvable replicator system: (i) it is stable under the presumed primitive Earth conditions, (ii) the monomeric building blocks of the informational polymer can be formed from available prebiotic compounds, (iii) the system is self-assembling and self-replicative and (iv) it is adaptive to changes in the environment and evolvable. Copyright © 2015 The Author. Published by Elsevier Ltd.. All rights reserved.

INITIATION AND REGULATION OF PARAMYXOVIRUS TRANSCRIPTION AND REPLICATION

PubMed Central

Noton, Sarah L.; Fearns, Rachel

2015-01-01

The paramyxovirus family has a genome consisting of a single strand of negative sense RNA. This genome acts as a template for two distinct processes: transcription to generate subgenomic, capped and polyadenylated mRNAs, and genome replication. These viruses only encode one polymerase. Thus, an intriguing question is, how does the viral polymerase initiate and become committed to either transcription or replication? By answering this we can begin to understand how these two processes are regulated. In this review article, we present recent findings from studies on the paramyxovirus, respiratory syncytial virus, which show how its polymerase is able to initiate transcription and replication from a single promoter. We discuss how these findings apply to other paramyxoviruses. Then, we examine how trans-acting proteins and promoter secondary structure might serve to regulate transcription and replication during different phases of the paramyxovirus replication cycle. PMID:25683441

Initiation and regulation of paramyxovirus transcription and replication.

PubMed

Noton, Sarah L; Fearns, Rachel

2015-05-01

The paramyxovirus family has a genome consisting of a single strand of negative sense RNA. This genome acts as a template for two distinct processes: transcription to generate subgenomic, capped and polyadenylated mRNAs, and genome replication. These viruses only encode one polymerase. Thus, an intriguing question is, how does the viral polymerase initiate and become committed to either transcription or replication? By answering this we can begin to understand how these two processes are regulated. In this review article, we present recent findings from studies on the paramyxovirus, respiratory syncytial virus, which show how its polymerase is able to initiate transcription and replication from a single promoter. We discuss how these findings apply to other paramyxoviruses. Then, we examine how trans-acting proteins and promoter secondary structure might serve to regulate transcription and replication during different phases of the paramyxovirus replication cycle. Copyright © 2015 Elsevier Inc. All rights reserved.

Towards Self-Replicating Chemical Systems Based on Cytidylic and Guanylic Acids

NASA Technical Reports Server (NTRS)

Kanavarioti, Anastassia

1999-01-01

This project was aimed towards a better understanding of template-directed reactions and, based on this, towards the development of efficient non-enzymatic RNA replicating systems. These systems could serve as models for the prebiotic synthesis of an RNA world. The major objectives of this project are: (a) To elucidate the mechanistic aspects of template-directed (TD) chemistry and (b) to identify active boundary regions, or conditions, environmental and other, that favor "organized chemistry" and stereo-selective polymerization of nucleotides. "Organized chemistry" may lead to enhanced polymerization efficiency which in turn is expected to facilitate the road towards a self-replicating chemical system based on all four nucleic acid bases.

Smarcal1-Mediated Fork Reversal Triggers Mre11-Dependent Degradation of Nascent DNA in the Absence of Brca2 and Stable Rad51 Nucleofilaments.

PubMed

Kolinjivadi, Arun Mouli; Sannino, Vincenzo; De Antoni, Anna; Zadorozhny, Karina; Kilkenny, Mairi; Técher, Hervé; Baldi, Giorgio; Shen, Rong; Ciccia, Alberto; Pellegrini, Luca; Krejci, Lumir; Costanzo, Vincenzo

2017-09-07

Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51 T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Both Chromosome Decondensation and Condensation Are Dependent on DNA Replication in C. elegans Embryos

PubMed Central

Sonneville, Remi; Craig, Gillian; Labib, Karim; Gartner, Anton; Blow, J. Julian

2015-01-01

Summary During cell division, chromatin alternates between a condensed state to facilitate chromosome segregation and a decondensed form when DNA replicates. In most tissues, S phase and mitosis are separated by defined G1 and G2 gap phases, but early embryogenesis involves rapid oscillations between replication and mitosis. Using Caenorhabditis elegans embryos as a model system, we show that chromosome condensation and condensin II concentration on chromosomal axes require replicated DNA. In addition, we found that, during late telophase, replication initiates on condensed chromosomes and promotes the rapid decondensation of the chromatin. Upon replication initiation, the CDC-45-MCM-GINS (CMG) DNA helicase drives the release of condensin I complexes from chromatin and the activation or displacement of inactive MCM-2–7 complexes, which together with the nucleoporin MEL-28/ELYS tethers condensed chromatin to the nuclear envelope, thereby promoting chromatin decondensation. Our results show how, in an early embryo, the chromosome-condensation cycle is functionally linked with DNA replication. PMID:26166571

Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli.

PubMed

Thomason, Lynn C; Costantino, Nina; Court, Donald L

2016-09-13

Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion. Bacteriophage homologous recombination systems are widely used for in vivo genetic engineering in bacteria. Single- or double-stranded linear DNA substrates containing short flanking homologies to chromosome targets are used to generate precise and accurate genetic modifications when introduced into bacteria expressing phage recombinases. Understanding the molecular mechanism of these recombination systems will facilitate improvements in the technology. Here, two phage-specific systems are shown to require exposure of complementary single-strand homologous targets for efficient recombination; these single-strand regions may be created during DNA replication or by single-strand exonuclease digestion of linear duplex DNA. Previously, in vitro studies reported that these recombinases promote the single-strand annealing of two complementary DNAs and also strand invasion of a single DNA strand into duplex DNA to create a three-stranded region. Here, in vivo experiments show that recombinase-mediated annealing of complementary single-stranded DNA is the predominant recombination pathway in E. coli. Copyright © 2016 Thomason et al.

Reflections on "Replicating Milgram" (Burger, 2009)

ERIC Educational Resources Information Center

Miller, Arthur G.

2009-01-01

In "Replicating Milgram: Would People Still Obey Today?" Jerry M. Burger (see record 2008-19206-001) reported a high base rate of obedience, comparable to that observed by Stanley Milgram (1974). Another condition, involving a defiant confederate, failed to significantly reduce obedience. This commentary discusses the primary contributions of…

SV40 Utilizes ATM Kinase Activity to Prevent Non-homologous End Joining of Broken Viral DNA Replication Products

PubMed Central

Sowd, Gregory A.; Mody, Dviti; Eggold, Joshua; Cortez, David; Friedman, Katherine L.; Fanning, Ellen

2014-01-01

Simian virus 40 (SV40) and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PKcs kinase activity, facilitates some aspects of double strand break (DSB) repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR) and do not colocalize with non-homologous end joining (NHEJ) factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PKcs and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5′ to 3′ end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication. PMID:25474690

Photoactivation of Akt1/GSK3β Isoform-Specific Signaling Axis Promotes Pancreatic β-Cell Regeneration.

PubMed

Huang, Lei; Jiang, Xiaoxiao; Gong, Longlong; Xing, Da

2015-08-01

Promotion of insulin-secreting β-cell regeneration in patients with diabetes is a promising approach for diabetes therapy, which can contribute to rescue the uncontrolled hyperglycemia. Low-power laser irradiation (LPLI) has been demonstrated to regulate multiple physiological processes both in vitro and in vivo through activation of various signaling pathways. In the present study, we showed that LPLI promoted β-cell replication and cell cycle progression through activation of Akt1/GSK3β isoform-specific signaling axis. Inhibition of PI3-K/Akt or GSK3 with specific inhibitors dramatically reduced or increased LPLI-induced β-cell replication, revealing Akt/GSK3 signaling axis was involved in β-cell replication and survival upon LPLI treatment. Furthermore, the results of shRNA-mediated knock down of Akt/GSK3 isoforms revealed that Akt1/GSK3β isoform-specific signaling axis regulated β-cell replication and survival in response to LPLI, but not Akt2/GSK3α. The mechanism by which LPLI promoted β-cell replication through Akt1/GSK3β signaling axis involved activation of β-catenin and down-regulation of p21. Taken together, these observations suggest that Akt1/GSK3β isoform signaling axis play a key role in β-cell replication and survival induced by LPLI. Moreover, our findings suggest that activation of Akt1/GSK3β isoform signaling axis by LPLI may provide guidance in practical applications for β-cell regenerative therapies. © 2015 Wiley Periodicals, Inc.

Cellular and molecular consequences of defective Fanconi anemia proteins in replication-coupled DNA repair: mechanistic insights

PubMed Central

Thompson, Larry H.; Hinz, John M.

2009-01-01

The Fanconi anemia (FA) molecular network consists of 15 “FANC” proteins, of which 13 are associated with mutations in patients with this cancer-prone chromosome instability disorder. Whereas historically the common phenotype associated with FA mutations is marked sensitivity to DNA interstrand crosslinking agents, the literature supports a more global role for FANC proteins in coping with diverse stresses encountered by replicative polymerases. We have attempted to reconcile and integrate numerous observations into a model in which FANC proteins coordinate the following physiological events during DNA crosslink repair: (a) activating a FANCM-ATR-dependent S-phase checkpoint; (b) mediating enzymatic replication-fork breakage and crosslink unhooking; (c) filling the resulting gap by translesion synthesis (TLS) by error-prone polymerase(s); and (d) restoring the resulting one-ended double-strand break by homologous recombination repair (HRR). The FANC core subcomplex (FANCA, B, C, E, F, G, L, FAAP100) promotes TLS for both crosslink and non-crosslink damage such as spontaneous oxidative base damage, UV-C photoproducts, and alkylated bases. TLS likely helps prevent stalled replication forks from breaking, thereby maintaining chromosome continuity. Diverse DNA damages and replication inhibitors result in monoubiquitination of the FANCD2-FANCI complex by the FANCL ubiquitin ligase activity of the core subcomplex upon its recruitment to chromatin by the FANCM-FAAP24 heterodimeric translocase. We speculate that this translocase activity acts as the primary damage sensor and helps remodel blocked replication forks to facilitate checkpoint activation and repair. Monoubiquitination of FANCD2-FANCI is needed for promoting HRR, in which the FANCD1/BRCA2 and FANCN/PALB2 proteins act at an early step. We conclude that the core subcomplex is required for both TLS and HRR occurring separately for non-crosslink damages and for both events during crosslink repair. The FANCJ/BRIP1/BACH1 helicase functions in association with BRCA1 and may remove structural barriers to replication, such as guanine quadruplex structures, and/or assist in crosslink unhooking. PMID:19622404

High-fidelity in vivo replication of DNA base shape mimics without Watson–Crick hydrogen bonds

PubMed Central

Delaney, James C.; Henderson, Paul T.; Helquist, Sandra A.; Morales, Juan C.; Essigmann, John M.; Kool, Eric T.

2003-01-01

We report studies testing the importance of Watson–Crick hydrogen bonding, base-pair geometry, and steric effects during DNA replication in living bacterial cells. Nonpolar DNA base shape mimics of thymine and adenine (abbreviated F and Q, respectively) were introduced into Escherichia coli by insertion into a phage genome followed by transfection of the vector into bacteria. Genetic assays showed that these two base mimics were bypassed with moderate to high efficiency in the cells and with very high efficiency under damage-response (SOS induction) conditions. Under both sets of conditions, the T-shape mimic (F) encoded genetic information in the bacteria as if it were thymine, directing incorporation of adenine opposite it with high fidelity. Similarly, the A mimic (Q) directed incorporation of thymine opposite itself with high fidelity. The data establish that Watson–Crick hydrogen bonding is not necessary for high-fidelity replication of a base pair in vivo. The results suggest that recognition of DNA base shape alone serves as the most powerful determinant of fidelity during transfer of genetic information in a living organism. PMID:12676985

BRCA2 and RAD51 promote double-strand break formation and cell death in response to gemcitabine.

PubMed

Jones, Rebecca M; Kotsantis, Panagiotis; Stewart, Grant S; Groth, Petra; Petermann, Eva

2014-10-01

Replication inhibitors cause replication fork stalling and double-strand breaks (DSB) that result from processing of stalled forks. During recovery from replication blocks, the homologous recombination (HR) factor RAD51 mediates fork restart and DSB repair. HR defects therefore sensitize cells to replication inhibitors, with clear implications for cancer therapy. Gemcitabine is a potent replication inhibitor used to treat cancers with mutations in HR genes such as BRCA2. Here, we investigate why, paradoxically, mutations in HR genes protect cells from killing by gemcitabine. Using DNA replication and DNA damage assays in mammalian cells, we show that even short gemcitabine treatments cause persistent replication inhibition. BRCA2 and RAD51 are recruited to chromatin early after removal of the drug, actively inhibit replication fork progression, and promote the formation of MUS81- and XPF-dependent DSBs that remain unrepaired. Our data suggest that HR intermediates formed at gemcitabine-stalled forks are converted into DSBs and thus contribute to gemcitabine-induced cell death, which could have implications for the treatment response of HR-deficient tumors. ©2014 American Association for Cancer Research.

BRCA2 and RAD51 promote double-strand break formation and cell death in response to Gemcitabine

PubMed Central

Jones, Rebecca M.; Kotsantis, Panagiotis; Stewart, Grant S.; Groth, Petra; Petermann, Eva

2014-01-01

Replication inhibitors cause replication fork stalling and double-strand breaks (DSBs) that result from processing of stalled forks. During recovery from replication blocks, the homologous recombination (HR) factor RAD51 mediates fork restart and DSB repair. HR defects therefore sensitise cells to replication inhibitors, with clear implications for cancer therapy. Gemcitabine is a potent replication inhibitor used to treat cancers with mutations in HR genes such as BRCA2. Here we investigate why, paradoxically, mutations in HR genes protect cells from killing by Gemcitabine. Using DNA replication and -damage assays in mammalian cells, we show that even short Gemcitabine treatments cause persistent replication inhibition. BRCA2 and RAD51 are recruited to chromatin early after removal of the drug, actively inhibit replication fork progression and promote the formation of MUS81- and XPF-dependent DSBs that remain unrepaired. Our data suggest that HR intermediates formed at Gemcitabine-stalled forks are converted into DSBs and thus contribute to Gemcitabine-induced cell death, which could have implications for the treatment response of HR-deficient tumours. PMID:25053826

Human CST Facilitates Genome-wide RAD51 Recruitment to GC-Rich Repetitive Sequences in Response to Replication Stress.

PubMed

Chastain, Megan; Zhou, Qing; Shiva, Olga; Fadri-Moskwik, Maria; Whitmore, Leanne; Jia, Pingping; Dai, Xueyu; Huang, Chenhui; Ye, Ping; Chai, Weihang

2016-08-02

The telomeric CTC1/STN1/TEN1 (CST) complex has been implicated in promoting replication recovery under replication stress at genomic regions, yet its precise role is unclear. Here, we report that STN1 is enriched at GC-rich repetitive sequences genome-wide in response to hydroxyurea (HU)-induced replication stress. STN1 deficiency exacerbates the fragility of these sequences under replication stress, resulting in chromosome fragmentation. We find that upon fork stalling, CST proteins form distinct nuclear foci that colocalize with RAD51. Furthermore, replication stress induces physical association of CST with RAD51 in an ATR-dependent manner. Strikingly, CST deficiency diminishes HU-induced RAD51 foci formation and reduces RAD51 recruitment to telomeres and non-telomeric GC-rich fragile sequences. Collectively, our findings establish that CST promotes RAD51 recruitment to GC-rich repetitive sequences in response to replication stress to facilitate replication restart, thereby providing insights into the mechanism underlying genome stability maintenance. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Reovirus Nonstructural Protein σNS Acts as an RNA-Stability Factor Promoting Viral Genome Replication.

PubMed

Zamora, Paula F; Hu, Liya; Knowlton, Jonathan J; Lahr, Roni M; Moreno, Rodolfo A; Berman, Andrea J; Prasad, B V Venkataram; Dermody, Terence S

2018-05-16

Viral nonstructural proteins, which are not packaged into virions, are essential for replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. Reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered that σNS increases RNA half-life using in vitro and cell-based RNA degradation experiments. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication. IMPORTANCE Following infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the Reoviridae family encode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different Reoviridae family viruses are diverged in primary sequence, these proteins are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Using in vitro and cell-culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the Reoviridae family assort and replicate their genomes. Copyright © 2018 American Society for Microbiology.

Critical Role of HAX-1 in Promoting Avian Influenza Virus Replication in Lung Epithelial Cells

PubMed Central

He, Ganlin; Cardona, Carol J.

2018-01-01

The PB1-F2 protein of influenza A virus has been considered a virulence factor, but its function in inducing apoptosis may be of disadvantage to viral replication. Host mechanisms to regulate PB1-F2-induced apoptosis remain unknown. We generated a PB1-F2-deficient avian influenza virus (AIV) H9N2 and found that the mutant virus replicated less efficiently in human lung epithelial cells. The PB1-F2-deficient virus produced less apoptotic cells, indicating that PB1-F2 of the H9N2 virus promotes apoptosis, occurring at the early stage of infection, in the lung epithelial cells. To understand how host cells regulate PB1-F2-induced apoptosis, we explored to identify cellular proteins interacting with PB1-F2 and found that HCLS1-associated protein X-1 (HAX-1), located mainly in the mitochondria as an apoptotic inhibitor, interacted with PB1-F2. Increased procaspase-9 activations, induced by PB1-F2, could be suppressed by HAX-1. In HAX-1 knockdown A549 cells, the replication of AIV H9N2 was suppressed in parallel to the activation of caspase-3 activation, which increased at the early stage of infection. We hypothesize that HAX-1 promotes AIV replication by interacting with PB1-F2, resulting in the suppression of apoptosis, prolonged cell survival, and enhancement of viral replication. Our data suggest that HAX-1 may be a promoting factor for AIV H9N2 replication through desensitizing PB1-F2 from its apoptotic induction in human lung epithelial cells. PMID:29576744

Replication stress affects the fidelity of nucleosome-mediated epigenetic inheritance

PubMed Central

Li, Wenzhu; Yi, Jia; Agbu, Pamela; Zhou, Zheng; Kelley, Richard L.; Jia, Songtao

2017-01-01

The fidelity of epigenetic inheritance or, the precision by which epigenetic information is passed along, is an essential parameter for measuring the effectiveness of the process. How the precision of the process is achieved or modulated, however, remains largely elusive. We have performed quantitative measurement of epigenetic fidelity, using position effect variegation (PEV) in Schizosaccharomyces pombe as readout, to explore whether replication perturbation affects nucleosome-mediated epigenetic inheritance. We show that replication stresses, due to either hydroxyurea treatment or various forms of genetic lesions of the replication machinery, reduce the inheritance accuracy of CENP-A/Cnp1 nucleosome positioning within centromere. Mechanistically, we demonstrate that excessive formation of single-stranded DNA, a common molecular abnormality under these conditions, might have correlation with the reduction in fidelity of centromeric chromatin duplication. Furthermore, we show that replication stress broadly changes chromatin structure at various loci in the genome, such as telomere heterochromatin expanding and mating type locus heterochromatin spreading out of the boundaries. Interestingly, the levels of inheritable expanding at sub-telomeric heterochromatin regions are highly variable among independent cell populations. Finally, we show that HU treatment of the multi-cellular organisms C. elegans and D. melanogaster affects epigenetically programmed development and PEV, illustrating the evolutionary conservation of the phenomenon. Replication stress, in addition to its demonstrated role in genetic instability, promotes variable epigenetic instability throughout the epigenome. PMID:28749973

DNA replication stress restricts ribosomal DNA copy number.

PubMed

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.

Synchronization of DNA array replication kinetics

NASA Astrophysics Data System (ADS)

Manturov, Alexey O.; Grigoryev, Anton V.

2016-04-01

In the present work we discuss the features of the DNA replication kinetics at the case of multiplicity of simultaneously elongated DNA fragments. The interaction between replicated DNA fragments is carried out by free protons that appears at the every nucleotide attachment at the free end of elongated DNA fragment. So there is feedback between free protons concentration and DNA-polymerase activity that appears as elongation rate dependence. We develop the numerical model based on a cellular automaton, which can simulate the elongation stage (growth of DNA strands) for DNA elongation process with conditions pointed above and we study the possibility of the DNA polymerases movement synchronization. The results obtained numerically can be useful for DNA polymerase movement detection and visualization of the elongation process in the case of massive DNA replication, eg, under PCR condition or for DNA "sequencing by synthesis" sequencing devices evaluation.

Genetic Determinants for Promoter Hypermethylation in the Lungs of Smokers: A Candidate Gene-Based Study

PubMed Central

Leng, Shuguang; Stidley, Christine A.; Liu, Yushi; Edlund, Christopher K.; Willink, Randall P.; Han, Younghun; Landi, Maria Teresa; Thun, Michael; Picchi, Maria A.; Bruse, Shannon E.; Crowell, Richard E.; Van Den Berg, David; Caporaso, Neil E.; Amos, Christopher I.; Siegfried, Jill M.; Tesfaigzi, Yohannes; Gilliland, Frank D.; Belinsky, Steven A.

2011-01-01

The detection of tumor suppressor gene promoter methylation in sputum-derived exfoliated cells predicts early lung cancer. Here we identified genetic determinants for this epigenetic process and examined their biological effects on gene regulation. A two-stage approach involving discovery and replication was employed to assess the association between promoter hypermethylation of a 12-gene panel and common variation in 40 genes involved in carcinogen metabolism, regulation of methylation, and DNA damage response in members of the Lovelace Smokers Cohort (n=1434). Molecular validation of three identified variants was conducted using primary bronchial epithelial cells. Association of study-wide significance (P<8.2×10−5) was identified for rs1641511, rs3730859, and rs1883264 in TP53, LIG1, and BIK, respectively. These SNPs were significantly associated with altered expression of the corresponding genes in primary bronchial epithelial cells. In addition, rs3730859 in LIG1 was also moderately associated with increased risk for lung cancer among Caucasian smokers. Together, our findings suggest that genetic variation in DNA replication and apoptosis pathways impacts the propensity for gene promoter hypermethylation in the aerodigestive tract of smokers. The incorporation of genetic biomarkers for gene promoter hypermethylation with clinical and somatic markers may improve risk assessment models for lung cancer. PMID:22139380

A switch between DNA polymerases δ and λ promotes error-free bypass of 8-oxo-G lesions

PubMed Central

Markkanen, Enni; Castrec, Benoît; Villani, Giuseppe; Hübscher, Ulrich

2012-01-01

7,8-Dihydro-8-oxoguanine (8-oxo-G) is a highly abundant and mutagenic lesion. Replicative DNA polymerases (pols) are slowed down at 8-oxo-G and insert both correct cytosine (C) and incorrect adenine (A) opposite 8-oxo-G, but they preferentially extend A:8-oxo-G mispairs. Nevertheless, 8-oxo-G bypass is fairly accurate in vivo. Thus, the question how correct bypass of 8-oxo-G lesions is accomplished despite the poor extension of C:8-oxo-G base pairs by replicative pols remains unanswered. Here we show that replicative pol δ pauses in front of 8-oxo-G and displays difficulties extending from correct C:8-oxo-G in contrast to extension from incorrect A:8-oxo-G. This leads to stalling of pol δ at 8-oxo-G after incorporation of correct C. This stalling at C:8-oxo-G can be overcome by a switch from pol δ to pols λ, β, or η, all of which are able to assist pol δ in 8-oxo-G bypass by translesion synthesis (TLS). Importantly, however, only pol λ selectively catalyzes the correct TLS past 8-oxo-G, whereas pols β and η show no selectivity and even preferentially enhance incorrect TLS. The selectivity of pol λ to promote the correct bypass depends on its N-terminal domain. Furthermore, pol λ−/− mouse embryonic fibroblast extracts display reduced 8-oxo-G TLS. Finally, the correct bypass of 8-oxo-G in gapped plasmids in mouse embryonic fibroblasts and HeLa cells is promoted in the presence of pol λ. Our findings suggest that even though 8-oxo-G is not a blocking lesion per se, correct replication over 8-oxo-G is promoted by a pol switch between pols δ and λ. PMID:23175785

A dual role of BRCA1 in two distinct homologous recombination mediated repair in response to replication arrest

PubMed Central

Feng, Zhihui; Zhang, Junran

2012-01-01

Homologous recombination (HR) is a major mechanism utilized to repair blockage of DNA replication forks. Here, we report that a sister chromatid exchange (SCE) generated by crossover-associated HR efficiently occurs in response to replication fork stalling before any measurable DNA double-strand breaks (DSBs). Interestingly, SCE produced by replication fork collapse following DNA DSBs creation is specifically suppressed by ATR, a central regulator of the replication checkpoint. BRCA1 depletion leads to decreased RPA2 phosphorylation (RPA2-P) following replication fork stalling but has no obvious effect on RPA2-P following replication fork collapse. Importantly, we found that BRCA1 promotes RAD51 recruitment and SCE induced by replication fork stalling independent of ATR. In contrast, BRCA1 depletion leads to a more profound defect in RAD51 recruitment and SCE induced by replication fork collapse when ATR is depleted. We concluded that BRCA1 plays a dual role in two distinct HR-mediated repair upon replication fork stalling and collapse. Our data established a molecular basis for the observation that defective BRCA1 leads to a high sensitivity to agents that cause replication blocks without being associated with DSBs, and also implicate a novel mechanism by which loss of cell cycle checkpoints promotes BRCA1-associated tumorigenesis via enhancing HR defect resulting from BRCA1 deficiency. PMID:21954437

Novel indole sulfides as potent HIV-1 NNRTIs.

PubMed

Brigg, Siobhan; Pribut, Nicole; Basson, Adriaan E; Avgenikos, Moscos; Venter, Reinhardt; Blackie, Margaret A; van Otterlo, Willem A L; Pelly, Stephen C

2016-03-15

In a previous communication we described a series of indole based NNRTIs which were potent inhibitors of HIV replication, both for the wild type and K103N strains of the virus. However, the methyl ether functionality on these compounds, which was crucial for potency, was susceptible to acid promoted indole assisted SN1 substitution. This particular problem did not bode well for an orally bioavailable drug. Here we describe bioisosteric replacement of this problematic functional group, leading to a series of compounds which are potent inhibitors of HIV replication, and are acid stable. Copyright © 2016 Elsevier Ltd. All rights reserved.

Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts.

PubMed

Strausfeld, U P; Howell, M; Descombes, P; Chevalier, S; Rempel, R E; Adamczewski, J; Maller, J L; Hunt, T; Blow, J J

1996-06-01

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define 'S-phase promoting factor' (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.

Randomized Control Trial of COMPASS for Improving Transition Outcomes of Students with Autism Spectrum Disorder.

PubMed

Ruble, Lisa A; McGrew, John H; Toland, Michael; Dalrymple, Nancy; Adams, Medina; Snell-Rood, Claire

2018-06-01

The postsecondary outcomes of individuals with autism spectrum disorder (ASD) are significantly worse than peers with other disabilities. One problem is the lack of empirically-supported transition planning interventions to guide services and help produce better outcomes. We applied an implementation science approach to adapt and modify an evidence-based consultation intervention originally tested with young children called the Collaborative Model for Promoting Competence and Success (COMPASS; Ruble et al., The collaborative model for promoting competence and success for students with ASD. Springer, New York, 2012a) and evaluate it for efficacy in a randomized controlled trial for transition-age youth. Results replicated findings with younger students with ASD that IEP outcomes were higher for COMPASS compared to the placebo control group (d = 2.1). Consultant fidelity was high and teacher adherence improved over time, replicating the importance of ongoing teacher coaching.

Architecture and inherent robustness of a bacterial cell-cycle control system.

PubMed

Shen, Xiling; Collier, Justine; Dill, David; Shapiro, Lucy; Horowitz, Mark; McAdams, Harley H

2008-08-12

A closed-loop control system drives progression of the coupled stalked and swarmer cell cycles of the bacterium Caulobacter crescentus in a near-mechanical step-like fashion. The cell-cycle control has a cyclical genetic circuit composed of four regulatory proteins with tight coupling to processive chromosome replication and cell division subsystems. We report a hybrid simulation of the coupled cell-cycle control system, including asymmetric cell division and responses to external starvation signals, that replicates mRNA and protein concentration patterns and is consistent with observed mutant phenotypes. An asynchronous sequential digital circuit model equivalent to the validated simulation model was created. Formal model-checking analysis of the digital circuit showed that the cell-cycle control is robust to intrinsic stochastic variations in reaction rates and nutrient supply, and that it reliably stops and restarts to accommodate nutrient starvation. Model checking also showed that mechanisms involving methylation-state changes in regulatory promoter regions during DNA replication increase the robustness of the cell-cycle control. The hybrid cell-cycle simulation implementation is inherently extensible and provides a promising approach for development of whole-cell behavioral models that can replicate the observed functionality of the cell and its responses to changing environmental conditions.

Helper-Free Foamy Virus Vectors

PubMed Central

TROBRIDGE, GRANT D.; RUSSELL, DAVID W.

2010-01-01

Retroviral vectors based on human foamy virus (HFV) have been developed and show promise as gene therapy vehicles. Here we describe a method for the production of HFV vector stocks free of detectable helper virus. The helper and vector plasmid constructs used both lack the HFV bel genes, so recombination between these constructs cannot create a wild-type virus. A fusion promoter that combines portions of the cytomegalovirus (CMV) immediate-early and HFV long terminal repeat (LTR) promoters was used to drive expression of both the helper and vector constructs. The CMV–LTR fusion promoter allows for HFV vector production in the absence of the Bel-1 trans-activator protein, which would otherwise be necessary for efficient transcription from the HFV LTR. Vector stocks containing either neomycin phosphotransferase or alkaline phosphatase reporter genes were produced by transient transfection at titers greater than 105 transducing units/ml. G418-resistant BHK-21 cells obtained by transduction with neo vectors contained randomly integrated HFV vector proviruses without detectable deletions or rearrangements. The vector stocks generated were free of replication-competent retrovirus (RCR), as determined by assays for LTR trans-activation and a marker rescue assay developed here for the detection of Bel-independent RCR. OVERVIEW SUMMARY Vectors based on human foamy virus have been developed but low titers and the presence of replication-competent retrovirus (RCR) in vector stocks have prevented their use in preclinical animal experiments. We have developed a transient transfection method that can be used to produce replication-incompetent HFV vector stocks at titers greater than 105/ml, and that does not produce contaminating RCR. The use of CMV-HFV LTR fusion promoters in the helper and vector constructs has circumvented the requirement for the HFV Bel-1 trans-activator protein. Consequently, the potential for generating wild-type HFV by recombination between helper and vector constructs during vector production has been eliminated. Here we describe HFV vector production using this Bel-independent system. PMID:9853518

DNA replication checkpoint promotes G1-S transcription by inactivating the MBF repressor Nrm1

PubMed Central

de Bruin, R. A. M.; Kalashnikova, T. I.; Aslanian, A.; Wohlschlegel, J.; Chahwan, C.; Yates, J. R.; Russell, P.; Wittenberg, C.

2008-01-01

The cell cycle transcriptional program imposes order on events of the cell-cycle and is a target for signals that regulate cell-cycle progression, including checkpoints required to maintain genome integrity. Neither the mechanism nor functional significance of checkpoint regulation of the cell-cycle transcription program are established. We show that Nrm1, an MBF-specific transcriptional repressor acting at the transition from G1 to S phase of the cell cycle, is at the nexus between the cell cycle transcriptional program and the DNA replication checkpoint in fission yeast. Phosphorylation of Nrm1 by the Cds1 (Chk2) checkpoint protein kinase, which is activated in response to DNA replication stress, promotes its dissociation from the MBF transcription factor. This leads to the expression of genes encoding components that function in DNA replication and repair pathways important for cell survival in response to arrested DNA replication. PMID:18682565

Polyploid Titan Cells Produce Haploid and Aneuploid Progeny To Promote Stress Adaptation

PubMed Central

Gerstein, Aleeza C.; Fu, Man Shun; Mukaremera, Liliane; Li, Zhongming; Ormerod, Kate L.; Fraser, James A.; Berman, Judith

2015-01-01

ABSTRACT Cryptococcus neoformans is a major life-threatening fungal pathogen. In response to the stress of the host environment, C. neoformans produces large polyploid titan cells. Titan cell production enhances the virulence of C. neoformans, yet whether the polyploid aspect of titan cells is specifically influential remains unknown. We show that titan cells were more likely to survive and produce offspring under multiple stress conditions than typical cells and that even their normally sized daughters maintained an advantage over typical cells in continued exposure to stress. Although polyploid titan cells generated haploid daughter cell progeny upon in vitro replication under nutrient-replete conditions, titan cells treated with the antifungal drug fluconazole produced fluconazole-resistant diploid and aneuploid daughter cells. Interestingly, a single titan mother cell was capable of generating multiple types of aneuploid daughter cells. The increased survival and genomic diversity of titan cell progeny promote rapid adaptation to new or high-stress conditions. PMID:26463162

Construction of a Food Grade Recombinant Bacillus subtilis Based on Replicative Plasmids with an Auxotrophic Marker for Biotransformation of d-Fructose to d-Allulose.

PubMed

He, Weiwei; Mu, Wanmeng; Jiang, Bo; Yan, Xin; Zhang, Tao

2016-04-27

A food grade recombinant Bacillus subtilis that produces d-psicose 3-epimerase (DPEase; EC 5.1.3.30) was constructed by transforming a replicative multicopy plasmid with a d-alanine racemase gene marker into B. subtilis 1A751 with the d-alanine racemase gene knocked out. The DPEase was expressed in B. subtilis without antibiotic resistance genes and without adding antibiotics during fermentation. Whole cells of the food grade recombinant B. subtilis were used to biotransform d-fructose to d-allulose. The two tandem promoters, including the HpaII and P43 promoters, increased expression levels compared to the use of one promoter, HpaII. For large-scale d-allulose production, the optimal enzyme dose was 40 enzyme activity units of dry cells per gram of d-fructose, which produced a 28.5% turnover yield in 60 min. The recombinant plasmid exhibited stability over 100 generations. This food grade recombinant B. subtilis may be used for large-scale d-allulose production in the food industry.

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

PubMed Central

Metz, Philippe; Chiramel, Abhilash; Chatel-Chaix, Laurent; Alvisi, Gualtiero; Bankhead, Peter; Mora-Rodríguez, Rodrigo; Long, Gang; Hamacher-Brady, Anne

2015-01-01

ABSTRACT Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV), several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. In addition, endolysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable overexpression of p62 significantly suppressed DENV replication, suggesting a novel role for p62 as a viral restriction factor. Overall, our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an antiviral role, which is countered by DENV. IMPORTANCE Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we used high-content, imaging-based flow cytometry to quantify autophagic flux and endolysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endolysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a proviral to an antiviral cellular process, which is counteracted by the virus. PMID:26018155

The metabolic sensors FXRα, PGC-1α, and SIRT1 cooperatively regulate hepatitis B virus transcription.

PubMed

Curtil, Claire; Enache, Liviu S; Radreau, Pauline; Dron, Anne-Gaëlle; Scholtès, Caroline; Deloire, Alexandre; Roche, Didier; Lotteau, Vincent; André, Patrice; Ramière, Christophe

2014-03-01

Hepatitis B virus (HBV) genome transcription is highly dependent on liver-enriched, metabolic nuclear receptors (NRs). Among others, NR farnesoid X receptor α (FXRα) enhances HBV core promoter activity and pregenomic RNA synthesis. Interestingly, two food-withdrawal-induced FXRα modulators, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and deacetylase SIRT1, have been found to be associated with HBV genomes ex vivo. Whereas PGC-1α induction was shown to increase HBV replication, the effect of SIRT1 on HBV transcription remains unknown. Here, we showed that, in hepatocarcinoma-derived Huh-7 cells, combined activation of FXRα by GW4064 and SIRT1 by activator 3 increased HBV core promoter-controlled luciferase expression by 25-fold, compared with a 10-fold increase with GW4064 alone. Using cell lines differentially expressing FXRα in overexpression and silencing experiments, we demonstrated that SIRT1 activated the core promoter in an FXRα- and PGC-1α-dependent manner. Maximal activation (>150-fold) was observed in FXRα- and PGC-1α-overexpressing Huh-7 cells treated with FXRα and SIRT1 activators. Similarly, in cells transfected with full-length HBV genomes, maximal induction (3.5-fold) of core promoter-controlled synthesis of 3.5-kb RNA was observed in the same conditions of transfection and treatments. Thus, we identified a subnetwork of metabolic factors regulating HBV replication, strengthening the hypothesis that transcription of HBV and metabolic genes is similarly controlled.

Important role of N108 residue in binding of bovine foamy virus transactivator Tas to viral promoters.

PubMed

Bing, Tiejun; Zhang, Suzhen; Liu, Xiaojuan; Liang, Zhibin; Shao, Peng; Zhang, Song; Qiao, Wentao; Tan, Juan

2016-06-30

Bovine foamy virus (BFV) encodes the transactivator BTas, which enhances viral gene transcription by binding to the long terminal repeat promoter and the internal promoter. In this study, we investigated the different replication capacities of two similar BFV full-length DNA clones, pBS-BFV-Y and pBS-BFV-B. Here, functional analysis of several chimeric clones revealed a major role for the C-terminal region of the viral genome in causing this difference. Furthermore, BTas-B, which is located in this C-terminal region, exhibited a 20-fold higher transactivation activity than BTas-Y. Sequence alignment showed that these two sequences differ only at amino acid 108, with BTas-B containing N108 and BTas-Y containing D108 at this position. Results of mutagenesis studies demonstrated that residue N108 is important for BTas binding to viral promoters. In addition, the N108D mutation in pBS-BFV-B reduced the viral replication capacity by about 1.5-fold. Our results suggest that residue N108 is important for BTas binding to BFV promoters and has a major role in BFV replication. These findings not only advances our understanding of the transactivation mechanism of BTas, but they also highlight the importance of certain sequence polymorphisms in modulating the replication capacity of isolated BFV clones.

Analysis of the HIV-1 LTR NF-kappaB-proximal Sp site III: evidence for cell type-specific gene regulation and viral replication.

PubMed

McAllister, J J; Phillips, D; Millhouse, S; Conner, J; Hogan, T; Ross, H L; Wigdahl, B

2000-09-01

It has been widely demonstrated that the human immunodeficiency virus type 1 (HIV-1) envelope, specifically the V3 loop of the gp120 spike, evolves to facilitate adaptation to different cellular populations within an infected host. Less energy has been directed at determining whether the viral promoter, designated the long terminal repeat (LTR), also exhibits this adaptive quality. Because of the unique nature of the cell populations infected during the course of HIV-1 infection, one might expect the opportunity for such adaptation to exist. This would permit select viral species to take advantage of the different array of conditions and factors influencing transcription within a given cell type. To investigate this hypothesis, the function of natural variants of the NF-kappaB-proximal Sp element (Sp site III) was examined in human cell line models of the two major cell types infected during the natural course of HIV-1 infection, T cells and monocytes. Utilizing the HIV-1 LAI molecular clone, which naturally contains a high-affinity Sp site III, substitution of low-affinity Sp sites in place of the natural site III element markedly decreased viral replication in Jurkat T cells. However, these substitutions had relatively small effects on viral replication in U-937 monocytic cells. Transient transfections of HIV-1 LAI-based LTR-luciferase constructs into these cell lines suggest that the large reduction in viral replication in Jurkat T cells, caused by low-affinity Sp site III variants, may result from reduced basal as well as Vpr- and Tat-activated LTR activities in Jurkat T cells compared to those in U-937 monocytic cells. When the function of Sp site III was examined in the context of HIV-1 YU-2-based LTR-luciferase constructs, substitution of a high-affinity element in place of the natural low-affinity element resulted in increased basal YU-2 LTR activity in Jurkat T cells and reduced activity in U-937 monocytic cells. These observations suggest that recruitment of Sp family members to Sp site III is of greater importance to the function of the viral promoter in the Jurkat T cell line as compared to the U-937 monocytic cell line. These observations also suggest that other regions of the LTR may compensate for Sp recruitment defects in specific cell populations. Copyright 2000 Academic Press.

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

PubMed Central

Bastia, Deepak; Srivastava, Pankaj; Zaman, Shamsu; Choudhury, Malay; Mohanty, Bidyut K.; Bacal, Julien; Langston, Lance D.; Pasero, Philippe; O’Donnell, Michael E.

2016-01-01

Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1–Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2–7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2–7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2–7 had to be phosphorylated for binding to phospho-Tof1–Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2–7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1–Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1–Ter complex. PMID:27298353

Act Healthy: promoting health behaviors and self-efficacy in the workplace.

PubMed

Schopp, Laura H; Bike, Denise H; Clark, Mary J; Minor, Marian A

2015-08-01

Chronic health conditions and multiple health risk factors afflict Americans and burden employers, but effective, affordable, workplace-based health promotion interventions have not been widely implemented. This is the first study to adapt the empirically validated Chronic Disease Self-Management Program for a general employee population in a workplace setting with an emphasis on disease prevention and health promotion. A quasi-experimental, wellness standard of care comparison, prospective cohort design was used among employee participants at a large University employer. Ninety-one individuals participated in the program. Participants reported significantly increased health behavior frequency and self-efficacy after the intervention, compared with their pre-intervention scores, and improvements were sustained at 3-month follow-up [self-rated abilities for health practices scale (SRA): F = 30.89, P < 0.001; health promoting lifestyle profile-II (HPLP-II): F = 36.30 P < 0.001]. Individuals in the intervention group reported improved self-efficacy and health behaviors compared with the wellness standard of care comparison group at post intervention (SRA: F = 12.45, P < 0.001; HPLP-II: F = 25.28, P < 0.001). Adapting lay-facilitated self-management for the workplace offers promise as a replicable, scalable, affordable model for culture change in organizations. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication

PubMed Central

Thai, Minh; Graham, Nicholas A; Braas, Daniel; Nehil, Michael; Komisopoulou, Evangelia; Kurdistani, Siavash K.; McCormick, Frank; Graeber, Thomas G.; Christofk, Heather R.

2014-01-01

SUMMARY Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. While recent studies have characterized virus-induced changes in host cell metabolism (Munger et al., 2008; Terry et al., 2012), the molecular mechanisms by which viruses reprogram cellular metabolism have remained elusive. Here we show that the gene product of adenovirus E4ORF1 is necessary for adenovirus-induced upregulation of host cell glucose metabolism and sufficient to promote enhanced glycolysis in cultured epithelial cells by activation of MYC. E4ORF1 localizes to the nucleus, binds to MYC, and enhances MYC binding to glycolytic target genes, resulting in elevated expression of specific glycolytic enzymes. E4ORF1 activation of MYC promotes increased nucleotide biosynthesis from glucose intermediates and enables optimal adenovirus replication in primary lung epithelial cells. Our findings show how a viral protein exploits host cell machinery to reprogram cellular metabolism and promote optimal progeny virion generation. PMID:24703700

Coupled Flip-Flop Model for REM Sleep Regulation in the Rat

PubMed Central

Dunmyre, Justin R.; Mashour, George A.; Booth, Victoria

2014-01-01

Recent experimental studies investigating the neuronal regulation of rapid eye movement (REM) sleep have identified mutually inhibitory synaptic projections among REM sleep-promoting (REM-on) and REM sleep-inhibiting (REM-off) neuronal populations that act to maintain the REM sleep state and control its onset and offset. The control mechanism of mutually inhibitory synaptic interactions mirrors the proposed flip-flop switch for sleep-wake regulation consisting of mutually inhibitory synaptic projections between wake- and sleep-promoting neuronal populations. While a number of synaptic projections have been identified between these REM-on/REM-off populations and wake/sleep-promoting populations, the specific interactions that govern behavioral state transitions have not been completely determined. Using a minimal mathematical model, we investigated behavioral state transition dynamics dictated by a system of coupled flip-flops, one to control transitions between wake and sleep states, and another to control transitions into and out of REM sleep. The model describes the neurotransmitter-mediated inhibitory interactions between a wake- and sleep-promoting population, and between a REM-on and REM-off population. We proposed interactions between the wake/sleep and REM-on/REM-off flip-flops to replicate the behavioral state statistics and probabilities of behavioral state transitions measured from experimental recordings of rat sleep under ad libitum conditions and after 24 h of REM sleep deprivation. Reliable transitions from REM sleep to wake, as dictated by the data, indicated the necessity of an excitatory projection from the REM-on population to the wake-promoting population. To replicate the increase in REM-wake-REM transitions observed after 24 h REM sleep deprivation required that this excitatory projection promote transient activation of the wake-promoting population. Obtaining the reliable wake-nonREM sleep transitions observed in the data required that activity of the wake-promoting population modulated the interaction between the REM-on and REM-off populations. This analysis suggests neuronal processes to be targeted in further experimental studies of the regulatory mechanisms of REM sleep. PMID:24722577

Coupled flip-flop model for REM sleep regulation in the rat.

PubMed

Dunmyre, Justin R; Mashour, George A; Booth, Victoria

2014-01-01

Recent experimental studies investigating the neuronal regulation of rapid eye movement (REM) sleep have identified mutually inhibitory synaptic projections among REM sleep-promoting (REM-on) and REM sleep-inhibiting (REM-off) neuronal populations that act to maintain the REM sleep state and control its onset and offset. The control mechanism of mutually inhibitory synaptic interactions mirrors the proposed flip-flop switch for sleep-wake regulation consisting of mutually inhibitory synaptic projections between wake- and sleep-promoting neuronal populations. While a number of synaptic projections have been identified between these REM-on/REM-off populations and wake/sleep-promoting populations, the specific interactions that govern behavioral state transitions have not been completely determined. Using a minimal mathematical model, we investigated behavioral state transition dynamics dictated by a system of coupled flip-flops, one to control transitions between wake and sleep states, and another to control transitions into and out of REM sleep. The model describes the neurotransmitter-mediated inhibitory interactions between a wake- and sleep-promoting population, and between a REM-on and REM-off population. We proposed interactions between the wake/sleep and REM-on/REM-off flip-flops to replicate the behavioral state statistics and probabilities of behavioral state transitions measured from experimental recordings of rat sleep under ad libitum conditions and after 24 h of REM sleep deprivation. Reliable transitions from REM sleep to wake, as dictated by the data, indicated the necessity of an excitatory projection from the REM-on population to the wake-promoting population. To replicate the increase in REM-wake-REM transitions observed after 24 h REM sleep deprivation required that this excitatory projection promote transient activation of the wake-promoting population. Obtaining the reliable wake-nonREM sleep transitions observed in the data required that activity of the wake-promoting population modulated the interaction between the REM-on and REM-off populations. This analysis suggests neuronal processes to be targeted in further experimental studies of the regulatory mechanisms of REM sleep.

Kis antitoxin couples plasmid R1 replication and parD (kis,kid) maintenance modules.

PubMed

López-Villarejo, Juan; Diago-Navarro, Elizabeth; Hernández-Arriaga, Ana María; Díaz-Orejas, Ramón

2012-03-01

The coupling between the replication and parD (kis, kid) maintenance modules of R1 has been revisited here by the isolation of a significant collection of conditional replication mutants in the pKN1562 mini-R1 plasmid, and in its derivative, pJLV01, specifically affected in the RNase activity of the Kid toxin. This new analysis aims to identify key factors in this coupling. For this purpose we have quantified and characterized the restriction introduced by parD to isolate conditional replication mutants of this plasmid, a signature of the modular coupling. This restriction depends on the RNase activity of the Kid toxin and it is relieved by either over-expression of the Kis antitoxin or by preventing its degradation by Lon and ClpAP proteases. Based on these data and on the correlation between copy numbers and parD transcriptional levels obtained in the different mutants, it is proposed that a reduction of Kis antitoxin levels in response to inefficient plasmid replication is the key factor for coupling plasmid replication and parD modules. Copyright © 2012 Elsevier Inc. All rights reserved.

Evaluative Priming in the Pronunciation Task.

PubMed

Klauer, Karl Christoph; Becker, Manuel; Spruyt, Adriaan

2016-01-01

We replicated and extended a study by Spruyt and Hermans (2008) in which picture primes engendered an evaluative-priming effect on the pronunciation of target words. As preliminary steps, we assessed data reproducibility of the original study, conducted Pilot Study I to identify highly semantically related prime-target pairs, reanalyzed the original data excluding such pairs, conducted Pilot Study II to demonstrate that we can replicate traditional associative priming effects in the pronunciation task, and conducted Pilot Study III to generate relatively unrelated sets of prime pictures and target words. The main study comprised three between-participants conditions: (1) a close replication of the original study, (2) the same condition excluding highly related prime-target pairs, and (3) a condition based on the relatively unrelated sets of prime pictures and target words developed in Pilot Study III. There was little evidence for an evaluative priming effect independent of semantic relatedness.

The Variables Associated With Health Promotion Behaviors Among Urban Black Women.

PubMed

Hepburn, Millie

2018-04-24

To improve understanding of variables impacting health promotion behaviors among urban Black women. A cross-sectional survey was used. Urban Black women (N = 132) between the ages of 30 to 64 years participated. The study was conducted in a U.S. metropolitan region in 2015. Health literacy (Newest Vital Sign [NVS]), self-efficacy (New General Self-Efficacy Scale [NGSE]), and readiness for change (Health Risk Instrument [HRI]) were correlated with health promotion behaviors (Health Promotion Lifestyle Profile II [HPLPII]). Univariate statistics addressed demographic characteristics; bivariate/simultaneous linear regression determined the relationships between the NVS, NGSE, and HRI to health promotion behaviors (HPLPII). Demographics: 72.6% completed high school and 25% completed college, and the mean body mass index (BMI) was >32. Positive correlations existed between each variable to health promotion behaviors: NVS (r = .244, p < .002), NGSE (r = .312, p < .001), HRI (r = .440, p < .001), and accounted for 29.8% of variances in health promotion behaviors. Education and health literacy were also correlated (r s = .414, p = .001). Although health literacy, self-efficacy, and readiness for change are associated with health promotion behaviors, readiness for change was the most highly correlated. The development and incorporation of interventions to promote health promotion behaviors should include readiness for change, health literacy, BMI, and education, especially among urban Black women in order to reduce critical health disparities. Community-based and culturally relevant strategies in promoting health that are integrated into existing lifestyles and designed to impact readiness for change will have the greatest impact on reducing health disparities both in the United States and in countries experiencing rapid urbanization. For example, healthy eating behaviors or increased physical activity may be best adopted when integrated into existing community-based spiritual or cultural events via trusted community leaders. Replication of this study in other populations of Black women will improve the generalizability of this study, both in the United States and globally. However, the addition of other demographic variables, such as a history of chronic conditions, military service, domestic or other violence, spirituality, and the availability of community resources, would strengthen the results in future studies. © 2018 Sigma Theta Tau International.

Modeling the Effects of Morphine on Simian Immunodeficiency Virus Dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vaidya, Naveen K.; Ribeiro, Ruy M.; Perelson, Alan S.

Complications of HIV-1 infection in individuals who utilize drugs of abuse is a significant problem, because these drugs have been associated with higher virus replication and accelerated disease progression as well as severe neuropathogenesis. To gain further insight it is important to quantify the effects of drugs of abuse on HIV-1 infection dynamics. Here, we develop a mathematical model that incorporates experimentally observed effects of morphine on inducing HIV-1 co-receptor expression. For comparison we also considered viral dynamic models with cytolytic or noncytolytic effector cell responses. Based on the small sample size Akaike information criterion, these models were inferior tomore » the new model based on changes in co-receptor expression. The model with morphine affecting co-receptor expression agrees well with the experimental data from simian immunodeficiency virus infections in morphine-addicted macaques. Our results show that morphine promotes a target cell subpopulation switch from a lower level of susceptibility to a state that is about 2-orders of magnitude higher in susceptibility to SIV infection. As a result, the proportion of target cells with higher susceptibility remains extremely high in morphine conditioning. Such a morphine-induced population switch not only has adverse effects on the replication rate, but also results in a higher steady state viral load and larger CD4 count drops. Moreover, morphine conditioning may pose extra obstacles to controlling viral load during antiretroviral therapy, such as pre-exposure prophylaxis and post infection treatments. In conclusion, this study provides, for the first time, a viral dynamics model, viral dynamics parameters, and related analytical and simulation results for SIV dynamics under drugs of abuse.« less

Modeling the Effects of Morphine on Simian Immunodeficiency Virus Dynamics

DOE PAGES

Vaidya, Naveen K.; Ribeiro, Ruy M.; Perelson, Alan S.; ...

2016-09-26

Complications of HIV-1 infection in individuals who utilize drugs of abuse is a significant problem, because these drugs have been associated with higher virus replication and accelerated disease progression as well as severe neuropathogenesis. To gain further insight it is important to quantify the effects of drugs of abuse on HIV-1 infection dynamics. Here, we develop a mathematical model that incorporates experimentally observed effects of morphine on inducing HIV-1 co-receptor expression. For comparison we also considered viral dynamic models with cytolytic or noncytolytic effector cell responses. Based on the small sample size Akaike information criterion, these models were inferior tomore » the new model based on changes in co-receptor expression. The model with morphine affecting co-receptor expression agrees well with the experimental data from simian immunodeficiency virus infections in morphine-addicted macaques. Our results show that morphine promotes a target cell subpopulation switch from a lower level of susceptibility to a state that is about 2-orders of magnitude higher in susceptibility to SIV infection. As a result, the proportion of target cells with higher susceptibility remains extremely high in morphine conditioning. Such a morphine-induced population switch not only has adverse effects on the replication rate, but also results in a higher steady state viral load and larger CD4 count drops. Moreover, morphine conditioning may pose extra obstacles to controlling viral load during antiretroviral therapy, such as pre-exposure prophylaxis and post infection treatments. In conclusion, this study provides, for the first time, a viral dynamics model, viral dynamics parameters, and related analytical and simulation results for SIV dynamics under drugs of abuse.« less

DNA replication stress restricts ribosomal DNA copy number

PubMed Central

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

The β2 clamp in the Mycobacterium tuberculosis DNA polymerase III αβ2ε replicase promotes polymerization and reduces exonuclease activity

PubMed Central

Gu, Shoujin; Li, Wenjuan; Zhang, Hongtai; Fleming, Joy; Yang, Weiqiang; Wang, Shihua; Wei, Wenjing; Zhou, Jie; Zhu, Guofeng; Deng, Jiaoyu; Hou, Jian; Zhou, Ying; Lin, Shiqiang; Zhang, Xian-En; Bi, Lijun

2016-01-01

DNA polymerase III (DNA pol III) is a multi-subunit replication machine responsible for the accurate and rapid replication of bacterial genomes, however, how it functions in Mycobacterium tuberculosis (Mtb) requires further investigation. We have reconstituted the leading-strand replication process of the Mtb DNA pol III holoenzyme in vitro, and investigated the physical and functional relationships between its key components. We verify the presence of an αβ2ε polymerase-clamp-exonuclease replicase complex by biochemical methods and protein-protein interaction assays in vitro and in vivo and confirm that, in addition to the polymerase activity of its α subunit, Mtb DNA pol III has two potential proofreading subunits; the α and ε subunits. During DNA replication, the presence of the β2 clamp strongly promotes the polymerization of the αβ2ε replicase and reduces its exonuclease activity. Our work provides a foundation for further research on the mechanism by which the replication machinery switches between replication and proofreading and provides an experimental platform for the selection of antimicrobials targeting DNA replication in Mtb. PMID:26822057

Self-Enhancement of Hepatitis C Virus Replication by Promotion of Specific Sphingolipid Biosynthesis

PubMed Central

Hirata, Yuichi; Ikeda, Kazutaka; Sudoh, Masayuki; Tokunaga, Yuko; Suzuki, Akemi; Weng, Leiyun; Ohta, Masatoshi; Tobita, Yoshimi; Okano, Ken; Ozeki, Kazuhisa; Kawasaki, Kenichi; Tsukuda, Takuo; Katsume, Asao; Aoki, Yuko; Umehara, Takuya; Sekiguchi, Satoshi; Toyoda, Tetsuya; Shimotohno, Kunitada; Soga, Tomoyoshi; Nishijima, Masahiro; Taguchi, Ryo; Kohara, Michinori

2012-01-01

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle. PMID:22916015

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

PubMed

Hirata, Yuichi; Ikeda, Kazutaka; Sudoh, Masayuki; Tokunaga, Yuko; Suzuki, Akemi; Weng, Leiyun; Ohta, Masatoshi; Tobita, Yoshimi; Okano, Ken; Ozeki, Kazuhisa; Kawasaki, Kenichi; Tsukuda, Takuo; Katsume, Asao; Aoki, Yuko; Umehara, Takuya; Sekiguchi, Satoshi; Toyoda, Tetsuya; Shimotohno, Kunitada; Soga, Tomoyoshi; Nishijima, Masahiro; Taguchi, Ryo; Kohara, Michinori

2012-01-01

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

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

PubMed

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.

P-body proteins regulate transcriptional rewiring to promote DNA replication stress resistance.

PubMed

Loll-Krippleber, Raphael; Brown, Grant W

2017-09-15

mRNA-processing (P-) bodies are cytoplasmic granules that form in eukaryotic cells in response to numerous stresses to serve as sites of degradation and storage of mRNAs. Functional P-bodies are critical for the DNA replication stress response in yeast, yet the repertoire of P-body targets and the mechanisms by which P-bodies promote replication stress resistance are unknown. In this study we identify the complete complement of mRNA targets of P-bodies during replication stress induced by hydroxyurea treatment. The key P-body protein Lsm1 controls the abundance of HHT1, ACF4, ARL3, TMA16, RRS1 and YOX1 mRNAs to prevent their toxic accumulation during replication stress. Accumulation of YOX1 mRNA causes aberrant downregulation of a network of genes critical for DNA replication stress resistance and leads to toxic acetaldehyde accumulation. Our data reveal the scope and the targets of regulation by P-body proteins during the DNA replication stress response.P-bodies form in response to stress and act as sites of mRNA storage and degradation. Here the authors identify the mRNA targets of P-bodies during DNA replication stress, and show that P-body proteins act to prevent toxic accumulation of these target transcripts.

E2F mediates induction of the Sp1-controlled promoter of the human DNA polymerase ɛ B-subunit gene POLE2

PubMed Central

Huang, Deqi; Jokela, Maarit; Tuusa, Jussi; Skog, Sven; Poikonen, Kari; Syväoja, Juhani E.

2001-01-01

The B-subunits of replicative DNA polymerases from Archaea to humans belong to the same protein family, suggesting that they share a common fundamental function. We report here the gene structure for the B-subunit of human DNA polymerase ɛ (POLE2), whose expression and transcriptional regulation is typical for replication proteins with some unique features. The 75 bp core promoter region, located within exon 1, contains an Sp1 element that is a critical determinant of promoter activity as shown by the luciferase reporter, electrophoretic mobility shift and DNase I footprinting assays. Two overlapping E2F elements adjacent to the Sp1 element are essential for full promoter activity and serum response. Binding sites for E2F1 and NF-1 reside immediately downstream from the core promoter region. Our results suggest that human POLE2 is regulated by two E2F–pocket protein complexes, one associated with Sp1 and the other with NF-1. So far, only one replicative DNA polymerase B-subunit gene promoter, POLA2 encoding the B-subunit of DNA polymerase α, has been characterized. Mitogenic activation of the POLE2 promoter by an E2F-mediated mechanism resembles that of POLA2, but the regulation of basal promoter activity is different between these two genes. PMID:11433027

EAT-UP™ Family-Centered Feeding Intervention to Promote Food Acceptance and Decrease Challenging Behaviors: A Single-Case Experimental Design Replicated across Three Families of Children with Autism Spectrum Disorder

ERIC Educational Resources Information Center

Cosbey, Joanna; Muldoon, Deirdre

2017-01-01

This study evaluated the effectiveness of a family-centered feeding intervention, Easing Anxiety Together with Understanding and Perseverance (EAT-UP™), for promoting food acceptance of children with autism spectrum disorder at home. A concurrent multiple-baseline design was used with systematic replication across three families. Baseline was…

Overexpression of pig selenoprotein S blocks OTA-induced promotion of PCV2 replication by inhibiting oxidative stress and p38 phosphorylation in PK15 cells.

PubMed

Gan, Fang; Hu, Zhihua; Huang, Yu; Xue, Hongxia; Huang, Da; Qian, Gang; Hu, Junfa; Chen, Xingxiang; Wang, Tian; Huang, Kehe

2016-04-12

Porcine circovirus type 2 (PCV2) is the primary cause of porcine circovirus disease, and ochratoxin A (OTA)-induced oxidative stress promotes PCV2 replication. In humans, selenoprotein S (SelS) has antioxidant ability, but it is unclear whether SelS affects viral infection. Here, we stably transfected PK15 cells with pig pCDNA3.1-SelS to overexpress SelS. Selenium (Se) at 2 or 4 μM and SelS overexpression blocked the OTA-induced increases of PCV2 DNA copy number and infected cell numbers. SelS overexpression also increased glutathione (GSH), NF-E2-related factor 2 (Nrf2) mRNA, and γ-glutamyl-cysteine synthetase mRNA levels; decreased reactive oxygen species (ROS) levels; and inhibited p38 phosphorylation in PCV2-infected PK15 cells, regardless of OTA treatment. Buthionine sulfoximine reversed all of the above SelS-induced changes. siRNA-mediated SelS knockdown decreased Nrf2 mRNA and GSH levels, increased ROS levels, and promoted PCV2 replication in OTA-treated PK15 cells. These data indicate that pig SelS blocks OTA-induced promotion of PCV2 replication by inhibiting the oxidative stress and p38 phosphorylation in PK15 cells.

Overexpression of pig selenoprotein S blocks OTA-induced promotion of PCV2 replication by inhibiting oxidative stress and p38 phosphorylation in PK15 cells

PubMed Central

Gan, Fang; Hu, Zhihua; Huang, Yu; Xue, Hongxia; Huang, Da; Qian, Gang; Hu, Junfa; Chen, Xingxiang; Wang, Tian; Huang, Kehe

2016-01-01

Porcine circovirus type 2 (PCV2) is the primary cause of porcine circovirus disease, and ochratoxin A (OTA)-induced oxidative stress promotes PCV2 replication. In humans, selenoprotein S (SelS) has antioxidant ability, but it is unclear whether SelS affects viral infection. Here, we stably transfected PK15 cells with pig pCDNA3.1-SelS to overexpress SelS. Selenium (Se) at 2 or 4 μM and SelS overexpression blocked the OTA-induced increases of PCV2 DNA copy number and infected cell numbers. SelS overexpression also increased glutathione (GSH), NF-E2-related factor 2 (Nrf2) mRNA, and γ-glutamyl-cysteine synthetase mRNA levels; decreased reactive oxygen species (ROS) levels; and inhibited p38 phosphorylation in PCV2-infected PK15 cells, regardless of OTA treatment. Buthionine sulfoximine reversed all of the above SelS-induced changes. siRNA-mediated SelS knockdown decreased Nrf2 mRNA and GSH levels, increased ROS levels, and promoted PCV2 replication in OTA-treated PK15 cells. These data indicate that pig SelS blocks OTA-induced promotion of PCV2 replication by inhibiting the oxidative stress and p38 phosphorylation in PK15 cells. PMID:26943035

Dicer functions as an antiviral system against human adenoviruses via cleavage of adenovirus-encoded noncoding RNA

PubMed Central

Machitani, Mitsuhiro; Sakurai, Fuminori; Wakabayashi, Keisaku; Tomita, Kyoko; Tachibana, Masashi; Mizuguchi, Hiroyuki

2016-01-01

In various organisms, including nematodes and plants, RNA interference (RNAi) is a defense system against virus infection; however, it is unclear whether RNAi functions as an antivirus system in mammalian cells. Rather, a number of DNA viruses, including herpesviruses, utilize post-transcriptional silencing systems for their survival. Here we show that Dicer efficiently suppresses the replication of adenovirus (Ad) via cleavage of Ad-encoding small RNAs (VA-RNAs), which efficiently promote Ad replication via the inhibition of eIF2α phosphorylation, to viral microRNAs (mivaRNAs). The Dicer knockdown significantly increases the copy numbers of VA-RNAs, leading to the efficient inhibition of eIF2α phosphorylation and the subsequent promotion of Ad replication. Conversely, overexpression of Dicer significantly inhibits Ad replication. Transfection with mivaRNA does not affect eIF2α phosphorylation or Ad replication. These results indicate that Dicer-mediated processing of VA-RNAs leads to loss of activity of VA-RNAs for enhancement of Ad replication and that Dicer functions as a defence system against Ad in mammalian cells. PMID:27273616

The effects of counting blessings on subjective well-being: a gratitude intervention in a Spanish sample.

PubMed

Martínez-Martí, María Luisa; Avia, María Dolores; Hernández-Lloreda, María José

2010-11-01

This study examined a gratitude intervention repeating Emmons and McCullough study (2003) in a Spanish sample, Participants were randomly assigned to one of three conditions (gratitude, hassles and any event) and kept daily records during 2 weeks of gratitude, affect, quality of relationships, physical and subjective well-being. We added design features to assess the intervention long-term impact (follow-up measures), and to improve the design control (pre-treatment measures). Following the cited authors' analysis, i.e., comparing groups only in the post-test, we replicated their results, finding differences in positive affect and gratitude between the gratitude condition and the hassles condition. However, when including both the pre and the follow-up measures in the analysis, results were replicated only partially, as the difference in gratitude disappeared. Moreover, the difference in positive affect between groups in the post-test seemed to be influenced mainly by a decrease in positive affect in the hassles group. Post-test differences between groups in positive affect disappeared in the follow-up. Gratitude interventions may have an effect on well-being, but we consider other methods to promote gratitude besides gratitude journals should be tested.

Delta-9 tetrahydrocannabinol (THC) inhibits lytic replication of gamma oncogenic herpesviruses in vitro.

PubMed

Medveczky, Maria M; Sherwood, Tracy A; Klein, Thomas W; Friedman, Herman; Medveczky, Peter G

2004-09-15

The major psychoactive cannabinoid compound of marijuana, delta-9 tetrahydrocannabinol (THC), has been shown to modulate immune responses and lymphocyte function. After primary infection the viral DNA genome of gamma herpesviruses persists in lymphoid cell nuclei in a latent episomal circular form. In response to extracellular signals, the latent virus can be activated, which leads to production of infectious virus progeny. Therefore, we evaluated the potential effects of THC on gamma herpesvirus replication. Tissue cultures infected with various gamma herpesviruses were cultured in the presence of increasing concentrations of THC and the amount of viral DNA or infectious virus yield was compared to those of control cultures. The effect of THC on Kaposi's Sarcoma Associated Herpesvirus (KSHV) and Epstein-Barr virus (EBV) replication was measured by the Gardella method and replication of herpesvirus saimiri (HVS) of monkeys, murine gamma herpesvirus 68 (MHV 68), and herpes simplex type 1 (HSV-1) was measured by yield reduction assays. Inhibition of the immediate early ORF 50 gene promoter activity was measured by the dual luciferase method. Micromolar concentrations of THC inhibit KSHV and EBV reactivation in virus infected/immortalized B cells. THC also strongly inhibits lytic replication of MHV 68 and HVS in vitro. Importantly, concentrations of THC that inhibit virus replication of gamma herpesviruses have no effect on cell growth or HSV-1 replication, indicating selectivity. THC was shown to selectively inhibit the immediate early ORF 50 gene promoter of KSHV and MHV 68. THC specifically targets viral and/or cellular mechanisms required for replication and possibly shared by these gamma herpesviruses, and the endocannabinoid system is possibly involved in regulating gamma herpesvirus latency and lytic replication. The immediate early gene ORF 50 promoter activity was specifically inhibited by THC. These studies may also provide the foundation for the development of antiviral strategies utilizing non-psychoactive derivatives of THC.

Does Promotion Orientation Help Explain Why Future-Orientated People Exercise and Eat Healthy?

PubMed

Milfont, Taciano L; Vilar, Roosevelt; Araujo, Rafaella C R; Stanley, Robert

2017-01-01

A study with United States undergraduate students showed individuals high in concern with future consequences engage in exercise and healthy eating because they adopt a promotion orientation, which represents the extent to which individuals are inclined to pursue positive gains. The present article reports a cross-cultural replication of the mediation findings with undergraduate samples from Brazil and New Zealand. Promotion orientation mediated the association between concern with future consequences and exercise attitudes in both countries, but the associations for healthy eating were not replicated-which could be explained by distinct obesity prevalence and eating habits in these socio-cultural contexts. We discuss theoretical and practical implications of the findings for promoting health behavior.

Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter

PubMed Central

Wang, Xiaohong; Liu, Haibin; Ge, Hui; Ajiro, Masahiko; Sharma, Nishi R.; Meyers, Craig; Morozov, Pavel; Tuschl, Thomas; Klar, Amar; Court, Donald

2017-01-01

ABSTRACT The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis-acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors. PMID:28559488

Bacterial-based Systems for Expression and Purification of Recombinant Lassa Virus Proteins of Immunological Relevance

DTIC Science & Technology

2008-06-06

sites. Abbreviations include: MBP gene (malE), MBP promoter (Ptac), philamentous phage origin of replication ( M13 ori), bacterial origin of replica...notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display

Skill-Based and Planned Active Play Versus Free-Play Effects on Fundamental Movement Skills in Preschoolers.

PubMed

Roach, Lindsay; Keats, Melanie

2018-01-01

Fundamental movement skill interventions are important for promoting physical activity, but the optimal intervention model for preschool children remains unclear. We compared two 8-week interventions, a structured skill-station and a planned active play approach, to a free-play control condition on pre- and postintervention fundamental movement skills. We also collected data regarding program attendance and perceived enjoyment. We found a significant interaction effect between intervention type and time. A Tukey honest significant difference analysis supported a positive intervention effect showing a significant difference between both interventions and the free-play control condition. There was a significant between-group difference in group attendance such that mean attendance was higher for both the free-play and planned active play groups relative to the structured skill-based approach. There were no differences in attendance between free-play and planned active play groups, and there were no differences in enjoyment ratings between the two intervention groups. In sum, while both interventions led to improved fundamental movement skills, the active play approach offered several logistical advantages. Although these findings should be replicated, they can guide feasible and sustainable fundamental movement skill programs within day care settings.

The Fanconi anemia pathway limits the severity of mutagenesis.

PubMed

Hinz, John M; Nham, Peter B; Salazar, Edmund P; Thompson, Larry H

2006-08-13

Fanconi anemia (FA) is a developmental and cancer predisposition disorder in which key, yet unknown, physiological events promoting chromosome stability are compromised. FA cells exhibit excess metaphase chromatid breaks and are universally hypersensitive to DNA interstrand crosslinking agents. Published mutagenesis data from single-gene mutation assays show both increased and decreased mutation frequencies in FA cells. In this review we discuss the data from the literature and from our isogenic fancg knockout hamster CHO cells, and interpret these data within the framework of a molecular model that accommodates these seemingly divergent observations. In FA cells, reduced rates of recovery of viable X-linked hypoxanthine phosphoribosyltransferase (hprt) mutants are characteristically observed for diverse mutagenic agents, but also in untreated cultures, indicating the relevance of the FA pathway for processing assorted DNA lesions. We ascribe these reductions to: (1) impaired mutagenic translesion synthesis within hprt during DNA replication and (2) lethality of mutant cells following replication fork breakage on the X chromosome, caused by unrepaired double-strand breaks or large deletions/translocations encompassing essential genes flanking hprt. These findings, along with studies showing increased spontaneous mutability of FA cells at two autosomal loci, support a model in which FA proteins promote both translesion synthesis at replication-blocking lesions and repair of broken replication forks by homologous recombination and DNA end joining. The essence of this model is that the FANC protein pathway serves to restrict the severity of mutational outcome by favoring base substitutions and small deletions over larger deletions and chromosomal rearrangements.

Differential principal component analysis of ChIP-seq.

PubMed

Ji, Hongkai; Li, Xia; Wang, Qian-fei; Ning, Yang

2013-04-23

We propose differential principal component analysis (dPCA) for analyzing multiple ChIP-sequencing datasets to identify differential protein-DNA interactions between two biological conditions. dPCA integrates unsupervised pattern discovery, dimension reduction, and statistical inference into a single framework. It uses a small number of principal components to summarize concisely the major multiprotein synergistic differential patterns between the two conditions. For each pattern, it detects and prioritizes differential genomic loci by comparing the between-condition differences with the within-condition variation among replicate samples. dPCA provides a unique tool for efficiently analyzing large amounts of ChIP-sequencing data to study dynamic changes of gene regulation across different biological conditions. We demonstrate this approach through analyses of differential chromatin patterns at transcription factor binding sites and promoters as well as allele-specific protein-DNA interactions.

Oncolytic adenovirus expressing interleukin-18 improves antitumor activity of dacarbazine for malignant melanoma.

PubMed

Yang, Chunhua; Cao, Hang; Liu, Ning; Xu, Kai; Ding, Meng; Mao, Li-Jun

2016-01-01

Conditionally replicating adenoviruses have emerged as novel therapeutic agents for cancer. This study aimed to evaluate synergistic antitumor activity of replication-competent adenovirus armed with interleukin (IL)-18 (ZD55-IL-18) and dacarbazine (DTIC) against melanoma. Melanoma A375 cells or nude mouse tumor xenografts were treated with ZD55-IL-18 alone or together with DTIC. The results showed that ZD55-IL-18 competently replicated in A375 cells and expressed IL-18, and these were not affected by DTIC. ZD55-IL-18 enhanced the cytotoxicity of DTIC accompanied by increased apoptosis. Moreover, ZD55-IL-18 and DTIC synergistically inhibited the growth but promoted the apoptosis of A375 xenografts and inhibited vascular endothelial growth factor expression and lung metastasis in xenografts of nude mice. In conclusion, this is the first study to show synergistic anticancer activity of ZD55-IL-18 and DTIC for malignant melanoma. Our results provide evidence that chemo-gene-viro therapeutic approach has greater potential for malignant cancers than conventional chemotherapy or gene therapy.

Oncolytic adenovirus expressing interleukin-18 improves antitumor activity of dacarbazine for malignant melanoma

PubMed Central

Yang, Chunhua; Cao, Hang; Liu, Ning; Xu, Kai; Ding, Meng; Mao, Li-jun

2016-01-01

Conditionally replicating adenoviruses have emerged as novel therapeutic agents for cancer. This study aimed to evaluate synergistic antitumor activity of replication-competent adenovirus armed with interleukin (IL)-18 (ZD55-IL-18) and dacarbazine (DTIC) against melanoma. Melanoma A375 cells or nude mouse tumor xenografts were treated with ZD55-IL-18 alone or together with DTIC. The results showed that ZD55-IL-18 competently replicated in A375 cells and expressed IL-18, and these were not affected by DTIC. ZD55-IL-18 enhanced the cytotoxicity of DTIC accompanied by increased apoptosis. Moreover, ZD55-IL-18 and DTIC synergistically inhibited the growth but promoted the apoptosis of A375 xenografts and inhibited vascular endothelial growth factor expression and lung metastasis in xenografts of nude mice. In conclusion, this is the first study to show synergistic anticancer activity of ZD55-IL-18 and DTIC for malignant melanoma. Our results provide evidence that chemo-gene-viro therapeutic approach has greater potential for malignant cancers than conventional chemotherapy or gene therapy. PMID:27895465

DNA transformations of Candida tropicalis with replicating and integrative vectors.

PubMed

Sanglard, D; Fiechter, A

1992-12-01

The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.

Metaresearch for Evaluating Reproducibility in Ecology and Evolution.

PubMed

Fidler, Fiona; Chee, Yung En; Wintle, Bonnie C; Burgman, Mark A; McCarthy, Michael A; Gordon, Ascelin

2017-03-01

Recent replication projects in other disciplines have uncovered disturbingly low levels of reproducibility, suggesting that those research literatures may contain unverifiable claims. The conditions contributing to irreproducibility in other disciplines are also present in ecology. These include a large discrepancy between the proportion of "positive" or "significant" results and the average statistical power of empirical research, incomplete reporting of sampling stopping rules and results, journal policies that discourage replication studies, and a prevailing publish-or-perish research culture that encourages questionable research practices. We argue that these conditions constitute sufficient reason to systematically evaluate the reproducibility of the evidence base in ecology and evolution. In some cases, the direct replication of ecological research is difficult because of strong temporal and spatial dependencies, so here, we propose metaresearch projects that will provide proxy measures of reproducibility.

A model for genesis of transcription systems.

PubMed

Burton, Zachary F; Opron, Kristopher; Wei, Guowei; Geiger, James H

2016-01-01

Repeating sequences generated from RNA gene fusions/ligations dominate ancient life, indicating central importance of building structural complexity in evolving biological systems. A simple and coherent story of life on earth is told from tracking repeating motifs that generate α/β proteins, 2-double-Ψ-β-barrel (DPBB) type RNA polymerases (RNAPs), general transcription factors (GTFs), and promoters. A general rule that emerges is that biological complexity that arises through generation of repeats is often bounded by solubility and closure (i.e., to form a pseudo-dimer or a barrel). Because the first DNA genomes were replicated by DNA template-dependent RNA synthesis followed by RNA template-dependent DNA synthesis via reverse transcriptase, the first DNA replication origins were initially 2-DPBB type RNAP promoters. A simplifying model for evolution of promoters/replication origins via repetition of core promoter elements is proposed. The model can explain why Pribnow boxes in bacterial transcription (i.e., (-12)TATAATG(-6)) so closely resemble TATA boxes (i.e., (-31)TATAAAAG(-24)) in archaeal/eukaryotic transcription. The evolution of anchor DNA sequences in bacterial (i.e., (-35)TTGACA(-30)) and archaeal (BRE(up); BRE for TFB recognition element) promoters is potentially explained. The evolution of BRE(down) elements of archaeal promoters is potentially explained.

Designing Place-Based Interventions for Sustainability and Replicability: The Case of GO! Austin/VAMOS! Austin

PubMed Central

Hussaini, Aliya; Pulido, Carmen Llanes; Basu, Semonti; Ranjit, Nalini

2018-01-01

Place-based health efforts account for the role of the community environment in shaping decisions and circumstances that affect population well-being. Such efforts, rooted as they are in the theory that health is socially determined, mobilize resources for health promotion that are not typically used, and offer a more informed and robust way of promoting health outcomes within a community. Common criticisms of place-based work include the difficulty of replication, since engagement is so specific to a place, and limited sustainability of the work, in the absence of continued institutional structures, both within the community and supporting structures outside the community, to keep these initiatives resilient. This paper describes a place-based initiative, GO! Austin/VAMOS! Austin (GAVA), which was designed to harness the strengths of place-based work—namely, its specificity to place and community. From the start, the project was designed to balance this specificity with a focus on developing and utilizing a standardized set of evidence-informed implementation and evaluation approaches and tools that were flexible enough to be modified for specific settings. This was accompanied by an emphasis on leadership and capacity building within resident leaders, which provided for informed intervention and demand building capacity, but also for longevity as partners, philanthropic, and otherwise, moved in and out of the work. PMID:29623272

Designing Place-Based Interventions for Sustainability and Replicability: The Case of GO! Austin/VAMOS! Austin.

PubMed

Hussaini, Aliya; Pulido, Carmen Llanes; Basu, Semonti; Ranjit, Nalini

2018-01-01

Place-based health efforts account for the role of the community environment in shaping decisions and circumstances that affect population well-being. Such efforts, rooted as they are in the theory that health is socially determined, mobilize resources for health promotion that are not typically used, and offer a more informed and robust way of promoting health outcomes within a community. Common criticisms of place-based work include the difficulty of replication , since engagement is so specific to a place, and limited sustainability of the work, in the absence of continued institutional structures, both within the community and supporting structures outside the community, to keep these initiatives resilient. This paper describes a place-based initiative, GO! Austin/VAMOS! Austin (GAVA), which was designed to harness the strengths of place-based work-namely, its specificity to place and community. From the start, the project was designed to balance this specificity with a focus on developing and utilizing a standardized set of evidence-informed implementation and evaluation approaches and tools that were flexible enough to be modified for specific settings. This was accompanied by an emphasis on leadership and capacity building within resident leaders, which provided for informed intervention and demand building capacity, but also for longevity as partners, philanthropic, and otherwise, moved in and out of the work.

The Medial Temporal Lobes are Critical for Reward-Based Decision Making under Conditions that Promote Episodic Future Thinking

PubMed Central

Palombo, Daniela J.; Keane, Margaret M.; Verfaellie, Mieke

2014-01-01

In the present study, we investigated the effect of medial temporal lobe (MTL) damage on human decision making in the context of reward-based intertemporal choice. During intertemporal choice, humans typically devalue (or discount) a future reward to account for its delayed arrival (e.g., preferring $30 now over $42 in 2 months), but this effect is attenuated when participants engage in episodic future thinking, i.e., project themselves into the future to imagine a specific event. We hypothesized that this attenuation would be selectively impaired in amnesic patients, who have deficits in episodic future thinking. Replicating previous work, in a standard intertemporal choice task, amnesic patients showed temporal discounting indices similar to healthy controls. Consistent with our hypothesis, while healthy controls demonstrated attenuated temporal discounting in a condition that required participants first to engage in episodic future thinking (e.g., to imagine spending $42 at a theatre in 2 months), amnesic patients failed to demonstrate this effect. Moreover, as expected, amnesic patients’ narratives were less episodically rich than those of controls. These findings extend the range of tasks that are shown to be MTL dependent to include not only memory-based decision-making tasks but also future-oriented ones. PMID:25284804

A CI-Independent Form of Replicative Inhibition: Turn Off of Early Replication of Bacteriophage Lambda

PubMed Central

Hayes, Sidney; Horbay, Monique A.; Hayes, Connie

2012-01-01

Several earlier studies have described an unusual exclusion phenotype exhibited by cells with plasmids carrying a portion of the replication region of phage lambda. Cells exhibiting this inhibition phenotype (IP) prevent the plating of homo-immune and hybrid hetero-immune lambdoid phages. We have attempted to define aspects of IP, and show that it is directed to repλ phages. IP was observed in cells with plasmids containing a λ DNA fragment including oop, encoding a short OOP micro RNA, and part of the lambda origin of replication, oriλ, defined by iteron sequences ITN1-4 and an adjacent high AT-rich sequence. Transcription of the intact oop sequence from its promoter, pO is required for IP, as are iterons ITN3–4, but not the high AT-rich portion of oriλ. The results suggest that IP silencing is directed to theta mode replication initiation from an infecting repλ genome, or an induced repλ prophage. Phage mutations suppressing IP, i.e., Sip, map within, or adjacent to cro or in O, or both. Our results for plasmid based IP suggest the hypothesis that there is a natural mechanism for silencing early theta-mode replication initiation, i.e. the buildup of λ genomes with oop + oriλ+ sequence. PMID:22590552

BRCA1 is Required for Post-replication Repair After UV-induced DNA Damage

PubMed Central

Pathania, Shailja; Nguyen, Jenna; Hill, Sarah J.; Scully, Ralph; Feunteun, Jean; Livingston, David M.

2011-01-01

BRCA1 contributes to the response to UV irradiation. Utilizing its BRCT motifs, it is recruited during S/G2 to UV-damaged sites in a DNA replication-dependent, but nucleotide excision repair- independent manner. More specifically, at UV- stalled replication forks, it promotes photoproduct excision, suppression of translesion synthesis, and the localization and activation of replication factor C complex (RFC) subunits. The last function, in turn, triggers post-UV checkpoint activation and post- replicative repair. These BRCA1 functions differ from those required for DSBR. PMID:21963239

The Replisome-Coupled E3 Ubiquitin Ligase Rtt101Mms22 Counteracts Mrc1 Function to Tolerate Genotoxic Stress

PubMed Central

Melnik, Andre; Wilson-Zbinden, Caroline; Schellhaas, René; Kastner, Lisa; Piwko, Wojciech; Dees, Martina; Picotti, Paola; Maric, Marija; Labib, Karim; Luke, Brian; Peter, Matthias

2016-01-01

Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1’s replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks. PMID:26849847

Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica.

PubMed

Shabbir Hussain, Murtaza; Gambill, Lauren; Smith, Spencer; Blenner, Mark A

2016-03-18

Eukaryotic promoters have a complex architecture to control both the strength and timing of gene transcription spanning up to thousands of bases from the initiation site. This complexity makes rational fine-tuning of promoters in fungi difficult to predict; however, this very same complexity enables multiple possible strategies for engineering promoter strength. Here, we studied promoter architecture in the oleaginous yeast, Yarrowia lipolytica. While recent studies have focused on upstream activating sequences, we systematically examined various components common in fungal promoters. Here, we examine several promoter components including upstream activating sequences, proximal promoter sequences, core promoters, and the TATA box in autonomously replicating expression plasmids and integrated into the genome. Our findings show that promoter strength can be fine-tuned through the engineering of the TATA box sequence, core promoter, and upstream activating sequences. Additionally, we identified a previously unreported oleic acid responsive transcription enhancement in the XPR2 upstream activating sequences, which illustrates the complexity of fungal promoters. The promoters engineered here provide new genetic tools for metabolic engineering in Y. lipolytica and provide promoter engineering strategies that may be useful in engineering other non-model fungal systems.

Social imitation versus strategic choice, or consensus versus cooperation, in the networked Prisoner's Dilemma

NASA Astrophysics Data System (ADS)

Vilone, Daniele; Ramasco, José J.; Sánchez, Angel; Miguel, Maxi San

2014-08-01

The interplay of social and strategic motivations in human interactions is a largely unexplored topic in collective social phenomena. Whether individuals' decisions are taken in a purely strategic basis or due to social pressure without a rational background crucially influences the model outcome. Here we study a networked Prisoner's Dilemma in which decisions are made either based on the replication of the most successful neighbor's strategy (unconditional imitation) or by pure social imitation following an update rule inspired by the voter model. The main effects of the voter dynamics are an enhancement of the final consensus, i.e., asymptotic states are generally uniform, and a promotion of cooperation in certain regions of the parameter space as compared to the outcome of purely strategic updates. Thus, voter dynamics acts as an interface noise and has a similar effect as a pure random noise; furthermore, its influence is mostly independent of the network heterogeneity. When strategic decisions are made following other update rules such as the replicator or Moran processes, the dynamic mixed state found under unconditional imitation for some parameters disappears, but an increase of cooperation in certain parameter regions is still observed. Comparing our results with recent experiments on the Prisoner's Dilemma, we conclude that such a mixed dynamics may explain moody conditional cooperation among the agents.

Social imitation versus strategic choice, or consensus versus cooperation, in the networked Prisoner's Dilemma.

PubMed

Vilone, Daniele; Ramasco, José J; Sánchez, Angel; San Miguel, Maxi

2014-08-01

The interplay of social and strategic motivations in human interactions is a largely unexplored topic in collective social phenomena. Whether individuals' decisions are taken in a purely strategic basis or due to social pressure without a rational background crucially influences the model outcome. Here we study a networked Prisoner's Dilemma in which decisions are made either based on the replication of the most successful neighbor's strategy (unconditional imitation) or by pure social imitation following an update rule inspired by the voter model. The main effects of the voter dynamics are an enhancement of the final consensus, i.e., asymptotic states are generally uniform, and a promotion of cooperation in certain regions of the parameter space as compared to the outcome of purely strategic updates. Thus, voter dynamics acts as an interface noise and has a similar effect as a pure random noise; furthermore, its influence is mostly independent of the network heterogeneity. When strategic decisions are made following other update rules such as the replicator or Moran processes, the dynamic mixed state found under unconditional imitation for some parameters disappears, but an increase of cooperation in certain parameter regions is still observed. Comparing our results with recent experiments on the Prisoner's Dilemma, we conclude that such a mixed dynamics may explain moody conditional cooperation among the agents.

Two synthetic Sp1-binding sites functionally substitute for the 21-base-pair repeat region to activate simian virus 40 growth in CV-1 cells.

PubMed Central

Lednicky, J; Folk, W R

1992-01-01

The 21-bp repeat region of simian virus 40 (SV40) activates viral transcription and DNA replication and contains binding sites for many cellular proteins, including Sp1, LSF, ETF, Ap2, Ap4, GT-1B, H16, and p53, and for the SV40 large tumor antigen. We have attempted to reduce the complexity of this region while maintaining its growth-promoting capacity. Deletion of the 21-bp repeat region from the SV40 genome delays the expression of viral early proteins and DNA replication and reduces virus production in CV-1 cells. Replacement of the 21-bp repeat region with two copies of DNA sequence motifs bound with high affinities by Sp1 promotes SV40 growth in CV-1 cells to nearly wild-type levels, but substitution by motifs bound less avidly by Sp1 or bound by other activator proteins does not restore growth. This indicates that Sp1 or a protein with similar sequence specificity is primarily responsible for the function of the 21-bp repeat region. We speculate about how Sp1 activates both SV40 transcription and DNA replication. Images PMID:1328672

Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes.

PubMed

Hilton, Benjamin A; Liu, Ji; Cartwright, Brian M; Liu, Yiyong; Breitman, Maya; Wang, Youjie; Jones, Rowdy; Tang, Hui; Rusinol, Antonio; Musich, Phillip R; Zou, Yue

2017-09-01

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that is caused by a point mutation in the LMNA gene, resulting in production of a truncated farnesylated-prelamin A protein (progerin). We previously reported that XPA mislocalized to the progerin-induced DNA double-strand break (DSB) sites, blocking DSB repair, which led to DSB accumulation, DNA damage responses, and early replication arrest in HGPS. In this study, the XPA mislocalization to DSBs occurred at stalled or collapsed replication forks, concurrent with a significant loss of PCNA at the forks, whereas PCNA efficiently bound to progerin. This PCNA sequestration likely exposed ds-ssDNA junctions at replication forks for XPA binding. Depletion of XPA or progerin each significantly restored PCNA at replication forks. Our results suggest that although PCNA is much more competitive than XPA in binding replication forks, PCNA sequestration by progerin may shift the equilibrium to favor XPA binding. Furthermore, we demonstrated that progerin-induced apoptosis could be rescued by XPA, suggesting that XPA-replication fork binding may prevent apoptosis in HGPS cells. Our results propose a mechanism for progerin-induced genome instability and accelerated replicative senescence in HGPS.-Hilton, B. A., Liu, J., Cartwright, B. M., Liu, Y., Breitman, M., Wang, Y., Jones, R., Tang, H., Rusinol, A., Musich, P. R., Zou, Y. Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes. © FASEB.

Promotion of Hendra Virus Replication by MicroRNA 146a

PubMed Central

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

Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication.

PubMed

Thai, Minh; Graham, Nicholas A; Braas, Daniel; Nehil, Michael; Komisopoulou, Evangelia; Kurdistani, Siavash K; McCormick, Frank; Graeber, Thomas G; Christofk, Heather R

2014-04-01

Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. Although recent studies have characterized virus-induced changes in host cell metabolism (Munger et al., 2008; Terry et al., 2012), the molecular mechanisms by which viruses reprogram cellular metabolism have remained elusive. Here, we show that the gene product of adenovirus E4ORF1 is necessary for adenovirus-induced upregulation of host cell glucose metabolism and sufficient to promote enhanced glycolysis in cultured epithelial cells by activation of MYC. E4ORF1 localizes to the nucleus, binds to MYC, and enhances MYC binding to glycolytic target genes, resulting in elevated expression of specific glycolytic enzymes. E4ORF1 activation of MYC promotes increased nucleotide biosynthesis from glucose intermediates and enables optimal adenovirus replication in primary lung epithelial cells. Our findings show how a viral protein exploits host cell machinery to reprogram cellular metabolism and promote optimal progeny virion generation. Copyright © 2014 Elsevier Inc. All rights reserved.

Physical signals for protein-DNA recognition

NASA Astrophysics Data System (ADS)

Cao, Xiao-Qin; Zeng, Jia; Yan, Hong

2009-09-01

This paper discovers consensus physical signals around eukaryotic splice sites, transcription start sites, and replication origin start and end sites on a genome-wide scale based on their DNA flexibility profiles calculated by three different flexibility models. These salient physical signals are localized highly rigid and flexible DNAs, which may play important roles in protein-DNA recognition by the sliding search mechanism. The found physical signals lead us to a detailed hypothetical view of the search process in which a DNA-binding protein first finds a genomic region close to the target site from an arbitrary starting location by three-dimensional (3D) hopping and intersegment transfer mechanisms for long distances, and subsequently uses the one-dimensional (1D) sliding mechanism facilitated by the localized highly rigid DNAs to accurately locate the target flexible binding site within 30 bp (base pair) short distances. Guided by these physical signals, DNA-binding proteins rapidly search the entire genome to recognize a specific target site from the 3D to 1D pathway. Our findings also show that current promoter prediction programs (PPPs) based on DNA physical properties may suffer from lots of false positives because other functional sites such as splice sites and replication origins have similar physical signals as promoters do.

Host Range Factor 1 from Lymantria dispar Nucleopolyhedrovirus (NPV) Is an Essential Viral Factor Required for Productive Infection of NPVs in IPLB-Ld652Y Cells Derived from L. dispar

PubMed Central

Ishikawa, Hiroki; Ikeda, Motoko; Felipe Alves, Cristiano A.; Thiem, Suzanne M.; Kobayashi, Michihiro

2004-01-01

Host range factor 1 (HRF-1) of Lymantria dispar multinucleocapsid nucleopolyhedrovirus promotes Autographa californica MNPV replication in nonpermissive Ld652Y cells derived from L. dispar. Here we demonstrate that restricted Hyphantria cunea NPV replication in Ld652Y cells was not due to apoptosis but was likely due to global protein synthesis arrest that could be restored by HRF-1. Our data also showed that HRF-1 promoted the production of progeny virions for two other baculoviruses, Bombyx mori NPV and Spodoptera exigua MNPV, whose replication in Ld652Y cells is limited to replication of viral DNA without successful production of infectious progeny virions. Thus, HRF-1 is an essential viral factor required for productive infection of NPVs in Ld652Y cells. PMID:15507661

Host range factor 1 from Lymantria dispar Nucleopolyhedrovirus (NPV) is an essential viral factor required for productive infection of NPVs in IPLB-Ld652Y cells derived from L. dispar.

PubMed

Ishikawa, Hiroki; Ikeda, Motoko; Alves, Cristiano A Felipe; Thiem, Suzanne M; Kobayashi, Michihiro

2004-11-01

Host range factor 1 (HRF-1) of Lymantria dispar multinucleocapsid nucleopolyhedrovirus promotes Autographa californica MNPV replication in nonpermissive Ld652Y cells derived from L. dispar. Here we demonstrate that restricted Hyphantria cunea NPV replication in Ld652Y cells was not due to apoptosis but was likely due to global protein synthesis arrest that could be restored by HRF-1. Our data also showed that HRF-1 promoted the production of progeny virions for two other baculoviruses, Bombyx mori NPV and Spodoptera exigua MNPV, whose replication in Ld652Y cells is limited to replication of viral DNA without successful production of infectious progeny virions. Thus, HRF-1 is an essential viral factor required for productive infection of NPVs in Ld652Y cells.

Generation of recombinant European bat lyssavirus type 1 and inter-genotypic compatibility of lyssavirus genotype 1 and 5 antigenome promoters.

PubMed

Orbanz, Jeannette; Finke, Stefan

2010-10-01

Bat lyssaviruses (Fam. Rhabdoviridae) represent a source for the infection of terrestial mammals and the development of rabies disease. Molecular differences in the replication of bat and non-bat lyssaviruses and their contribution to pathogenicity, however, are unknown. One reason for this is the lack of reverse genetics systems for bat-restricted lyssaviruses. To investigate bat lyssavirus replication and host adaptation, we developed a reverse genetics system for European bat lyssavirus type 1 (EBLV-1; genotype 5). This was achieved by co-transfection of HEK-293T cells with a full-length EBLV-1 genome cDNA and expression plasmids for EBLV-1 proteins, resulting in recombinant EBLV-1 (rEBLV-1). Replication of rEBLV-1 was comparable to that of parental virus, showing that rEBLV-1 is a valid tool to investigate EBLV-1 replication functions. In a first approach, we tested whether the terminal promoter sequences of EBLV-1 are genotype-specific. Although genotype 1 (rabies virus) minigenomes were successfully amplified by EBLV-1 helper virus, in the context of the complete virus, only the antigenome promoter (AGP) sequence of EBLV-1 was replaceable, as indicated by comparable replication of rEBLV-1 and the chimeric virus. These analyses demonstrate that the terminal AGPs of genotype 1 and genotype 5 lyssaviruses are compatible with those of the heterologous genotype.

The Large Marseillevirus Explores Different Entry Pathways by Forming Giant Infectious Vesicles.

PubMed

Arantes, Thalita Souza; Rodrigues, Rodrigo Araújo Lima; Dos Santos Silva, Ludmila Karen; Oliveira, Graziele Pereira; de Souza, Helton Luís; Khalil, Jacques Y B; de Oliveira, Danilo Bretas; Torres, Alice Abreu; da Silva, Luis Lamberti; Colson, Philippe; Kroon, Erna Geessien; da Fonseca, Flávio Guimarães; Bonjardim, Cláudio Antônio; La Scola, Bernard; Abrahão, Jônatas Santos

2016-06-01

Triggering the amoebal phagocytosis process is a sine qua non condition for most giant viruses to initiate their replication cycle and consequently to promote their progeny formation. It is well known that the amoebal phagocytosis process requires the recognition of particles of >500 nm, and most amoebal giant viruses meet this requirement, such as mimivirus, pandoravirus, pithovirus, and mollivirus. However, in the context of the discovery of amoebal giant viruses in the last decade, Marseillevirus marseillevirus (MsV) has drawn our attention, because despite its ability to successfully replicate in Acanthamoeba, remarkably it does not fulfill the >500-nm condition, since it presents an ∼250-nm icosahedrally shaped capsid. We deeply investigated the MsV cycle by using a set of methods, including virological, molecular, and microscopic (immunofluorescence, scanning electron microscopy, and transmission electron microscopy) assays. Our results revealed that MsV is able to form giant vesicles containing dozens to thousands of viral particles wrapped by membranes derived from amoebal endoplasmic reticulum. Remarkably, our results strongly suggested that these giant vesicles are able to stimulate amoebal phagocytosis and to trigger the MsV replication cycle by an acidification-independent process. Also, we observed that MsV entry may occur by the phagocytosis of grouped particles (without surrounding membranes) and by an endosome-stimulated pathway triggered by single particles. Taken together, not only do our data deeply describe the main features of MsV replication cycle, but this is the first time, to our knowledge, that the formation of giant infective vesicles related to a DNA virus has been described. Triggering the amoebal phagocytosis process is a sine qua non condition required by most giant viruses to initiate their replication cycle. This process requires the recognition of particles of >500 nm, and many giant viruses meet this requirement. However, MsV is unusual, as despite having particles of ∼250 nm it is able to replicate in Acanthamoeba Our results revealed that MsV is able to form giant vesicles, containing dozens to thousands of viral particles, wrapped in membranes derived from amoebal endoplasmic reticulum. Remarkably, our results strongly suggest that these giant vesicles are able to stimulate phagocytosis using an acidification-independent process. Our work not only describes the main features of the MsV replication cycle but also describes, for the first time to our knowledge, the formation of huge infective vesicles in a large DNA viruses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Promoting Teachers' Social and Emotional Competence: A Replication Study of the Cultivating Awareness and Resilience in Education (CARE) Program

ERIC Educational Resources Information Center

Jennings, Patricia A.; Brown, Joshua L.; Frank, Jennifer; Tanler, Regin; Doyle, Sebrina; Rasheed, Damira; DeWeese, Anna; Greenberg, Mark

2014-01-01

The present study, which takes place in a high-poverty section of a large urban area of the northeastern United States, is based upon the prosocial classroom theoretical model that emphasizes the significance of teachers' social and emotional competence (SEC) and well-being in the development and maintenance of supportive teacher-student…

WDR5 Facilitates Human Cytomegalovirus Replication by Promoting Capsid Nuclear Egress.

PubMed

Yang, Bo; Liu, Xi-Juan; Yao, Yongxuan; Jiang, Xuan; Wang, Xian-Zhang; Yang, Hong; Sun, Jin-Yan; Miao, Yun; Wang, Wei; Huang, Zhen-Li; Wang, Yanyi; Tang, Qiyi; Rayner, Simon; Britt, William J; McVoy, Michael A; Luo, Min-Hua; Zhao, Fei

2018-05-01

WD repeat-containing protein 5 (WDR5) is essential for assembling the VISA-associated complex to induce a type I interferon antiviral response to Sendai virus infection. However, the roles of WDR5 in DNA virus infections are not well described. Here, we report that human cytomegalovirus exploits WDR5 to facilitate capsid nuclear egress. Overexpression of WDR5 in fibroblasts slightly enhanced the infectious virus yield. However, WDR5 knockdown dramatically reduced infectious virus titers with only a small decrease in viral genome replication or gene expression. Further investigation of late steps of viral replication found that WDR5 knockdown significantly impaired formation of the viral nuclear egress complex and induced substantially fewer infoldings of the inner nuclear membrane. In addition, fewer capsids were associated with these infoldings, and there were fewer capsids in the cytoplasm. Restoration of WDR5 partially reversed these effects. These results suggest that WDR5 knockdown impairs the nuclear egress of capsids, which in turn decreases virus titers. These findings reveal an important role for a host factor whose function(s) is usurped by a viral pathogen to promote efficient replication. Thus, WDR5 represents an interesting regulatory mechanism and a potential antiviral target. IMPORTANCE Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 170 open reading frames and exploits numerous cellular factors to facilitate its replication. HCMV infection increases protein levels of WD repeat-containing protein 5 (WDR5) during infection, overexpression of WDR5 enhances viral replication, and knockdown of WDR5 dramatically attenuates viral replication. Our results indicate that WDR5 promotes the nuclear egress of viral capsids, the depletion of WDR5 resulting in a significant decrease in production of infectious virions. This is the first report that WDR5 favors HCMV, a DNA virus, replication and highlights a novel target for antiviral therapy. Copyright © 2018 American Society for Microbiology.

A Necessary Condition for Coexistence of Autocatalytic Replicators in a Prebiotic Environment

PubMed Central

Hernandez, Andres F.; Grover, Martha A.

2013-01-01

A necessary, but not sufficient, mathematical condition for the coexistence of short replicating species is presented here. The mathematical condition is obtained for a prebiotic environment, simulated as a fed-batch reactor, which combines monomer recycling, variable reaction order and a fixed monomer inlet flow with two replicator types and two monomer types. An extensive exploration of the parameter space in the model validates the robustness and efficiency of the mathematical condition, with nearly 1.7% of parameter sets meeting the condition and half of those exhibiting sustained coexistence. The results show that it is possible to generate a condition of coexistence, where two replicators sustain a linear growth simultaneously for a wide variety of chemistries, under an appropriate environment. The presence of multiple monomer types is critical to sustaining the coexistence of multiple replicator types. PMID:25369813

A necessary condition for coexistence of autocatalytic replicators in a prebiotic environment.

PubMed

Hernandez, Andres F; Grover, Martha A

2013-07-24

A necessary, but not sufficient, mathematical condition for the coexistence of short replicating species is presented here. The mathematical condition is obtained for a prebiotic environment, simulated as a fed-batch reactor, which combines monomer recycling, variable reaction order and a fixed monomer inlet flow with two replicator types and two monomer types. An extensive exploration of the parameter space in the model validates the robustness and efficiency of the mathematical condition, with nearly 1.7% of parameter sets meeting the condition and half of those exhibiting sustained coexistence. The results show that it is possible to generate a condition of coexistence, where two replicators sustain a linear growth simultaneously for a wide variety of chemistries, under an appropriate environment. The presence of multiple monomer types is critical to sustaining the coexistence of multiple replicator types.

Seeing and Being Green? The Effect of Money Priming on Willingness to Perform Sustainable Actions, Social Connectedness, and Prosociality.

PubMed

Capaldi, Colin A; Zelenski, John M

2016-01-01

This investigation attempted to conceptually replicate/extend research that suggests that reminders of money can inhibit prosociality, promote self-sufficiency, and influence political beliefs. Based on these results, we hypothesized that money primes would decrease willingness to engage in sustainable actions. We also predicted that people would distribute points less prosocially and feel less socially connected when money was primed. Individuals were recruited from an undergraduate participant pool and MTurk. Meta-analytic results across the two samples revealed that money priming did not have a significant impact on willingness to act sustainably, but it did cause participants to distribute points less prosocially and report lower social connectedness than individuals in the control condition. While effects were smaller than those reported in Vohs, Mead, and Goode (2006), this study still offers support for the detrimental impact of reminders of money on interpersonal relations.

Drosophila nuclear factor DREF regulates the expression of the mitochondrial DNA helicase and mitochondrial transcription factor B2 but not the mitochondrial translation factor B1

PubMed Central

Fernández-Moreno, Miguel A.; Hernández, Rosana; Adán, Cristina; Roberti, Marina; Bruni, Francesco; Polosa, Paola Loguercio; Cantatore, Palmiro; Matsushima, Yuichi; Kaguni, Laurie S.; Garesse, Rafael

2016-01-01

DREF [DRE (DNA replication-related element)-binding factor] controls the transcription of numerous genes in Drosophila, many involved in nuclear DNA (nDNA) replication and cell proliferation, three in mitochondrial DNA (mtDNA) replication and two in mtDNA transcription termination. In this work, we have analysed the involvement of DREF in the expression of the known remaining genes engaged in the minimal mtDNA replication (d-mtDNA helicase) and transcription (the activator d-mtTFB2) machineries and of a gene involved in mitochondrial mRNA translation (d-mtTFB1). We have identified their transcriptional initiation sites and DRE sequences in their promoter regions. Gel-shift and chromatin immunoprecipitation assays demonstrate that DREF interacts in vitro and in vivo with the d-mtDNA helicase and d-mtTFB2, but not with the d-mtTFB1 promoters. Transient transfection assays in Drosophila S2 cells with mutated DRE motifs and truncated promoter regions show that DREF controls the transcription of d-mtDNA helicase and d-mtTFB2, but not that of d-mtTFB1. RNA interference of DREF in S2 cells reinforces these results showing a decrease in the mRNA levels of d-mtDNA helicase and d-mtTFB2 and no changes in those of the d-mtTFB1. These results link the genetic regulation of nuclear DNA replication with the genetic control of mtDNA replication and transcriptional activation in Drosophila. PMID:23916463

The lytic origin of herpesvirus papio is highly homologous to Epstein-Barr virus ori-Lyt: evolutionary conservation of transcriptional activation and replication signals.

PubMed Central

Ryon, J J; Fixman, E D; Houchens, C; Zong, J; Lieberman, P M; Chang, Y N; Hayward, G S; Hayward, S D

1993-01-01

Herpesvirus papio (HVP) is a B-lymphotropic baboon virus with an estimated 40% homology to Epstein-Barr virus (EBV). We have cloned and sequenced ori-Lyt of herpesvirus papio and found a striking degree of nucleotide homology (89%) with ori-Lyt of EBV. Transcriptional elements form an integral part of EBV ori-Lyt. The promoter and enhancer domains of EBV ori-Lyt are conserved in herpesvirus papio. The EBV ori-Lyt promoter contains four binding sites for the EBV lytic cycle transactivator Zta, and the enhancer includes one Zta and two Rta response elements. All five of the Zta response elements and one of the Rta motifs are conserved in HVP ori-Lyt, and the HVP DS-L leftward promoter and the enhancer were activated in transient transfection assays by the EBV Zta and Rta transactivators. The EBV ori-Lyt enhancer contains a palindromic sequence, GGTCAGCTGACC, centered on a PvuII restriction site. This sequence, with a single base change, is also present in the HVP ori-Lyt enhancer. DNase I footprinting demonstrated that the PvuII sequence was bound by a protein present in a Raji nuclear extract. Mobility shift and competition assays using oligonucleotide probes identified this sequence as a binding site for the cellular transcription factor MLTF. Mutagenesis of the binding site indicated that MLTF contributes significantly to the constitutive activity of the ori-Lyt enhancer. The high degree of conservation of cis-acting signal sequences in HVP ori-Lyt was further emphasized by the finding that an HVP ori-Lyt-containing plasmid was replicated in Vero cells by a set of cotransfected EBV replication genes. The central domain of EBV ori-Lyt contains two related AT-rich palindromes, one of which is partially duplicated in the HVP sequence. The AT-rich palindromes are functionally important cis-acting motifs. Deletion of these palindromes severely diminished replication of an ori-Lyt target plasmid. Images PMID:8389916

FANCD2 limits replication stress and genome instability in cells lacking BRCA2

PubMed Central

Buffa, Francesca M.; McDermott, Ultan; Tarsounas, Madalena

2016-01-01

The tumor suppressor BRCA2 plays a key role in genome integrity by promoting replication fork stability and homologous recombination (HR) DNA repair. Here we report that human cancer cells lacking BRCA2 rely on the Fanconi anemia protein FANCD2 to limit replication fork progression and genomic instability. Our results identify a novel role for FANCD2 in limiting constitutive replication stress in BRCA2-deficient cells, which impacts on cell survival and treatment responses. PMID:27322732

Investigation of the Causes of Breast Cancer at the Cellular Level: Isolation of In Vivo Binding Sites of the Human Origin Recognition Complex

DTIC Science & Technology

2002-08-01

We study the process of DNA replication in proliferating human cells. Our efforts are directed to the identification and characterization of proteins...that promote DNA replication (initiators) as well as the DNA sequences recognized by them (replicators) . We have focused in a group of initiator...to be a critical factor for the coordination of DNA replication with the cell division cycle. hOrclp levels are higher between the exit of mitosis and

Metaresearch for Evaluating Reproducibility in Ecology and Evolution

PubMed Central

Fidler, Fiona; Chee, Yung En; Wintle, Bonnie C.; Burgman, Mark A.; McCarthy, Michael A.; Gordon, Ascelin

2017-01-01

Abstract Recent replication projects in other disciplines have uncovered disturbingly low levels of reproducibility, suggesting that those research literatures may contain unverifiable claims. The conditions contributing to irreproducibility in other disciplines are also present in ecology. These include a large discrepancy between the proportion of “positive” or “significant” results and the average statistical power of empirical research, incomplete reporting of sampling stopping rules and results, journal policies that discourage replication studies, and a prevailing publish-or-perish research culture that encourages questionable research practices. We argue that these conditions constitute sufficient reason to systematically evaluate the reproducibility of the evidence base in ecology and evolution. In some cases, the direct replication of ecological research is difficult because of strong temporal and spatial dependencies, so here, we propose metaresearch projects that will provide proxy measures of reproducibility. PMID:28596617

Leveraging Genomic Annotations and Pleiotropic Enrichment for Improved Replication Rates in Schizophrenia GWAS

PubMed Central

Wang, Yunpeng; Thompson, Wesley K.; Schork, Andrew J.; Holland, Dominic; Chen, Chi-Hua; Bettella, Francesco; Desikan, Rahul S.; Li, Wen; Witoelar, Aree; Zuber, Verena; Devor, Anna; Nöthen, Markus M.; Rietschel, Marcella; Chen, Qiang; Werge, Thomas; Cichon, Sven; Weinberger, Daniel R.; Djurovic, Srdjan; O’Donovan, Michael; Visscher, Peter M.; Andreassen, Ole A.; Dale, Anders M.

2016-01-01

Most of the genetic architecture of schizophrenia (SCZ) has not yet been identified. Here, we apply a novel statistical algorithm called Covariate-Modulated Mixture Modeling (CM3), which incorporates auxiliary information (heterozygosity, total linkage disequilibrium, genomic annotations, pleiotropy) for each single nucleotide polymorphism (SNP) to enable more accurate estimation of replication probabilities, conditional on the observed test statistic (“z-score”) of the SNP. We use a multiple logistic regression on z-scores to combine information from auxiliary information to derive a “relative enrichment score” for each SNP. For each stratum of these relative enrichment scores, we obtain nonparametric estimates of posterior expected test statistics and replication probabilities as a function of discovery z-scores, using a resampling-based approach that repeatedly and randomly partitions meta-analysis sub-studies into training and replication samples. We fit a scale mixture of two Gaussians model to each stratum, obtaining parameter estimates that minimize the sum of squared differences of the scale-mixture model with the stratified nonparametric estimates. We apply this approach to the recent genome-wide association study (GWAS) of SCZ (n = 82,315), obtaining a good fit between the model-based and observed effect sizes and replication probabilities. We observed that SNPs with low enrichment scores replicate with a lower probability than SNPs with high enrichment scores even when both they are genome-wide significant (p < 5x10-8). There were 693 and 219 independent loci with model-based replication rates ≥80% and ≥90%, respectively. Compared to analyses not incorporating relative enrichment scores, CM3 increased out-of-sample yield for SNPs that replicate at a given rate. This demonstrates that replication probabilities can be more accurately estimated using prior enrichment information with CM3. PMID:26808560

Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota

PubMed Central

Kirouac, Kevin N.; Ling, Hong

2011-01-01

The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι’s biological role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA containing an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equilibrium. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the additional O8 atom of the lesion base, which influences nucleotide selection, enzymatic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase. PMID:21300901

Oncolytic adenovirus that overproduces ADP and replicates selectively in tumors due to hTERT promoter-regulated E4 gene expression.

PubMed

Kuppuswamy, M; Spencer, J F; Doronin, K; Tollefson, A E; Wold, W S M; Toth, K

2005-11-01

We have constructed a novel oncolytic adenovirus (Ad) vector, named VRX-011, in which the replication of the vector is targeted to cancer cells by the replacement of the wild-type Ad E4 promoter with the human telomerase reverse transcriptase (hTERT) promoter. Genes in the Ad E4 transcription unit are essential for Ad replication; therefore, VRX-011 will grow efficiently only in cells in which the hTERT promoter is active, that is, in a wide range of cancer and immortalized cells but not in most somatic cells. Consistent with these expectations, VRX-011 replicated efficiently in all cancer cell lines examined, while its growth was restricted in various primary and normal cells. VRX-011 overexpresses ADP (also known as E3-11.6K), an Ad protein required for efficient cell lysis and release of virions from cells at late stages of infection. This overexpression enhances cell-to-cell spread and could significantly increase antitumor efficacy. In a xenograft model in nude mice, both intratumoral and intravenous administration of VRX-011 effectively suppressed the growth of subcutaneous Hep3B human liver tumors. Also, intravenous delivery of VRX-011 greatly reduced the number and size of A549 human lung cancer cell nodules in a disseminated lung tumor model in nude mice. Importantly, tail vein administration of different doses of VRX-011 in C57BL/6 mice showed minimal liver toxicity. Considering its broad range of lytic replication in cancer cells, its attenuated phenotype in primary cells, its efficacy in suppressing xenografts, and its low toxicity in mouse liver, VRX-011 is a promising candidate for further evaluation as an anticancer therapeutic.

Inhibition of adenovirus replication by the E1A antisense transcript initiated from hsp70 and VA-1 promoters.

PubMed

Miroshnichenko, O I; Borisenko, A S; Ponomareva, T I; Tikhonenko, T I

1990-03-01

The E1A region of the adenoviral genome, important for initiation of virus infection and activation of other viral genes, was chosen as a target for engineering antisense RNA (asRNA) to inhibit adenovirus 5 (Ad5) replication in COS-1 cell culture in vitro. The hsp70 promoter, taken from the appropriate heat-shock-protein gene of Drosophila melanogaster, and the VA-1 RNA promoter, derived from the Ad5 gene coding for low-molecular-mass VA-1 RNA and recognized by RNA polymerase III were used as regulatory elements of transcription. The two types of recombinant constructs contained E1A fragments of 710 bp (hsp70 constructs) or 380 or 740 bp (VA-1 RNA constructs) in reverse orientation relative to the promoter position, as well as a transcription termination signal, the SV40 ori, and the gene controlling Geneticin (antibiotic G418) resistance (G418R). After selection of transfected COS-1 cells in the presence of G418, a number of stable G418R cell lines were raised which expressed engineered asRNAs. Plating of Ad5 suspensions of known titre on monolayers of transfected COS-1 cells clearly showed strong inhibition of adenovirus replication by asRNAs: 75% with the hsp70 promoter and 90% with the VA-1 RNA promoter.

Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture.

PubMed

Komoto, Satoshi; Kanai, Yuta; Fukuda, Saori; Kugita, Masanori; Kawagishi, Takahiro; Ito, Naoto; Sugiyama, Makoto; Matsuura, Yoshiharu; Kobayashi, Takeshi; Taniguchi, Koki

2017-11-01

The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches. Copyright © 2017 American Society for Microbiology.

The von Hippel-Lindau (VHL) tumor-suppressor gene is down-regulated by selenium deficiency in Caco-2 cells and rat colon mucosa.

PubMed

Uthus, Eric; Begaye, Adrienne; Ross, Sharon; Zeng, Huawei

2011-08-01

To test the hypothesis that selenium affects DNA methylation and hence gene regulation, we employed a methylation array (Panomics) in the human colonic epithelial Caco-2 cell model. The array profiles DNA methylation from promoter regions of 82 human genes. After conditioning cells to repeatedly reduced concentrations of fetal bovine serum, a serum-free culture was established. Se-methylselenocysteine (SeMSC) was added at 0 (deficient Se) or 250 (control Se) nM to cells maintained in DMEM. After 7 days, cells were collected and stored at -80 °C until analysis; experiments were replicated three times. Glutathione peroxidase activity was significantly decreased in cells grown in low SeMSC. Cells grown in 250 nM SeMSC had maximal GPx activity. Genomic DNA from cells grown in the low-SeMSC media and media containing 250 nM SeMSC was incubated with methylation-binding protein followed by isolation of methylated DNA. The methylated DNA was labeled with biotin and hybridized to the methylation array. Thus, genes with promoter methylation will produce a higher chemiluminescence signal than those genes with no promoter methylation. Of the genes profiled, the von Hippel-Lindau (VHL) gene was most different as indicated by quantification following chemiluminescence detection demonstrating that the promoter region of VHL was hypermethylated in cells from the low-SeMSC media. To determine whether promoter methylation affected transcription, we isolated RNA from replicate samples and performed real-time RT PCR. VHL (mRNA) was down-regulated (fold change significantly <1) in cells grown in low SeMSC compared to cells grown in 250 nM SeMSC (control; fold change = 1). We also show that (mRNA) Vhl expression is significantly reduced in mucosa from rats fed a diet deficient in Se. Our results suggest that low Se status affects DNA promoter region methylation and that this can result in down-regulation of the tumor suppressor gene VHL.

The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage

PubMed Central

Fu, Haiqing; Martin, Melvenia M.; Regairaz, Marie; Huang, Liang; You, Yang; Lin, Chi-Mei; Ryan, Michael; Kim, RyangGuk; Shimura, Tsutomu; Pommier, Yves; Aladjem, Mirit I.

2015-01-01

The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81 deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81 deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins. PMID:25879486

TakeCARE, a Video to Promote Bystander Behavior on College Campuses: Replication and Extension.

PubMed

Jouriles, Ernest N; Sargent, Kelli S; Salis, Katie Lee; Caiozzo, Christina; Rosenfield, David; Cascardi, Michele; Grych, John H; O'Leary, K Daniel; McDonald, Renee

2017-08-01

Previous research has demonstrated that college students who view TakeCARE, a video bystander program designed to encourage students to take action to prevent sexual and relationship violence (i.e., bystander behavior), display more bystander behavior relative to students who view a control video. The current study aimed to replicate and extend these findings by testing two different methods of administering TakeCARE and examining moderators of TakeCARE's effects on bystander behavior. Students at four universities ( n = 557) were randomly assigned to one of three conditions: (a) view TakeCARE in a monitored computer lab, (b) view TakeCARE at their own convenience after receiving an email link to the video, or (c) view a video about study skills (control group). Participants completed measures of bystander behavior at baseline and at a 1-month follow-up. Participants in both TakeCARE conditions reported more bystander behavior at follow-up assessments, compared with participants in the control condition. The beneficial effect of TakeCARE did not differ significantly across administration methods. However, the effects of TakeCARE on bystander behavior were moderated by students' perceptions of campus responsiveness to sexual violence, with more potent effects when students perceived their institution as responsive to reports of sexual violence.

[Efficacy of mental health prevention and promotion strategies in higher education].

PubMed

Martineau, Marc; Beauchamp, Guy; Marcotte, Diane

Recent studies inform of increases in reported mental health problems in higher education students worldwide, with suicide and homicide being the most dramatic outcomes. Improving first-hand intervention and implementing mental health prevention and promotion strategies in colleges and universities are amongst the most commonly mentioned means of addressing these concerns. While institutions increasingly favor mental health promotion, most programs have not been properly evaluated and most strategies cannot be replicated. The article analyses results obtained from literature reviews and meta-analysis focusing on mental health prevention and promotion strategies targeting college and university students. Mindfulness, cognitive-behavioral and relaxation strategies, as well as social ability training, appeared to be the most effective if they were practiced under supervision. The implementation of supervised mental health prevention strategies within a setting-based systematic and multifactorial promotion frame could significantly decrease mental health problems in higher education students.

Identification of significant features by the Global Mean Rank test.

PubMed

Klammer, Martin; Dybowski, J Nikolaj; Hoffmann, Daniel; Schaab, Christoph

2014-01-01

With the introduction of omics-technologies such as transcriptomics and proteomics, numerous methods for the reliable identification of significantly regulated features (genes, proteins, etc.) have been developed. Experimental practice requires these tests to successfully deal with conditions such as small numbers of replicates, missing values, non-normally distributed expression levels, and non-identical distributions of features. With the MeanRank test we aimed at developing a test that performs robustly under these conditions, while favorably scaling with the number of replicates. The test proposed here is a global one-sample location test, which is based on the mean ranks across replicates, and internally estimates and controls the false discovery rate. Furthermore, missing data is accounted for without the need of imputation. In extensive simulations comparing MeanRank to other frequently used methods, we found that it performs well with small and large numbers of replicates, feature dependent variance between replicates, and variable regulation across features on simulation data and a recent two-color microarray spike-in dataset. The tests were then used to identify significant changes in the phosphoproteomes of cancer cells induced by the kinase inhibitors erlotinib and 3-MB-PP1 in two independently published mass spectrometry-based studies. MeanRank outperformed the other global rank-based methods applied in this study. Compared to the popular Significance Analysis of Microarrays and Linear Models for Microarray methods, MeanRank performed similar or better. Furthermore, MeanRank exhibits more consistent behavior regarding the degree of regulation and is robust against the choice of preprocessing methods. MeanRank does not require any imputation of missing values, is easy to understand, and yields results that are easy to interpret. The software implementing the algorithm is freely available for academic and commercial use.

DigOut: viewing differential expression genes as outliers.

PubMed

Yu, Hui; Tu, Kang; Xie, Lu; Li, Yuan-Yuan

2010-12-01

With regards to well-replicated two-conditional microarray datasets, the selection of differentially expressed (DE) genes is a well-studied computational topic, but for multi-conditional microarray datasets with limited or no replication, the same task is not properly addressed by previous studies. This paper adopts multivariate outlier analysis to analyze replication-lacking multi-conditional microarray datasets, finding that it performs significantly better than the widely used limit fold change (LFC) model in a simulated comparative experiment. Compared with the LFC model, the multivariate outlier analysis also demonstrates improved stability against sample variations in a series of manipulated real expression datasets. The reanalysis of a real non-replicated multi-conditional expression dataset series leads to satisfactory results. In conclusion, a multivariate outlier analysis algorithm, like DigOut, is particularly useful for selecting DE genes from non-replicated multi-conditional gene expression dataset.

Replication of a chronic hepatitis B virus genotype F1b construct.

PubMed

Hernández, Sergio; Jiménez, Gustavo; Alarcón, Valentina; Prieto, Cristian; Muñoz, Francisca; Riquelme, Constanza; Venegas, Mauricio; Brahm, Javier; Loyola, Alejandra; Villanueva, Rodrigo A

2016-03-01

Genotype F is one of the less-studied genotypes of human hepatitis B virus, although it is widely distributed in regions of Central and South American. Our previous studies have shown that HBV genotype F is prevalent in Chile, and phylogenetic analysis of its full-length sequence amplified from the sera of chronically infected patients identified it as HBV subgenotype F1b. We have previously reported the full-length sequence of a HBV molecular clone obtained from a patient chronically infected with genotype F1b. In this report, we established a system to study HBV replication based on hepatoma cell lines transfected with full-length monomers of the HBV genome. Culture supernatants were analyzed after transfection and found to contain both HBsAg and HBeAg viral antigens. Consistently, fractionated cell extracts revealed the presence of viral replication, with both cytoplasmic and nuclear DNA intermediates. Analysis of HBV-transfected cells by indirect immunofluorescence or immunoelectron microscopy revealed the expression of viral antigens and cytoplasmic viral particles, respectively. To test the functionality of the ongoing viral replication further at the level of chromatinized cccDNA, transfected cells were treated with a histone deacetylase inhibitor, and this resulted in increased viral replication. This correlated with changes posttranslational modifications of histones at viral promoters. Thus, the development of this viral replication system for HBV genotype F will facilitate studies on the regulation of viral replication and the identification of new antiviral drugs.

Mutant p53 perturbs DNA replication checkpoint control through TopBP1 and Treslin.

PubMed

Liu, Kang; Lin, Fang-Tsyr; Graves, Joshua D; Lee, Yu-Ju; Lin, Weei-Chin

2017-05-09

Accumulating evidence supports the gain-of-function of mutant forms of p53 (mutp53s). However, whether mutp53 directly perturbs the DNA replication checkpoint remains unclear. Previously, we have demonstrated that TopBP1 forms a complex with mutp53s and mediates their gain-of-function through NF-Y and p63/p73. Akt phosphorylates TopBP1 and induces its oligomerization, which inhibits its ATR-activating function. Here we show that various contact and conformational mutp53s bypass Akt to induce TopBP1 oligomerization and attenuate ATR checkpoint response during replication stress. The effect on ATR response caused by mutp53 can be exploited in a synthetic lethality strategy, as depletion of another ATR activator, DNA2, in mutp53-R273H-expressing cancer cells renders cells hypersensitive to cisplatin. Expression of mutp53-R273H also makes cancer cells more sensitive to DNA2 depletion or DNA2 inhibitors. In addition to ATR-activating function during replication stress, TopBP1 interacts with Treslin in a Cdk-dependent manner to initiate DNA replication during normal growth. We find that mutp53 also interferes with TopBP1 replication function. Several contact, but not conformational, mutp53s enhance the interaction between TopBP1 and Treslin and promote DNA replication despite the presence of a Cdk2 inhibitor. Together, these data uncover two distinct mechanisms by which mutp53 enhances DNA replication: ( i ) Both contact and conformational mutp53s can bind TopBP1 and attenuate the checkpoint response to replication stress, and ( ii ) during normal growth, contact (but not conformational) mutp53s can override the Cdk2 requirement to promote replication by facilitating the TopBP1/Treslin interaction.

Msh2-Msh3 Interferes with Okazaki Fragment Processing to Promote Trinucleotide Repeat Expansions

PubMed Central

Kantartzis, Athena; Williams, Gregory M.; Balakrishnan, Lata; Roberts, Rick L.; Surtees, Jennifer A.; Bambara, Robert A.

2012-01-01

Summary Trinucleotide repeat (TNR) expansions are the underlying cause of more than forty neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington’s disease. Although genetic evidence has attributed the cause of these diseases to errors in DNA replication and/or repair, clear molecular mechanisms have not been described. We have focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. We further provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki fragment processing by flap endonuclease1 (Rad27) and DNA Ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging strand DNA replication. PMID:22938864

Msh2-Msh3 interferes with Okazaki fragment processing to promote trinucleotide repeat expansions.

PubMed

Kantartzis, Athena; Williams, Gregory M; Balakrishnan, Lata; Roberts, Rick L; Surtees, Jennifer A; Bambara, Robert A

2012-08-30

Trinucleotide repeat (TNR) expansions are the underlying cause of more than 40 neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington's disease. Although genetic evidence points to errors in DNA replication and/or repair as the cause of these diseases, clear molecular mechanisms have not been described. Here, we focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. Furthermore, we provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki fragment processing by flap endonuclease1 (Rad27) and DNA ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging-strand DNA replication. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

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

PubMed

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.

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism

PubMed Central

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 MA; 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 AM; Simpson, Michael A; Cortez, David; Alkuraya, Fowzan S; Mathew, Christopher G; Jackson, Andrew P; Stewart, Grant S

2017-01-01

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 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 stabilises forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATR-dependent signalling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity, and potentiating chromosomal instability. Hypomorphic mutations substantially reduce DONSON protein levels and impair fork stability in patient cells, consistent with defective DNA replication underlying the disease phenotype. In summary, we identify mutations in DONSON as a common cause of microcephalic dwarfism, and establish DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability. PMID:28191891

Replication Stress: A Lifetime of Epigenetic Change

PubMed Central

Khurana, Simran; Oberdoerffer, Philipp

2015-01-01

DNA replication is essential for cell division. Challenges to the progression of DNA polymerase can result in replication stress, promoting the stalling and ultimately collapse of replication forks. The latter involves the formation of DNA double-strand breaks (DSBs) and has been linked to both genome instability and irreversible cell cycle arrest (senescence). Recent technological advances have elucidated many of the factors that contribute to the sensing and repair of stalled or broken replication forks. In addition to bona fide repair factors, these efforts highlight a range of chromatin-associated changes at and near sites of replication stress, suggesting defects in epigenome maintenance as a potential outcome of aberrant DNA replication. Here, we will summarize recent insight into replication stress-induced chromatin-reorganization and will speculate on possible adverse effects for gene expression, nuclear integrity and, ultimately, cell function. PMID:26378584

Assembly of Slx4 signaling complexes behind DNA replication forks.

PubMed

Balint, Attila; Kim, TaeHyung; Gallo, David; Cussiol, Jose Renato; Bastos de Oliveira, Francisco M; Yimit, Askar; Ou, Jiongwen; Nakato, Ryuichiro; Gurevich, Alexey; Shirahige, Katsuhiko; Smolka, Marcus B; Zhang, Zhaolei; Brown, Grant W

2015-08-13

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress. © 2015 The Authors.

Elongator complex is critical for cell cycle progression and leaf patterning in Arabidopsis.

PubMed

Xu, Deyang; Huang, Weihua; Li, Yang; Wang, Hua; Huang, Hai; Cui, Xiaofeng

2012-03-01

The mitotic cell cycle in higher eukaryotes is of pivotal importance for organ growth and development. Here, we report that Elongator, an evolutionarily conserved histone acetyltransferase complex, acts as an important regulator of mitotic cell cycle to promote leaf patterning in Arabidopsis. Mutations in genes encoding Elongator subunits resulted in aberrant cell cycle progression, and the altered cell division affects leaf polarity formation. The defective cell cycle progression is caused by aberrant DNA replication and increased DNA damage, which activate the DNA replication checkpoint to arrest the cell cycle. Elongator interacts with proliferating cell nuclear antigen (PCNA) and is required for efficient histone 3 (H3) and H4 acetylation coupled with DNA replication. Levels of chromatin-bound H3K56Ac and H4K5Ac known to associate with replicons during DNA replication were reduced in the mutants of both Elongator and chromatin assembly factor 1 (CAF-1), another protein complex that physically interacts with PCNA for DNA replication-coupled chromatin assembly. Disruptions of CAF-1 also led to severe leaf polarity defects, which indicated that Elongator and CAF-1 act, at least partially, in the same pathway to promote cell cycle progression. Collectively, our results demonstrate that Elongator is an important regulator of mitotic cell cycle, and the Elongator pathway plays critical roles in promoting leaf polarity formation. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Stress-induced premature senescence (SIPS)--influence of SIPS on radiotherapy.

PubMed

Suzuki, Masatoshi; Boothman, David A

2008-03-01

Replicative senescence is a fundamental feature in normal human diploid cells and results from dysfunctional telomeres at the Hayflick cell division limit. Ionizing radiation (IR) prematurely induces the same phenotypes as replicative senescence prior to the Hayflick limit. This process is known as stress-induced premature senescence (SIPS). Since the cell cycle is irreversibly arrested in SIPS-induced cells, even if they are stimulated by various growth factors, it is thought that SIPS is a form of cell death, irreversibly eliminating replicating cells. IR-induced-focus formation of DNA repair proteins, a marker of DNA damage, is detected in SIPS as well as replicative senescent cells. Furthermore, both processes persistently induce cell cycle checkpoint mechanisms, indicating DNA damage created by ionizing radiation induces SIPS in normal cells, possibly by the same mechanisms as those occurring in replicative senescence. Interestingly, IR induces SIPS not only in normal cells, but also in tumor cells. Due to the expression of telomerase in tumor cells, telomere-dependent replicative senescence does not occur. However, SIPS is induced under certain conditions after IR exposure. Thus, cell death triggered by IR can be attributed to apoptosis or SIPS in tumor cells. However, metabolic function remains intact in SIPS-induced cancer cells, and recent studies show that senescence eliminate cells undergoing SIPS secrete various kinds of factors outside the cell, changing the microenvironment. Evidence using co-culture systems containing normal senescent stromal cells and epithelial tumor cells show that factors secreted from senescent stroma cells promote the growth of tumor epithelial cells both in vitro and in vivo. Thus, regulation of factors secreted from SIPS-induced stromal cells, as well as tumor cells, may affect radiotherapy.

Replication of type 5 adenovirus promotes middle ear infection by Streptococcus pneumoniae in the chinchilla model of otitis media

PubMed Central

Murrah, Kyle A.; Turner, Roberta L.; Pang, Bing; Perez, Antonia C.; Reimche, Jennifer L.; King, Lauren B.; Wren, John; Gandhi, Uma; Swords, W. Edward; Ornelles, David A.

2015-01-01

Adenoviral infection is a major risk factor for otitis media. We hypothesized that adenovirus promotes bacterial ascension into the middle ear through the disruption of normal function in the Eustachian tubes due to inflammation-induced changes. An intranasal infection model of the chinchilla was used to test the ability of type 5 adenovirus to promote middle ear infection by Streptococcus pneumoniae. The hyperinflammatory adenovirus mutant dl327 and the nonreplicating adenovirus mutant H5wt300ΔpTP were used to test the role of inflammation and viral replication, respectively, in promotion of pneumococcal middle ear infection. Precedent infection with adenovirus resulted in a significantly greater incidence of middle ear disease by S. pneumoniae as compared to nonadenovirus infected animals. Infection with the adenovirus mutant dl327 induced a comparable degree of bacterial ascension into the middle ear as did infection with the wild-type virus. By contrast, infection with the nonreplicating adenovirus mutant H5wt300ΔpTP resulted in less extensive middle ear infection compared to the wild-type adenovirus. We conclude that viral replication is necessary for adenoviral-induced pneumococcal middle ear disease. PMID:25251686

Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante

Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteinsmore » retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. We suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.« less

Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

DOE PAGES

Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante; ...

2015-09-08

Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteinsmore » retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. We suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.« less

THE FORK AND THE KINASE: A DNA REPLICATION TALE FROM A CHK1 PERSPECTIVE

PubMed Central

González Besteiro, Marina A.; Gottifredi, Vanesa

2014-01-01

Replication fork progression is being continuously hampered by exogenously introduced and naturally occurring DNA lesions and other physical obstacles. The checkpoint kinase 1 (Chk1) is activated at replication forks that encounter damaged-DNA. Chk1 inhibits the initiation of new replication factories and stimulates the firing of dormant origins (those in the vicinity of stalled forks). Chk1 also avoids fork collapse into DSBs (double strand breaks) and promotes fork elongation. At the molecular level, the current model considers stalled forks as the site of Chk1 activation and the nucleoplasm as the location where Chk1 phosphorylates target proteins. This model certainly serves to explain how Chk1 modulates origin firing, but how Chk1 controls the fate of stalled forks is less clear. Interestingly, recent reports demonstrating that Chk1 phosphorylates chromatin-bound proteins and even holds kinase-independent functions might shed light on how Chk1 contributes to the elongation of damaged DNA. Such findings unveil a puzzling connection between Chk1 and DNA-lesion bypass, which might be central to promoting fork elongation and checkpoint attenuation. In summary, the multifaceted and versatile functions of Chk1 at ongoing forks and replication origins determine the extent and quality of the cellular response to replication stress. PMID:25795119

Common variants at the promoter region of the APOM confer a risk of rheumatoid arthritis

PubMed Central

Hu, Hae-Jin; Jin, Eun-Heui; Yim, Seon-Hee; Yang, So-Young; Jung, Seung-Hyun; Shin, Seung-Hun; Kim, Wan-Uk; Shim, Seung-Cheol; Kim, Tai-Gyu

2011-01-01

Although the genetic component in the etiology of rheumatoid arthritis (RA) has been consistently suggested, many novel genetic loci remain to uncover. To identify RA risk loci, we performed a genome-wide association study (GWAS) with 100 RA cases and 600 controls using Affymetrix SNP array 5.0. The candidate risk locus (APOM gene) was re-sequenced to discover novel promoter and coding variants in a group of the subjects. Replication was performed with the independent case-control set comprising of 578 RAs and 711 controls. Through GWAS, we identified a novel SNP associated with RA at the APOM gene in the MHC class III region on 6p21.33 (rs805297, odds ratio (OR) = 2.28, P = 5.20 × 10-7). Three more polymorphisms were identified at the promoter region of the APOM by the re-sequencing. For the replication, we genotyped the four SNP loci in the independent case-control set. The association of rs805297 identified by GWAS was successfully replicated (OR = 1.40, P = 6.65 × 10-5). The association became more significant in the combined analysis of discovery and replication sets (OR = 1.56, P = 2.73 ± 10-10). The individuals with the rs805297 risk allele (A) at the promoter region showed a significantly lower level of APOM expression compared with those with the protective allele (C) homozygote. In the logistic regressions by the phenotype status, the homozygote risk genotype (A/A) consistently showed higher ORs than the heterozygote one (A/C) for the phenotype-positive RAs. These results indicate that APOM promoter polymorphisms are significantly associated with the susceptibility to RA. PMID:21844665

Microscopic Observation of Self-Propagation of Calcifying Nanoparticles (Nanobacteria)

NASA Technical Reports Server (NTRS)

Mathew, Grace; McKay, David S.; Ciftcioglu, Neva

2007-01-01

Biologists typically define living organisms as carbon and water-based cellular forms with :self-replication" as the fundamental trait of the life process. However, this standard dictionary definition of life does not help scientists to categorize self-replicators like viruses, prions, proteons and artificial life. CNP also named nanobacteria were discovered in early 1990s as about 100 nanometer-sized bacteria-like particles with unique apatite mineral-shells around them, and found to be associated with pathological-calcification related diseases. Although CNP have been isolated and cultured from mammalian blood and diseased calcified tissues, and their biomineralizing properties well established, their biological nature and self-replicating capability have always been severely challenged. The terms "self-replication", "self-assembly" or "self-propagation" have been widely used for all systems including nanomachines, crystals, computer viruses and memes. In a simple taxonomy, all biological and non-biological "self replicators", have been classified into "living" or "nonliving" based on the properties of the systems and the amount of support they require to self-replicate. To enhance our understanding about self-replicating nature of CNP, we have investigated their growth in specific culture conditions using conventional inverted light microscope and BioStation IM, Nikon s latest time-lapse imaging system. Their morphological structure was examined using scanning (SEM) and transmission (TEM) electron microscopy. This present study, in conjunction with previous findings of metabolic activity, antibiotic sensitivity, antibody specificity, morphological aspects and infectivity, all concomitantly validate CNP as living self-replicators.

Zinc Finger-Containing Cellular Transcription Corepressor ZBTB25 Promotes Influenza Virus RNA Transcription and Is a Target for Zinc Ejector Drugs.

PubMed

Chen, Shu-Chuan; Jeng, King-Song; Lai, Michael M C

2017-10-15

Influenza A virus (IAV) replication relies on an intricate interaction between virus and host cells. How the cellular proteins are usurped for IAV replication remains largely obscure. The aim of this study was to search for novel and potential cellular factors that participate in IAV replication. ZBTB25, a transcription repressor of a variety of cellular genes, was identified by an RNA interference (RNAi) genomic library screen. Depletion of ZBTB25 significantly reduced IAV production. Conversely, overexpression of ZBTB25 enhanced it. ZBTB25 interacted with the viral RNA-dependent RNA polymerase (RdRp) protein and modulated its transcription activity. In addition, ZBTB25 also functioned as a viral RNA (vRNA)-binding protein, binding preferentially to the U-rich sequence within the 5' untranslated region (UTR) of vRNA. Both protein-protein and protein-RNA interactions involving ZBTB25 facilitated viral RNA transcription and replication. In addition, ZBTB25 suppressed interferon production, further enhancing viral replication. ZBTB25-associated functions required an intact zinc finger domain and posttranslational SUMO-1 modification of ZBTB25. Furthermore, treatment with disulfiram (a zinc ejector) of ZBTB25-overexpressing cells showed significantly reduced IAV production as a result of reduced RNA synthesis. Our findings indicate that IAV usurps ZBTB25 for IAV RNA synthesis and serves as a novel and potential therapeutic antiviral target. IMPORTANCE IAV-induced seasonal influenza causes severe illness and death in high-risk populations. However, IAV has developed resistance to current antiviral drugs due to its high mutation rate. Therefore, development of drugs targeting cellular factors required for IAV replication is an attractive alternative for IAV therapy. Here, we discovered a cellular protein, ZBTB25, that enhances viral RdRp activity by binding to both viral RdRp and viral RNA to stimulate viral RNA synthesis. A unique feature of ZBTB25 in the regulation of viral replication is its dual transcription functions, namely, promoting viral RNA transcription through binding to the U-rich region of vRNA and suppressing cellular interferon production. ZBTB25 contains a zinc finger domain that is required for RNA-inhibitory activity by chelating zinc ions. Disulfiram treatment disrupts the zinc finger functions, effectively repressing IAV replication. Based on our findings, we demonstrate that ZBTB25 regulates IAV RNA transcription and replication and serves as a promising antiviral target for IAV treatment. Copyright © 2017 American Society for Microbiology.

Zinc Finger-Containing Cellular Transcription Corepressor ZBTB25 Promotes Influenza Virus RNA Transcription and Is a Target for Zinc Ejector Drugs

PubMed Central

Chen, Shu-Chuan; Jeng, King-Song

2017-01-01

ABSTRACT Influenza A virus (IAV) replication relies on an intricate interaction between virus and host cells. How the cellular proteins are usurped for IAV replication remains largely obscure. The aim of this study was to search for novel and potential cellular factors that participate in IAV replication. ZBTB25, a transcription repressor of a variety of cellular genes, was identified by an RNA interference (RNAi) genomic library screen. Depletion of ZBTB25 significantly reduced IAV production. Conversely, overexpression of ZBTB25 enhanced it. ZBTB25 interacted with the viral RNA-dependent RNA polymerase (RdRp) protein and modulated its transcription activity. In addition, ZBTB25 also functioned as a viral RNA (vRNA)-binding protein, binding preferentially to the U-rich sequence within the 5′ untranslated region (UTR) of vRNA. Both protein-protein and protein-RNA interactions involving ZBTB25 facilitated viral RNA transcription and replication. In addition, ZBTB25 suppressed interferon production, further enhancing viral replication. ZBTB25-associated functions required an intact zinc finger domain and posttranslational SUMO-1 modification of ZBTB25. Furthermore, treatment with disulfiram (a zinc ejector) of ZBTB25-overexpressing cells showed significantly reduced IAV production as a result of reduced RNA synthesis. Our findings indicate that IAV usurps ZBTB25 for IAV RNA synthesis and serves as a novel and potential therapeutic antiviral target. IMPORTANCE IAV-induced seasonal influenza causes severe illness and death in high-risk populations. However, IAV has developed resistance to current antiviral drugs due to its high mutation rate. Therefore, development of drugs targeting cellular factors required for IAV replication is an attractive alternative for IAV therapy. Here, we discovered a cellular protein, ZBTB25, that enhances viral RdRp activity by binding to both viral RdRp and viral RNA to stimulate viral RNA synthesis. A unique feature of ZBTB25 in the regulation of viral replication is its dual transcription functions, namely, promoting viral RNA transcription through binding to the U-rich region of vRNA and suppressing cellular interferon production. ZBTB25 contains a zinc finger domain that is required for RNA-inhibitory activity by chelating zinc ions. Disulfiram treatment disrupts the zinc finger functions, effectively repressing IAV replication. Based on our findings, we demonstrate that ZBTB25 regulates IAV RNA transcription and replication and serves as a promising antiviral target for IAV treatment. PMID:28768860

Insulator protein Su(Hw) recruits SAGA and Brahma complexes and constitutes part of Origin Recognition Complex-binding sites in the Drosophila genome

PubMed Central

Vorobyeva, Nadezhda E.; Mazina, Marina U.; Golovnin, Anton K.; Kopytova, Daria V.; Gurskiy, Dmitriy Y.; Nabirochkina, Elena N.; Georgieva, Sofia G.; Georgiev, Pavel G.; Krasnov, Aleksey N.

2013-01-01

Despite increasing data on the properties of replication origins, molecular mechanisms underlying origin recognition complex (ORC) positioning in the genome are still poorly understood. The Su(Hw) protein accounts for the activity of best-studied Drosophila insulators. Here, we show that Su(Hw) recruits the histone acetyltransferase complex SAGA and chromatin remodeler Brahma to Su(Hw)-dependent insulators, which gives rise to regions with low nucleosome density and creates conditions for ORC binding. Depletion in Su(Hw) leads to a dramatic drop in the levels of SAGA, Brahma and ORC subunits and a significant increase in nucleosome density on Su(Hw)-dependent insulators, whereas artificial Su(Hw) recruitment itself is sufficient for subsequent SAGA, Brahma and ORC binding. In contrast to the majority of replication origins that associate with promoters of active genes, Su(Hw)-binding sites constitute a small proportion (6%) of ORC-binding sites that are localized preferentially in transcriptionally inactive chromatin regions termed BLACK and BLUE chromatin. We suggest that the key determinants of ORC positioning in the genome are DNA-binding proteins that constitute different DNA regulatory elements, including insulators, promoters and enhancers. Su(Hw) is the first example of such a protein. PMID:23609538

CDK1 promotes nascent DNA synthesis and induces resistance of cancer cells to DNA-damaging therapeutic agents

PubMed Central

Liao, Hongwei; Ji, Fang; Geng, Xinwei; Xing, Meichun; Li, Wen; Chen, Zhihua; Shen, Huahao; Ying, Songmin

2017-01-01

Cyclin dependent kinase 1 (CDK1) is essential for cell viability and plays a vital role in many biological events including cell cycle control, DNA damage repair, and checkpoint activation. Here, we identify an unanticipated role for CDK1 in promoting nascent DNA synthesis during S-phase. We report that a short duration of CDK1 inhibition, which does not perturb cell cycle progression, triggers a replication-associated DNA damage response (DDR). This DDR is associated with a disruption of replication fork progression and leads to genome instability. Moreover, we show that compromised CDK1 activity dramatically increases the efficacy of chemotherapeutic agents that kill cancer cells through perturbing DNA replication, including Olaparib, an FDA approved PARP inhibitor. Our study has revealed an important role for CDK1 in the DNA replication program, and suggests that the therapeutic targeting CDK1 may be a novel approach for combination chemotherapy. PMID:29207595

Focal adhesion kinase (FAK) regulates polymerase activity of multiple influenza A virus subtypes.

PubMed

Elbahesh, Husni; Bergmann, Silke; Russell, Charles J

2016-12-01

Influenza A viruses (IAVs) cause numerous pandemics and yearly epidemics resulting in ~500,000 annual deaths globally. IAV modulates cellular signaling pathways at every step of the infection cycle. Focal adhesion kinase (FAK) has been shown to play a critical role in endosomal trafficking of influenza A viruses, yet it is unclear how FAK kinase activity regulates IAV replication. Using mini-genomes derived from H1N1, H5N1 and H7N9 viruses, we dissected RNA replication by IAVs independent of viral entry or release. Our results show FAK activity promotes efficient IAV polymerase activity and inhibiting FAK activity with a chemical inhibitor or a kinase-dead mutant significantly reduces IAV polymerase activity. Using co-immunoprecipitations and proximity ligation assays, we observed interactions between FAK and the viral nucleoprotein, supporting a direct role of FAK in IAV replication. Altogether, the data indicates that FAK kinase activity is important in promoting IAV replication by regulating its polymerase activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX

PubMed Central

Clynes, David; Jelinska, Clare; Xella, Barbara; Ayyub, Helena; Scott, Caroline; Mitson, Matthew; Taylor, Stephen; Higgs, Douglas R.; Gibbons, Richard J.

2015-01-01

Fifteen per cent of cancers maintain telomere length independently of telomerase by the homologous recombination (HR)-associated alternative lengthening of telomeres (ALT) pathway. A unifying feature of these tumours are mutations in ATRX. Here we show that expression of ectopic ATRX triggers a suppression of the pathway and telomere shortening. Importantly ATRX-mediated ALT suppression is dependent on the histone chaperone DAXX. Re-expression of ATRX is associated with a reduction in replication fork stalling, a known trigger for HR and loss of MRN from telomeres. A G-quadruplex stabilizer partially reverses the effect of ATRX, inferring ATRX may normally facilitate replication through these sequences that, if they persist, promote ALT. We propose that defective telomere chromatinization through loss of ATRX promotes the persistence of aberrant DNA secondary structures, which in turn present a barrier to DNA replication, leading to replication fork stalling, collapse, HR and subsequent recombination-mediated telomere synthesis in ALT cancers. PMID:26143912

MYC-induced reprogramming of glutamine catabolism supports optimal virus replication

PubMed Central

Thai, Minh; Thaker, Shivani K.; Feng, Jun; Du, Yushen; Hu, Hailiang; Ting Wu, Ting; Graeber, Thomas G.; Braas, Daniel; Christofk, Heather R.

2015-01-01

Viruses rewire host cell glucose and glutamine metabolism to meet the bioenergetic and biosynthetic demands of viral propagation. However, the mechanism by which viruses reprogram glutamine metabolism and the metabolic fate of glutamine during adenovirus infection have remained elusive. Here, we show MYC activation is necessary for adenovirus-induced upregulation of host cell glutamine utilization and increased expression of glutamine transporters and glutamine catabolism enzymes. Adenovirus-induced MYC activation promotes increased glutamine uptake, increased use of glutamine in reductive carboxylation and increased use of glutamine in generating hexosamine pathway intermediates and specific amino acids. We identify glutaminase (GLS) as a critical enzyme for optimal adenovirus replication and demonstrate that GLS inhibition decreases replication of adenovirus, herpes simplex virus 1 and influenza A in cultured primary cells. Our findings show that adenovirus-induced reprogramming of glutamine metabolism through MYC activation promotes optimal progeny virion generation, and suggest that GLS inhibitors may be useful therapeutically to reduce replication of diverse viruses. PMID:26561297

Genome Cyclization as Strategy for Flavivirus RNA Replication

PubMed Central

Villordo, Sergio M.; Gamarnik, Andrea V.

2017-01-01

Long-range and local RNA-RNA contacts in viral RNA genomes result in tertiary structures that modulate the function of enhancers, promoters, and silencers during translation, RNA replication, and encapsidation. In the case of flaviviruses, the presence of inverted complementary sequences at the 5′ and 3′ ends of the genome mediate long-range RNA interactions and RNA cyclization. The circular conformation of flavivirus genomes was demonstrated to be essential for RNA amplification. New ideas about the mechanisms by which circular genomes participate in flavivirus replication have emerged in the last few years. Here, we will describe the latest information about cis-acting elements involved in flavivirus genome cyclization, RNA promoter elements required for viral polymerase recognition, and how these elements together coordinate viral RNA synthesis. PMID:18703097

Replication Fork Protection Factors Controlling R-Loop Bypass and Suppression.

PubMed

Chang, Emily Yun-Chia; Stirling, Peter C

2017-01-14

Replication-transcription conflicts have been a well-studied source of genome instability for many years and have frequently been linked to defects in RNA processing. However, recent characterization of replication fork-associated proteins has revealed that defects in fork protection can directly or indirectly stabilize R-loop structures in the genome and promote transcription-replication conflicts that lead to genome instability. Defects in essential DNA replication-associated activities like topoisomerase, or the minichromosome maintenance (MCM) helicase complex, as well as fork-associated protection factors like the Fanconi anemia pathway, both appear to mitigate transcription-replication conflicts. Here, we will highlight recent advances that support the concept that normal and robust replisome function itself is a key component of mitigating R-loop coupled genome instability.

Chromatin Constrains the Initiation and Elongation of DNA Replication.

PubMed

Devbhandari, Sujan; Jiang, Jieqing; Kumar, Charanya; Whitehouse, Iestyn; Remus, Dirk

2017-01-05

Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Role of Chemokines and Trafficking of Immune Cells in Parasitic Infections

PubMed Central

McGovern, Kathryn E.; Wilson, Emma H.

2014-01-01

Parasites are diverse eukaryotic pathogens that can have complex life cycles. Their clearance, or control within a mammalian host requires the coordinated effort of the immune system. The cell types recruited to areas of infection can combat the disease, promote parasite replication and survival, or contribute to disease pathology. Location and timing of cell recruitment can be crucial. In this review, we explore the role chemokines play in orchestrating and balancing the immune response to achieve optimal control of parasite replication without promoting pathology. PMID:25383073

Supersoft lithography: Candy-based fabrication of soft silicone microstructures

PubMed Central

Moraes, Christopher; Labuz, Joseph M.; Shao, Yue; Fu, Jianping; Takayama, Shuichi

2015-01-01

We designed a fabrication technique able to replicate microstructures in soft silicone materials (E < 1 kPa). Sugar-based ‘hard candy’ recipes from the confectionery industry were modified to be compatible with silicone processing conditions, and used as templates for replica molding. Microstructures fabricated in soft silicones can then be easily released by dissolving the template in water. We anticipate that this technique will be of particular importance in replicating physiologically soft, microstructured environments for cell culture, and demonstrate a first application in which intrinsically soft microstructures are used to measure forces generated by fibroblast-laden contractile tissues. PMID:26245893

Supersoft lithography: candy-based fabrication of soft silicone microstructures.

PubMed

Moraes, Christopher; Labuz, Joseph M; Shao, Yue; Fu, Jianping; Takayama, Shuichi

2015-01-01

We designed a fabrication technique able to replicate microstructures in soft silicone materials (E < 1 kPa). Sugar-based 'hard candy' recipes from the confectionery industry were modified to be compatible with silicone processing conditions, and used as templates for replica molding. Microstructures fabricated in soft silicones can then be easily released by dissolving the template in water. We anticipate that this technique will be of particular importance in replicating physiologically soft, microstructured environments for cell culture, and demonstrate a first application in which intrinsically soft microstructures are used to measure forces generated by fibroblast-laden contractile tissues.

Compartmentalized partnered replication for the directed evolution of genetic parts and circuits.

PubMed

Abil, Zhanar; Ellefson, Jared W; Gollihar, Jimmy D; Watkins, Ella; Ellington, Andrew D

2017-12-01

Compartmentalized partnered replication (CPR) is an emulsion-based directed evolution method based on a robust and modular phenotype-genotype linkage. In contrast to other in vivo directed evolution approaches, CPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPR is based on gene circuits in which the selection of a 'partner' function from a library leads to the production of a thermostable polymerase. After library preparation, bacteria produce partner proteins that can potentially lead to enhancement of transcription, translation, gene regulation, and other aspects of cellular metabolism that reinforce thermostable polymerase production. Individual cells are then trapped in water-in-oil emulsion droplets in the presence of primers and dNTPs, followed by the recovery of the partner genes via emulsion PCR. In this step, droplets with cells expressing partner proteins that promote polymerase production will produce higher copy numbers of the improved partner gene. The resulting partner genes can subsequently be recloned for the next round of selection. Here, we present a step-by-step guideline for the procedure by providing examples of (i) selection of T7 RNA polymerases that recognize orthogonal promoters and (ii) selection of tRNA for enhanced amber codon suppression. A single round of CPR should take ∼3-5 d, whereas a whole directed evolution can be performed in 3-10 rounds, depending on selection efficiency.

Regulation of early gene expression from the bovine papillomavirus genome in transiently transfected C127 cells.

PubMed Central

Szymanski, P; Stenlund, A

1991-01-01

Expression of bovine papillomavirus (BPV) early gene products is required for viral DNA replication and establishment of the transformed phenotype. By the use of a highly efficient electroporation system, we have examined for the first time the transcriptional activity of BPV promoters in their natural genomic context in a replication-permissive cell line. We have determined that a qualitatively distinct stage of transcription is not detectable prior to DNA replication in transiently transfected cells. This suggests that the transcriptional activity of the BPV genome in stably transformed cells represents the early stage of BPV gene expression. Quantitative differences in promoter activity between transiently transfected and stably transformed cells suggest that subtle changes in gene expression may control progression of the viral life cycle. Deletion analysis demonstrated that the E2 transactivator protein stimulates all of the early promoters through sequences located in the upstream regulatory region. This E2-dependent enhancer was found to be highly redundant, and particular E2 binding sites did not display a preference for particular promoters. Despite this dependence on a common cis-acting sequence, the various promoters displayed different sensitivities to the E2 transactivator. The findings that E2 regulates all promoters and, with the exception of the E2 repressors, that no other known viral gene product appears to affect transcription indicate that the E2 system functions as the master regulator of BPV early gene expression. Images PMID:1656065

An oncolytic adenovirus enhances antiangiogenic and antitumoral effects of a replication-deficient adenovirus encoding endostatin by rescuing its selective replication in nasopharyngeal carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ran-yi, E-mail: liuranyi@mail.sysu.edu.cn; Zhou, Ling; Zhang, Yan-ling

2013-12-13

Highlights: •H101 promotes endostatin expression by Ad-Endo via rescuing Ad-Endo replication. •H101 rescued Ad-Endo replication by supplying E1A and E1B19k proteins. •Ad-Endo enhanced the cytotoxicity of H101 in NPC cells. •Ad-Endo and oncolytic Ad H101 have synergistic antitumor effects on NPC. -- Abstract: A replication-deficient adenovirus (Ad) encoding secreted human endostatin (Ad-Endo) has been demonstrated to have promising antiangiogenic and antitumoral effects. The E1B55k-deleted Ad H101 can selectively lyse cancer cells. In this study, we explored the antitumor effects and cross-interactions of Ad-Endo and H101 on nasopharyngeal carcinoma (NPC). The results showed that H101 dramatically promoted endostatin expression by Ad-Endomore » via rescuing Ad-Endo replication in NPC cells, and the expressed endostatin proteins significantly inhibited the proliferation of human umbilical vein endothelial cells. E1A and E1B19k products are required for the rescuing of H101 to Ad-Endo replication in CNE-1 and CNE-2 cells, but not in C666-1 cells. On the other hand, Ad-Endo enhanced the cytotoxicity of H101 by enhancing Ad replication in NPC cells. The combination of H101 and Ad-Endo significantly inhibited CNE-2 xenografts growth through the increased endostatin expression and Ad replication. These findings indicate that the combination of Ad-Endo gene therapy and oncolytic Ad therapeutics could be promising in comprehensive treatment of NPC.« less

Fatty acid translocase promoted hepatitis B virus replication by upregulating the levels of hepatic cytosolic calcium.

PubMed

Huang, Jian; Zhao, Lei; Yang, Ping; Chen, Zhen; Ruan, Xiong Z; Huang, Ailong; Tang, Ni; Chen, Yaxi

2017-09-15

Hepatitis B virus (HBV) is designated a "metabolovirus" due to the intimate connection between the virus and host metabolism. The nutrition state of the host plays a relevant role in the severity of HBV infection. Metabolic syndrome (MS) is prone to increasing HBV DNA loads and accelerating the progression of liver disease in patients with chronic hepatitis B (CHB). Cluster of differentiation 36 (CD36), also named fatty acid translocase, is known to facilitate long-chain fatty acid uptake and contribute to the development of MS. We recently found that CD36 overexpression enhanced HBV replication. In this study, we further explored the mechanism by which CD36 overexpression promotes HBV replication. Our data showed that CD36 overexpression increased HBV replication, and CD36 knockdown inhibited HBV replication. RNA sequencing found some of the differentially expressed genes were involved in calcium ion homeostasis. CD36 overexpression elevated the cytosolic calcium level, and CD36 knockdown decreased the cytosolic calcium level. Calcium chelator BAPTA-AM could override the HBV replication increased by CD36 overexpression, and the calcium activator thapsigargin could improve the HBV replication reduced by CD36 knockdown. We further found that CD36 overexpression activated Src kinase, which plays an important role in the regulation of the store-operated Ca 2+ channel. An inhibitor of Src kinase (SU6656) significantly reduced the CD36-induced HBV replication. We identified a novel link between CD36 and HBV replication, which is associated with cytosolic calcium and the Src kinase pathway. CD36 may represent a potential therapeutic target for the treatment of CHB patients with MS. Copyright © 2017 Elsevier Inc. All rights reserved.

RPA binds histone H3-H4 and functions in DNA replication-coupled nucleosome assembly.

PubMed

Liu, Shaofeng; Xu, Zhiyun; Leng, He; Zheng, Pu; Yang, Jiayi; Chen, Kaifu; Feng, Jianxun; Li, Qing

2017-01-27

DNA replication-coupled nucleosome assembly is essential to maintain genome integrity and retain epigenetic information. Multiple involved histone chaperones have been identified, but how nucleosome assembly is coupled to DNA replication remains elusive. Here we show that replication protein A (RPA), an essential replisome component that binds single-stranded DNA, has a role in replication-coupled nucleosome assembly. RPA directly binds free H3-H4. Assays using a synthetic sequence that mimics freshly unwound single-stranded DNA at replication fork showed that RPA promotes DNA-(H3-H4) complex formation immediately adjacent to double-stranded DNA. Further, an RPA mutant defective in H3-H4 binding exhibited attenuated nucleosome assembly on nascent chromatin. Thus, we propose that RPA functions as a platform for targeting histone deposition to replication fork, through which RPA couples nucleosome assembly with ongoing DNA replication. Copyright © 2017, American Association for the Advancement of Science.

Kaposi's Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N6-Adenosine Methylation To Promote Lytic Replication

PubMed Central

Chen, E. Ricky; Nilsen, Timothy W.

2017-01-01

ABSTRACT N6-adenosine methylation (m6A) is the most common posttranscriptional RNA modification in mammalian cells. We found that most transcripts encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) genome undergo m6A modification. The levels of m6A-modified mRNAs increased substantially upon stimulation for lytic replication. The blockage of m6A inhibited splicing of the pre-mRNA encoding the replication transcription activator (RTA), a key KSHV lytic switch protein, and halted viral lytic replication. We identified several m6A sites in RTA pre-mRNA crucial for splicing through interactions with YTH domain containing 1 (YTHDC1), an m6A nuclear reader protein, in conjunction with serine/arginine-rich splicing factor 3 (SRSF3) and SRSF10. Interestingly, RTA induced m6A and enhanced its own pre-mRNA splicing. Our results not only demonstrate an essential role of m6A in regulating RTA pre-mRNA splicing but also suggest that KSHV has evolved a mechanism to manipulate the host m6A machinery to its advantage in promoting lytic replication. IMPORTANCE KSHV productive lytic replication plays a pivotal role in the initiation and progression of Kaposi's sarcoma tumors. Previous studies suggested that the KSHV switch from latency to lytic replication is primarily controlled at the chromatin level through histone and DNA modifications. The present work reports for the first time that KSHV genome-encoded mRNAs undergo m6A modification, which represents a new mechanism at the posttranscriptional level in the control of viral replication. PMID:28592530

Expression of microRNA let-7a positively correlates with hepatitis B virus replication in hepatocellular carcinoma tissues

PubMed Central

Chen, Jian; Liu, Jie; Luo, Zhongguang; Jiang, Weiru; Huang, Jianping; Qiu, Zhibing; Yue, Wenjie; Wu, Lijun

2017-01-01

Let-7a miRNA is downregulated in various cancers. However, in hepatocellular carcinoma (HCC) patients infected with hepatitis B virus (HBV), the relationship between let-7a and HBV replication has not been fully elucidated. Liver specimens were collected from 23 HCC patients with chronically active HBV. The serum levels of the HBV antigens hepatitis B surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg), and the HBV antibodies, anti-HBs, anti-HBe and anti-hepatitis B core antigen (anti-HBc) were measured using the microparticle enzyme immunoassay. Let-7a levels and HBV DNA copy numbers were measured by quantitative real-time PCR (qRT-PCR) and analyzed statistically. A let-7a specific antisense oligonucleotide was introduced to the HBV-producing cell line HepG2.2.15 and a change in HBV DNA copy numbers was assessed by qRT-PCR. HCC patients with highly active HBV replication (>106 DNA copies/mL) showed higher levels of serum HBsAg and anti-HBc than patients with less active HBV replication (<103 DNA copies/mL). The level of let-7a was lower in malignant tissues than in adjacent normal tissues. However, patients with highly active HBV replication demonstrated a significantly higher level of let-7a in hepatocarcinoma tissue than patients with less active HBV replication (P < 0.05). A higher level of let-7a was observed in the HBV-producing cell line HepG2.2.15 than in HepG2 cells (P < 0.05), and let-7a down-regulation by antisense oligonucleotides led to a reduction in HBV DNA copy numbers (P < 0.05), indicating a correlation between the let-7a level and HBV replication. Down-regulation of let-7a reduces HBV replication and could prevent the development of HCC, suggesting it could be an effective therapeutic treatment for HBV infection. Impact statement Although interferon and nucleic acid analogues effectively suppress HBV replication in HBV patients, there is no treatment which eradicates the virus. Moreover, the therapeutic effect can be reduced by virus mutations or drug resistance. Let-7a is a miRNA initially found in the nematode as a master regulator of developmental processes, but also exists in humans. It has been reported that the transcription of let-7a was much lower in HCC than in normal liver tissues and specific miRNA could directly promote virus replication. Therefore we hypothesized that transcription of let-7a promotes HBV replication, which might compromise the therapeutic effects of antivirus treatments. In our present study, we demonstrated a correlation between let-7a transcription and HBV replication in surgical specimens obtained from patients with HCC, as well as in HCC cell lines. Our finding might be the base for a new approach to improve HBV infection treatments in the future. PMID:28440732

Single-Case Experimental Designs to Evaluate Novel Technology-Based Health Interventions

PubMed Central

Cassidy, Rachel N; Raiff, Bethany R

2013-01-01

Technology-based interventions to promote health are expanding rapidly. Assessing the preliminary efficacy of these interventions can be achieved by employing single-case experiments (sometimes referred to as n-of-1 studies). Although single-case experiments are often misunderstood, they offer excellent solutions to address the challenges associated with testing new technology-based interventions. This paper provides an introduction to single-case techniques and highlights advances in developing and evaluating single-case experiments, which help ensure that treatment outcomes are reliable, replicable, and generalizable. These advances include quality control standards, heuristics to guide visual analysis of time-series data, effect size calculations, and statistical analyses. They also include experimental designs to isolate the active elements in a treatment package and to assess the mechanisms of behavior change. The paper concludes with a discussion of issues related to the generality of findings derived from single-case research and how generality can be established through replication and through analysis of behavioral mechanisms. PMID:23399668

Association of an APOC3 promoter variant with type 2 diabetes risk and need for insulin treatment in lean persons.

PubMed

van Hoek, M; van Herpt, T W; Dehghan, A; Hofman, A; Lieverse, A G; van Duijn, C M; Witteman, J C M; Sijbrands, E J G

2011-06-01

An APOC3 promoter haplotype has been previously associated with type 1 diabetes. In this population-based study, we investigated whether APOC3 polymorphisms increase type 2 diabetes risk and need for insulin treatment in lean participants. In the Rotterdam Study, a population-based prospective cohort (n = 7,983), Cox and logistic regression models were used to analyse the associations and interactive effects of APOC3 promoter variants (-482C > T, -455T > C) and BMI on type 2 diabetes risk and insulin treatment. Analyses were followed by replication in an independent case-control sample (1,817 cases, 2,292 controls) and meta-analysis. In lean participants, the -482T allele was associated with increased risk of prevalent and incident type 2 diabetes: OR -482CT 1.47 (95% CI 1.13-1.92), -482TT 1.40 (95% CI 0.83-2.35), p = 0.009 for trend; HR -482CT 1.35 (95% CI 0.96-1.89), -482TT 1.68 (95% CI 0.91-3.1), p = 0.03 for trend, respectively. These results were confirmed by replication. Meta-analysis was highly significant (-482T meta-analysis p = 1.1 × 10(-4)). A borderline significant interaction was observed for insulin use among participants with type 2 diabetes (-482CT*BMI p = 0.06, -455TC*BMI p = 0.02). At a population-based level, the influence of APOC3 promoter variants on type 2 diabetes risk varies with the level of adiposity. Lean carriers of the -482T allele had increased type 2 diabetes risk, while such an effect was not observed in overweight participants. Conversely, in overweight participants the -455C allele seemed protective against type 2 diabetes. The interaction of the variants with need for insulin treatment may indicate beta cell involvement in lean participants. Our findings suggest overlap in the genetic backgrounds of type 1 diabetes and type 2 diabetes in lean patients.

Bean Yellow Dwarf Virus replicons for high-level transgene expression in transgenic plants and cell cultures.

PubMed

Zhang, Xiuren; Mason, Hugh

2006-02-05

A novel stable transgenic plant expression system was developed using elements of the replication machinery of Bean Yellow Dwarf Virus (BeYDV). The system contains two transgenes: 1) The BeYDV replicon vector with an expression cassette flanked by cis-acting DNA elements of BeYDV, and 2) The viral replication initiator protein (Rep) controlled by an alcohol-inducible promoter. When Rep expression was triggered by treatment with ethanol, it induced release of the BeYDV replicon from stably integrated T-DNA and episomal replication to high copy number. Replicon amplification resulted in substantially increased transgene mRNA levels (up to 80-fold) and translation products (up to 10-fold) after induction of Rep expression by ethanol treatment in tobacco NT1 cells and leaves of whole potato plants. Thus, the BeYDV stable transformant replicon system is a powerful tool for plant-based production of recombinant proteins. (c) 2005 Wiley Periodicals, Inc.

Continuous in vitro evolution of bacteriophage RNA polymerase promoters

NASA Technical Reports Server (NTRS)

Breaker, R. R.; Banerji, A.; Joyce, G. F.

1994-01-01

Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of both RNA and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts. Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.

Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

PubMed

Xin, Qi-Lin; Deng, Cheng-Lin; Chen, Xi; Wang, Jun; Wang, Shao-Bo; Wang, Wei; Deng, Fei; Zhang, Bo; Xiao, Gengfu; Zhang, Lei-Ke

2017-06-15

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate antiviral responses. Identification of host proteins involved in the ZIKV replication process may lead to the discovery of antiviral targets. In this study, the first quantitative proteomic analysis of ZIKV-infected cells was performed to investigate host proteins involved in the ZIKV replication process. Bioinformatics analysis highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the UPS, bortezomib, can inhibit ZIKV infection in vivo Our study not only illustrated how host cells respond to ZIKV infection but also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. Copyright © 2017 American Society for Microbiology.

Benchmarking of a T-wave alternans detection method based on empirical mode decomposition.

PubMed

Blanco-Velasco, Manuel; Goya-Esteban, Rebeca; Cruz-Roldán, Fernando; García-Alberola, Arcadi; Rojo-Álvarez, José Luis

2017-07-01

T-wave alternans (TWA) is a fluctuation of the ST-T complex occurring on an every-other-beat basis of the surface electrocardiogram (ECG). It has been shown to be an informative risk stratifier for sudden cardiac death, though the lack of gold standard to benchmark detection methods has promoted the use of synthetic signals. This work proposes a novel signal model to study the performance of a TWA detection. Additionally, the methodological validation of a denoising technique based on empirical mode decomposition (EMD), which is used here along with the spectral method, is also tackled. The proposed test bed system is based on the following guidelines: (1) use of open source databases to enable experimental replication; (2) use of real ECG signals and physiological noise; (3) inclusion of randomized TWA episodes. Both sensitivity (Se) and specificity (Sp) are separately analyzed. Also a nonparametric hypothesis test, based on Bootstrap resampling, is used to determine whether the presence of the EMD block actually improves the performance. The results show an outstanding specificity when the EMD block is used, even in very noisy conditions (0.96 compared to 0.72 for SNR = 8 dB), being always superior than that of the conventional SM alone. Regarding the sensitivity, using the EMD method also outperforms in noisy conditions (0.57 compared to 0.46 for SNR=8 dB), while it decreases in noiseless conditions. The proposed test setting designed to analyze the performance guarantees that the actual physiological variability of the cardiac system is reproduced. The use of the EMD-based block in noisy environment enables the identification of most patients with fatal arrhythmias. Copyright © 2017 Elsevier B.V. All rights reserved.

RecQL4 is required for the association of Mcm10 and Ctf4 with replication origins in human cells

PubMed Central

Im, Jun-Sub; Park, Soon-Young; Cho, Won-Ho; Bae, Sung-Ho; Hurwitz, Jerard; Lee, Joon-Kyu

2015-01-01

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells. PMID:25602958

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism.

PubMed

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.

Human FAN1 promotes strand incision in 5'-flapped DNA complexed with RPA.

PubMed

Takahashi, Daisuke; Sato, Koichi; Hirayama, Emiko; Takata, Minoru; Kurumizaka, Hitoshi

2015-09-01

Fanconi anaemia (FA) is a human infantile recessive disorder. Seventeen FA causal proteins cooperatively function in the DNA interstrand crosslink (ICL) repair pathway. Dual DNA strand incisions around the crosslink are critical steps in ICL repair. FA-associated nuclease 1 (FAN1) is a DNA structure-specific endonuclease that is considered to be involved in DNA incision at the stalled replication fork. Replication protein A (RPA) rapidly assembles on the single-stranded DNA region of the stalled fork. However, the effect of RPA on the FAN1-mediated DNA incision has not been determined. In this study, we purified human FAN1, as a bacterially expressed recombinant protein. FAN1 exhibited robust endonuclease activity with 5'-flapped DNA, which is formed at the stalled replication fork. We found that FAN1 efficiently promoted DNA incision at the proper site of RPA-coated 5'-flapped DNA. Therefore, FAN1 possesses the ability to promote the ICL repair of 5'-flapped DNA covered by RPA. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

CLB5-dependent activation of late replication origins in S. cerevisiae.

PubMed

Donaldson, A D; Raghuraman, M K; Friedman, K L; Cross, F R; Brewer, B J; Fangman, W L

1998-08-01

Replication origins in chromosomes are activated at specific times during the S phase. We show that the B-type cyclins are required for proper execution of this temporal program. clb5 cells activate early origins but not late origins, explaining the previously described long clb5 S phase. Origin firing appears normal in cIb6 mutants. In clb5 clb6 double mutant cells, the late origin firing defect is suppressed, accounting for the normal duration of the phase despite its delayed onset. Therefore, Clb5p promotes the timely activation of early and late origins, but Clb6p can activate only early origins. In clb5 clb6 mutants, the other B-type cyclins (Clb1-4p) promote an S phase during which both early and late replication origins fire.

The fork and the kinase: a DNA replication tale from a CHK1 perspective.

PubMed

González Besteiro, Marina A; Gottifredi, Vanesa

2015-01-01

Replication fork progression is being continuously hampered by exogenously introduced and naturally occurring DNA lesions and other physical obstacles. Checkpoint kinase 1 (Chk1) is activated at replication forks that encounter damaged DNA. Subsequently, Chk1 inhibits the initiation of new replication factories and stimulates the firing of dormant origins (those in the vicinity of stalled forks). Chk1 also avoids fork collapse into DSBs (double strand breaks) and promotes fork elongation. At the molecular level, the current model considers stalled forks as the site of Chk1 activation and the nucleoplasm as the location where Chk1 phosphorylates target proteins. This model certainly serves to explain how Chk1 modulates origin firing, but how Chk1 controls the fate of stalled forks is less clear. Interestingly, recent reports demonstrating that Chk1 phosphorylates chromatin-bound proteins and even holds kinase-independent functions might shed light on how Chk1 contributes to the elongation of damaged DNA. Indeed, such findings have unveiled a puzzling connection between Chk1 and DNA lesion bypass, which might be central to promoting fork elongation and checkpoint attenuation. In summary, Chk1 is a multifaceted and versatile signaling factor that acts at ongoing forks and replication origins to determine the extent and quality of the cellular response to replication stress. Copyright © 2014 Elsevier B.V. All rights reserved.

Laser controlled singlet oxygen generation in mitochondria to promote mitochondrial DNA replication in vitro.

PubMed

Zhou, Xin; Wang, Yupei; Si, Jing; Zhou, Rong; Gan, Lu; Di, Cuixia; Xie, Yi; Zhang, Hong

2015-11-18

Reports have shown that a certain level of reactive oxygen species (ROS) can promote mitochondrial DNA (mtDNA) replication. However, it is unclear whether it is the mitochondrial ROS that stimulate mtDNA replication and this requires further investigation. Here we employed a photodynamic system to achieve controlled mitochondrial singlet oxygen ((1)O2) generation. HeLa cells incubated with 5-aminolevulinic acid (ALA) were exposed to laser irradiation to induce (1)O2 generation within mitochondria. Increased mtDNA copy number was detected after low doses of 630 nm laser light in ALA-treated cells. The stimulated mtDNA replication was directly linked to mitochondrial (1)O2 generation, as verified using specific ROS scavengers. The stimulated mtDNA replication was regulated by mitochondrial transcription factor A (TFAM) and mtDNA polymerase γ. MtDNA control region modifications were induced by (1)O2 generation in mitochondria. A marked increase in 8-Oxoguanine (8-oxoG) level was detected in ALA-treated cells after irradiation. HeLa cell growth stimulation and G1-S cell cycle transition were also observed after laser irradiation in ALA-treated cells. These cellular responses could be due to a second wave of ROS generation detected in mitochondria. In summary, we describe a controllable method of inducing mtDNA replication in vitro.

The FANC pathway is activated by adenovirus infection and promotes viral replication-dependent recombination

PubMed Central

Cherubini, Gioia; Naim, Valeria; Caruso, Paola; Burla, Romina; Bogliolo, Massimo; Cundari, Enrico; Benihoud, Karim; Saggio, Isabella; Rosselli, Filippo

2011-01-01

Deciphering the crosstalk between a host cell and a virus during infection is important not only to better define viral biology but also to improve our understanding of cellular processes. We identified the FANC pathway as a helper of viral replication and recombination by searching for cellular targets that are modified by adenovirus (Ad) infection and are involved in its outcome. This pathway, which is involved in the DNA damage response and checkpoint control, is altered in Fanconi anaemia, a rare cancer predisposition syndrome. We show here that Ad5 infection activates the FANC pathway independent of the classical DNA damage response. Infection with a non-replicating Ad shows that the presence of viral DNA is not sufficient to induce the monoubiquitination of FANCD2 but still activates the DNA damage response coordinated by phospho-NBS1 and phospho-CHK1. E1A expression alone fails to induce FANCD2 monoubiquitination, indicating that a productive viral infection and/or replication is required for FANC pathway activation. Our data indicate that Ad5 infection induces FANCD2 activation to promote its own replication. Specifically, we show that FANCD2 is involved in the recombination process that accompanies viral DNA replication. This study provides evidence of a DNA damage-independent function of the FANC pathway and identifies a cellular system involved in Ad5 recombination. PMID:21421559

Genomic amplification of the human DHFR/MSH3 locus remodels mismatch recognition and repair activities.

PubMed

Drummond, J T

1999-01-01

Mismatch recognition in human cells is mediated by two heterodimers, MutS alpha and MutS beta. MutS alpha appears to shoulder primary responsibility for mismatch correction during replication, based on its relative abundance and ability to recognize a broad spectrum of base-base and base-insertion mismatches. Because MutS alpha and MutS beta share a common component, MSH2, conditions that influence the expression or degradation of MSH3 or MSH6 can redistribute the profile of mismatch recognition and repair. MSH3 is linked by a shared promoter with DHFR, connecting two pathways with key roles in DNA metabolism. In a classic example of gene amplification, the DHFR (and MSH3) locus can become amplified to several hundred copies in the presence of methotrexate. Under these conditions, MutS beta forms at the expense of MutS alpha, and the mutation rate in these tumor cells rises more than 100-fold. The implications for cancer chemotherapy include a potential increase in mutability when tumors are treated with methotrexate, which could increase the frequency of subsequent mutations that influence the tumor's drug sensitivity or aggressiveness. Because processing certain types of DNA damage by the mismatch repair pathway has also been implicated in tumor sensitivity to agents such as cisplatin, changes in expression at the DHFR/MSH3 locus may have further relevance to the outcome of multi-drug treatment regimens.

Thermodynamic Basis for the Emergence of Genomes during Prebiotic Evolution

PubMed Central

Woo, Hyung-June; Vijaya Satya, Ravi; Reifman, Jaques

2012-01-01

The RNA world hypothesis views modern organisms as descendants of RNA molecules. The earliest RNA molecules must have been random sequences, from which the first genomes that coded for polymerase ribozymes emerged. The quasispecies theory by Eigen predicts the existence of an error threshold limiting genomic stability during such transitions, but does not address the spontaneity of changes. Following a recent theoretical approach, we applied the quasispecies theory combined with kinetic/thermodynamic descriptions of RNA replication to analyze the collective behavior of RNA replicators based on known experimental kinetics data. We find that, with increasing fidelity (relative rate of base-extension for Watson-Crick versus mismatched base pairs), replications without enzymes, with ribozymes, and with protein-based polymerases are above, near, and below a critical point, respectively. The prebiotic evolution therefore must have crossed this critical region. Over large regions of the phase diagram, fitness increases with increasing fidelity, biasing random drifts in sequence space toward ‘crystallization.’ This region encloses the experimental nonenzymatic fidelity value, favoring evolutions toward polymerase sequences with ever higher fidelity, despite error rates above the error catastrophe threshold. Our work shows that experimentally characterized kinetics and thermodynamics of RNA replication allow us to determine the physicochemical conditions required for the spontaneous crystallization of biological information. Our findings also suggest that among many potential oligomers capable of templated replication, RNAs may have evolved to form prebiotic genomes due to the value of their nonenzymatic fidelity. PMID:22693440

Helicase promotes replication re-initiation from an RNA transcript.

PubMed

Sun, Bo; Singh, Anupam; Sultana, Shemaila; Inman, James T; Patel, Smita S; Wang, Michelle D

2018-06-13

To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. After encountering an obstacle, a progressing replisome often aborts DNA synthesis but continues to unwind. However, little is known about how DNA synthesis is resumed downstream of an obstacle. Here, we examine the consequences of a non-replicating replisome collision with a co-directional RNA polymerase (RNAP). Using single-molecule and ensemble methods, we find that T7 helicase interacts strongly with a non-replicating T7 DNA polymerase (DNAP) at a replication fork. As the helicase advances, the associated DNAP also moves forward. The presence of the DNAP increases both helicase's processivity and unwinding rate. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. These observations exhibit T7 helicase's novel role in replication re-initiation.

Getting it done at the ends: Pif1 family DNA helicases and telomeres.

PubMed

Geronimo, Carly L; Zakian, Virginia A

2016-08-01

It is widely appreciated that the ends of linear DNA molecules cannot be fully replicated by the conventional replication apparatus. Less well known is that semi-conservative replication of telomeric DNA also presents problems for DNA replication. These problems likely arise from the atypical chromatin structure of telomeres, the GC-richness of telomeric DNA that makes it prone to forming DNA secondary structures, and from RNA-DNA hybrids, formed by transcripts of one or both DNA strands. Given the different aspects of telomeres that complicate their replication, it is not surprising that multiple DNA helicases promote replication of telomeric DNA. This review focuses on one such class of DNA helicases, the Pif1 family of 5'-3' DNA helicases. In budding and fission yeasts, Pif1 family helicases impact both telomerase-mediated and semi-conservative replication of telomeric DNA as well as recombination-mediated telomere lengthening. Copyright © 2016. Published by Elsevier B.V.

Getting it done at the ends: Pif1 family DNA helicases and telomeres

PubMed Central

Geronimo, Carly L.; Zakian, Virginia A.

2017-01-01

It is widely appreciated that the ends of linear DNA molecules cannot be fully replicated by the conventional replication apparatus. Less well known is that semi-conservative replication of telomeric DNA also presents problems for DNA replication. These problems likely arise from the atypical chromatin structure of telomeres, the GC-richness of telomeric DNA that makes it prone to forming DNA secondary structures, and from RNA-DNA hybrids, formed by transcripts of one or both DNA strands. Given the different aspects of telomeres that complicate their replication, it is not surprising that multiple DNA helicases promote replication of telomeric DNA. This review focuses on one such class of DNA helicases, the Pif1 family of 5′–3′ DNA helicases. In budding and fission yeasts, Pif1 family helicases impact both telomerase-mediated and semi-conservative replication of telomeric DNA as well as recombination-mediated telomere lengthening. PMID:27233114

Strand displacement amplification as an in vitro model for rolling-circle replication: deletion formation and evolution during serial transfer.

PubMed Central

Walter, N G; Strunk, G

1994-01-01

Strand displacement amplification is an isothermal DNA amplification reaction based on a restriction endonuclease nicking its recognition site and a polymerase extending the nick at its 3' end, displacing the downstream strand. The reaction resembles rolling-circle replication of single-stranded phages and small plasmids. The displaced sense strand serves as target for an antisense reaction and vice versa, resulting in exponential growth and the autocatalytic nature of this in vitro reaction as long as the template is the limiting agent. We describe the optimization of strand displacement amplification for in vitro evolution experiments under serial transfer conditions. The reaction was followed and controlled by use of the fluorescent dye thiazole orange binding to the amplified DNA. We were able to maintain exponential growth conditions with a doubling time of 3.0 min throughout 100 transfers or approximately 350 molecular generations by using an automatic handling device. Homology of in vitro amplification with rolling-circle replication was mirrored by the occurring evolutionary processes. Deletion events most likely caused by a slipped mispairing mechanism as postulated for in vivo replication took place. Under our conditions, the mutation rate was high and a molecular quasi-species formed with a mutant lacking internal hairpin formation ability and thus outgrowing all other species under dGTP/dCTP deficiency. Images PMID:8058737

Pregnancy Research on Osteopathic Manipulation Optimizing Treatment Effects: The PROMOTE Study Protocol.

PubMed

Hensel, Kendi L; Carnes, Michael S; Stoll, Scott T

2016-11-01

The structural and physiologic changes in a woman's body during pregnancy can predispose pregnant women to low back pain and its associated disability, as well as to complications of pregnancy, labor, and delivery. Anecdotal and empirical evidence has indicated that osteopathic manipulative treatment (OMT) may be efficacious in improving pain and functionality in women who are pregnant. Based on that premise, the Pregnancy Research on Osteopathic Manipulation Optimizing Treatment Effects (PROMOTE) study was designed as a prospective, randomized, placebo-controlled, and blinded clinical trial to evaluate the efficacy of an OMT protocol for pain during third-trimester pregnancy. The OMT protocol developed for the PROMOTE study was based on physiologic theory and the concept of the interrelationship of structure and function. The 12 well-defined, standardized OMT techniques used in the protocol are commonly taught at osteopathic medical schools in the United States. These techniques can be easily replicated as a 20-minute protocol applied in conjunction with usual prenatal care, thus making it feasible to implement into clinical practice. This article presents an overview of the study design and treatment protocols used in the PROMOTE study.

Host Pah1p phosphatidate phosphatase limits viral replication by regulating phospholipid synthesis

PubMed Central

Zhang, Zhenlu; He, Guijuan; Catanzaro, Nicholas; Wu, Zujian; Xie, Lianhui

2018-01-01

Replication of positive-strand RNA viruses [(+)RNA viruses] takes place in membrane-bound viral replication complexes (VRCs). Formation of VRCs requires virus-mediated manipulation of cellular lipid synthesis. Here, we report significantly enhanced brome mosaic virus (BMV) replication and much improved cell growth in yeast cells lacking PAH1 (pah1Δ), the sole yeast ortholog of human LIPIN genes. PAH1 encodes Pah1p (phosphatidic acid phosphohydrolase), which converts phosphatidate (PA) to diacylglycerol that is subsequently used for the synthesis of the storage lipid triacylglycerol. Inactivation of Pah1p leads to altered lipid composition, including high levels of PA, total phospholipids, ergosterol ester, and free fatty acids, as well as expansion of the nuclear membrane. In pah1Δ cells, BMV replication protein 1a and double-stranded RNA localized to the extended nuclear membrane, there was a significant increase in the number of VRCs formed, and BMV genomic replication increased by 2-fold compared to wild-type cells. In another yeast mutant that lacks both PAH1 and DGK1 (encodes diacylglycerol kinase converting diacylglycerol to PA), which has a normal nuclear membrane but maintains similar lipid compositional changes as in pah1Δ cells, BMV replicated as efficiently as in pah1Δ cells, suggesting that the altered lipid composition was responsible for the enhanced BMV replication. We further showed that increased levels of total phospholipids play an important role because the enhanced BMV replication required active synthesis of phosphatidylcholine, the major membrane phospholipid. Moreover, overexpression of a phosphatidylcholine synthesis gene (CHO2) promoted BMV replication. Conversely, overexpression of PAH1 or plant PAH1 orthologs inhibited BMV replication in yeast or Nicotiana benthamiana plants. Competing with its host for limited resources, BMV inhibited host growth, which was markedly alleviated in pah1Δ cells. Our work suggests that Pah1p promotes storage lipid synthesis and thus represses phospholipid synthesis, which in turn restricts both viral replication and cell growth during viral infection. PMID:29649282

Dynamic Architecture of Eukaryotic DNA Replication Forks In Vivo, Visualized by Electron Microscopy.

PubMed

Zellweger, Ralph; Lopes, Massimo

2018-01-01

The DNA replication process can be heavily perturbed by several different conditions of genotoxic stress, particularly relevant for cancer onset and therapy. The combination of psoralen crosslinking and electron microscopy has proven instrumental to reveal the fine architecture of in vivo DNA replication intermediates and to uncover their remodeling upon specific conditions of genotoxic stress. The replication structures are stabilized in vivo (by psoralen crosslinking) prior to extraction and enrichment procedures, allowing their visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize and interpret in vivo replication intermediates of eukaryotic genomic DNA, and includes an improved method for enrichment of replication intermediates, compared to previously used BND-cellulose columns.

RENP Replication Unlikely Without Federal Support. Technical Appendices Supporting RMC Report UR 327.

ERIC Educational Resources Information Center

Errecart, Michael T.

The Response to Educational Needs Project (RENP) focuses on training teachers as a vehicle for promoting student achievement in a compensatory education program. This document supplements a report on RENP replication and provides information on cost analysis, methodology, and sample and data collection. In Appendix A the following questions are…

DNA Replication Origins in Immunoglobulin Switch Regions Regulate Class Switch Recombination in an R-Loop-Dependent Manner.

PubMed

Wiedemann, Eva-Maria; Peycheva, Mihaela; Pavri, Rushad

2016-12-13

Class switch recombination (CSR) at the immunoglobulin heavy chain (IgH) locus generates antibody isotypes. CSR depends on double-strand breaks (DSBs) induced by activation-induced cytidine deaminase (AID). Although DSB formation and repair machineries are active in G1 phase, efficient CSR is dependent on cell proliferation and S phase entry; however, the underlying mechanisms are obscure. Here, we show that efficient CSR requires the replicative helicase, the Mcm complex. Mcm proteins are enriched at IgH switch regions during CSR, leading to assembly of facultative replication origins that require Mcm helicase function for productive CSR. Assembly of CSR-associated origins is facilitated by R loops and promotes the physical proximity (synapsis) of recombining switch regions, which is reduced by R loop inhibition or Mcm complex depletion. Thus, R loops contribute to replication origin specification that promotes DSB resolution in CSR. This suggests a mechanism for the dependence of CSR on S phase and cell division. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication.

PubMed

Bharaj, Preeti; Atkins, Colm; Luthra, Priya; Giraldo, Maria Isabel; Dawes, Brian E; Miorin, Lisa; Johnson, Jeffrey R; Krogan, Nevan J; Basler, Christopher F; Freiberg, Alexander N; Rajsbaum, Ricardo

2017-09-15

Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis, because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here, we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV. Copyright © 2017 American Society for Microbiology.

The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication

PubMed Central

Bharaj, Preeti; Atkins, Colm; Luthra, Priya; Giraldo, Maria Isabel; Dawes, Brian E.; Miorin, Lisa; Johnson, Jeffrey R.; Krogan, Nevan J.; Basler, Christopher F.; Freiberg, Alexander N.

2017-01-01

ABSTRACT Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis, because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here, we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV. PMID:28679761

Agrobacterium tumefaciens supports DNA replication of diverse geminivirus types.

PubMed

Selth, Luke A; Randles, John W; Rezaian, M Ali

2002-04-10

We have previously shown that the soil-borne plant pathogen Agrobacterium tumefaciens supports the replication of tomato leaf curl geminivirus (Australian isolate) (TLCV) DNA. However, the reproducibility of this observation with other geminiviruses has been questioned. Here, we show that replicative DNA forms of three other geminiviruses also accumulate at varying levels in Agrobacterium. Geminiviral DNA constructs that lacked the ability to replicate in Agrobacterium were rendered replication-competent by changing their configuration so that two copies of the viral ori were present. Furthermore, we report that low-level replication of TLCV DNA can occur in Escherichia coli containing a dimeric TLCV construct in a high copy number plasmid. These findings were reinforced by expression studies using beta-glucuronidase which revealed that all six TLCV promoters are active in Agrobacterium, and two are functional in E. coli.

Back to the Origin

PubMed Central

Evertts, Adam G.

2012-01-01

In bacteria, replication is a carefully orchestrated event that unfolds the same way for each bacterium and each cell division. The process of DNA replication in bacteria optimizes cell growth and coordinates high levels of simultaneous replication and transcription. In metazoans, the organization of replication is more enigmatic. The lack of a specific sequence that defines origins of replication has, until recently, severely limited our ability to define the organizing principles of DNA replication. This question is of particular importance as emerging data suggest that replication stress is an important contributor to inherited genetic damage and the genomic instability in tumors. We consider here the replication program in several different organisms including recent genome-wide analyses of replication origins in humans. We review recent studies on the role of cytosine methylation in replication origins, the role of transcriptional looping and gene gating in DNA replication, and the role of chromatin’s 3-dimensional structure in DNA replication. We use these new findings to consider several questions surrounding DNA replication in metazoans: How are origins selected? What is the relationship between replication and transcription? How do checkpoints inhibit origin firing? Why are there early and late firing origins? We then discuss whether oncogenes promote cancer through a role in DNA replication and whether errors in DNA replication are important contributors to the genomic alterations and gene fusion events observed in cancer. We conclude with some important areas for future experimentation. PMID:23634256

Inhibition of adenovirus 5 replication in COS-1 cells by antisense RNAs against the viral E1a region.

PubMed

Miroshnichenko, O I; Ponomareva, T I; Tikchonenko, T I

1989-12-07

To study the effect of antisense E1a RNA (asRNA) on adenovirus development, two types of adenovirus 5 E1a antisense constructs have been engineered. One was complementary to the viral DNA region [nucleotide (nt) positions 500-720] regulated by the metallothionein-I promoter, and the other was complementary to the DNA regions (nt positions 630-1570) under control of the long terminal repeat Moloney mouse leukosis virus promoter. Both asRNA constructs were cloned into a plasmid containing the simian virus 40 origin of replication, the gene controlling geneticin (G418) resistance (G418R), and other regulatory elements. The COS-1 cells, which contained up to 100 copies of the engineered plasmids, synthesized antiviral asRNAs, which provided 71 to over 95% inhibition of adenoviral replication, in comparison to the control cells not synthesizing asRNAs.

Reactive oxygen species regulate DNA copy number in isolated yeast mitochondria by triggering recombination-mediated replication.

PubMed

Hori, Akiko; Yoshida, Minoru; Shibata, Takehiko; Ling, Feng

2009-02-01

Mitochondrial DNA (mtDNA) encodes proteins that are essential for cellular ATP production. Reactive oxygen species (ROS) are respiratory byproducts that damage mtDNA and other cellular components. In Saccharomyces cerevisiae, the oxidized base excision-repair enzyme Ntg1 introduces a double-stranded break (DSB) at the mtDNA replication origin ori5; this DSB initiates the rolling-circle mtDNA replication mediated by the homologous DNA pairing protein Mhr1. Thus, ROS may play a role in the regulation of mtDNA copy number. Here, we show that the treatment of isolated mitochondria with low concentrations of hydrogen peroxide increased mtDNA copy number in an Ntg1- and Mhr1-dependent manner. This treatment elevated the DSB levels at ori5 of hypersuppressive [rho(-)] mtDNA only if Ntg1 was active. In vitro Ntg1-treatment of hypersuppressive [rho(-)] mtDNA extracted from hydrogen peroxide-treated mitochondria revealed increased oxidative modifications at ori5 loci. We also observed that purified Ntg1 created breaks in single-stranded DNA harboring oxidized bases, and that ori5 loci have single-stranded character. Furthermore, chronic low levels of hydrogen peroxide increased in vivo mtDNA copy number. We therefore propose that ROS act as a regulator of mtDNA copy number, acting through the Mhr1-dependent initiation of rolling-circle replication promoted by Ntg1-induced DSB in the single-stranded regions at ori5.

HNRNPLL stabilizes mRNAs for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells.

PubMed

Sakuma, Keiichiro; Sasaki, Eiichi; Kimura, Kenya; Komori, Koji; Shimizu, Yasuhiro; Yatabe, Yasushi; Aoki, Masahiro

2018-06-05

HNRNPLL (heterogeneous nuclear ribonucleoprotein L-like), an RNA-binding protein that regulates alternative splicing of pre-mRNAs, has been shown to regulate differentiation of lymphocytes, as well as metastasis of colorectal cancer cells. Here we show that HNRNPLL promotes cell cycle progression and hence proliferation of colorectal cancer cells. Functional annotation analysis of those genes whose expression levels were changed by three-fold or more in RNA sequencing analysis between SW480 cells overexpressing HNRNPLL and those knocked down for HNRNPLL revealed enrichment of DNA replication-related genes by HNRNPLL overexpression. Among 13 genes detected in the DNA replication pathway, PCNA, RFC3, and FEN1 showed reproducible upregulation by HNRNPLL overexpression both at mRNA and protein levels in SW480 and HT29 cells. Importantly, knockdown of any of these genes alone suppressed the proliferation promoting effect induced by HNRNPLL overexpression. RNA-immunoprecipitation assay presented a binding of FLAG-tagged HNRNPLL to mRNA of these genes, and HNRNPLL overexpression significantly suppressed the downregulation of these genes during 12 hours of actinomycin D treatment, suggesting a role of HNRNPLL in mRNA stability. Finally, analysis of a public RNA sequencing dataset of clinical samples suggested a link between overexpression of HNRNPLL and that of PCNA, RFC3, and FEN1. This link was further supported by immunohistochemistry of colorectal cancer clinical samples, whereas expression of CDKN1A, which is known to inhibit the cooperative function of PCNA, RFC3, and FEN1, was negatively associated with HNRNPLL expression. These results indicate that HNRNPLL stabilizes mRNAs encoding regulators of DNA replication and promotes colorectal cancer cell proliferation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positive-strand RNA virus

PubMed Central

Flather, Dylan; Semler, Bert L.

2015-01-01

The compartmentalization of DNA replication and gene transcription in the nucleus and protein production in the cytoplasm is a defining feature of eukaryotic cells. The nucleus functions to maintain the integrity of the nuclear genome of the cell and to control gene expression based on intracellular and environmental signals received through the cytoplasm. The spatial separation of the major processes that lead to the expression of protein-coding genes establishes the necessity of a transport network to allow biomolecules to translocate between these two regions of the cell. The nucleocytoplasmic transport network is therefore essential for regulating normal cellular functioning. The Picornaviridae virus family is one of many viral families that disrupt the nucleocytoplasmic trafficking of cells to promote viral replication. Picornaviruses contain positive-sense, single-stranded RNA genomes and replicate in the cytoplasm of infected cells. As a result of the limited coding capacity of these viruses, cellular proteins are required by these intracellular parasites for both translation and genomic RNA replication. Being of messenger RNA polarity, a picornavirus genome can immediately be translated upon entering the cell cytoplasm. However, the replication of viral RNA requires the activity of RNA-binding proteins, many of which function in host gene expression, and are consequently localized to the nucleus. As a result, picornaviruses disrupt nucleocytoplasmic trafficking to exploit protein functions normally localized to a different cellular compartment from which they translate their genome to facilitate efficient replication. Furthermore, picornavirus proteins are also known to enter the nucleus of infected cells to limit host-cell transcription and down-regulate innate antiviral responses. The interactions of picornavirus proteins and host-cell nuclei are extensive, required for a productive infection, and are the focus of this review. PMID:26150805

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

PubMed

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.

An oncolytic adenovirus vector combining enhanced cell-to-cell spreading, mediated by the ADP cytolytic protein, with selective replication in cancer cells with deregulated wnt signaling.

PubMed

Toth, Karoly; Djeha, Hakim; Ying, Baoling; Tollefson, Ann E; Kuppuswamy, Mohan; Doronin, Konstantin; Krajcsi, Peter; Lipinski, Kai; Wrighton, Christopher J; Wold, William S M

2004-05-15

We have constructed a novel oncolytic adenovirus (Ad) vector named VRX-009 that combines enhanced cell spread with tumor-specific replication. Enhanced spread, which could significantly increase antitumor efficacy, is mediated by overexpression of the Ad cytolytic protein named ADP (also known as E3-11.6K). Replication of VRX-009 is restricted to cells with a deregulated wnt signal transduction pathway by replacement of the wild-type Ad E4 promoter with a synthetic promoter consisting of five consensus binding sites for the T-cell factor transcription factor. Tumor-selective replication is indicated by several lines of evidence. VRX-009 expresses E4ORF3, a representative Ad E4 protein, only in colon cancer cell lines. Furthermore, VRX-009 replicates preferentially in colon cancer cell lines as evidenced by virus productivity 2 orders of magnitude higher in SW480 colon cancer cells than in A549 lung cancer cells. Replication in primary human bronchial epithelial cells and human umbilical vein endothelial cells was also significantly lower than in SW480 cells. When tested in human tumor xenografts in nude mice, VRX-009 effectively suppressed the growth of SW480 colon tumors but not of A549 lung tumors. VRX-009 may provide greater level of antitumor efficacy than standard oncolytic Ad vectors in tumors in which a defect in wnt signaling increases the level of nuclear beta-catenin.

Host control of plasmid replication: requirement for the sigma factor sigma 32 in transcription of mini-F replication initiator gene.

PubMed Central

Wada, C; Imai, M; Yura, T

1987-01-01

Replication of F factor or mini-F plasmid is strongly inhibited in the rpoH (htpR) mutants of Escherichia coli deficient in the sigma factor (sigma 32) known to be required for heat shock gene expression. Transcription of the mini-F repE gene encoding a replication initiator protein (E protein) was examined by operon fusion and by direct determination of repE mRNA. The synthesis rate and the level of repE mRNA were found to increase transiently upon temperature upshift (30 degrees C to 42 degrees C) in wild-type cells but to decrease rapidly in the rpoH mutants. Thus sigma 32 appeared to be directly involved in transcription of repE whose product, E protein, in turn activates DNA replication from the mini-F ori2 region. This scheme of host-controlled plasmid replication is further supported by the analysis of transcription in vitro: RNA synthesis can be initiated from the repE promoter by a minor form of RNA polymerase containing sigma 32 but not by the major polymerase containing the normal sigma factor sigma 70. The sigma 32-mediated transcription from the repE promoter is strongly inhibited by the E protein. We conclude that transcription of the mini-F repE gene is mediated by the host transcription factor sigma 32 and is negatively controlled by its own product. Images PMID:2447584

The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products.

PubMed

Rattay, Stephanie; Trilling, Mirko; Megger, Dominik A; Sitek, Barbara; Meyer, Helmut E; Hengel, Hartmut; Le-Trilling, Vu Thuy Khanh

2015-08-01

Transcription of mouse cytomegalovirus (MCMV) immediate early ie1 and ie3 is controlled by the major immediate early promoter/enhancer (MIEP) and requires differential splicing. Based on complete loss of genome replication of an MCMV mutant carrying a deletion of the ie3-specific exon 5, the multifunctional IE3 protein (611 amino acids; pIE611) is considered essential for viral replication. Our analysis of ie3 transcription resulted in the identification of novel ie3 isoforms derived from alternatively spliced ie3 transcripts. Construction of an IE3-hemagglutinin (IE3-HA) virus by insertion of an in-frame HA epitope sequence allowed detection of the IE3 isoforms in infected cells, verifying that the newly identified transcripts code for proteins. This prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using Δie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication. To determine the role of pIE611 for viral gene expression during MCMV infection in an unbiased global approach, we used label-free quantitative mass spectrometry to delineate pIE611-dependent changes of the MCMV proteome. Interestingly, further analysis revealed transcriptional as well as posttranscriptional regulation of MCMV gene products by pIE611. Cytomegaloviruses are pathogenic betaherpesviruses persisting in a lifelong latency from which reactivation can occur under conditions of immunosuppression, immunoimmaturity, or inflammation. The switch from latency to reactivation requires expression of immediate early genes. Therefore, understanding of immediate early gene regulation might add insights into viral pathogenesis. The mouse cytomegalovirus (MCMV) immediate early 3 protein (611 amino acids; pIE611) is considered essential for viral replication. The identification of novel protein isoforms derived from alternatively spliced ie3 transcripts prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using Δie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication and delineated pIE611-dependent changes of the MCMV proteome. Our findings have fundamental implications for the interpretation of earlier studies on pIE3 functions and highlight the complex orchestration of MCMV gene regulation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Cellular microRNA-miR-548g-3p modulates the replication of dengue virus.

PubMed

Wen, Weitao; He, Zhenjian; Jing, Qinlong; Hu, Yiwen; Lin, Cuiji; Zhou, Rui; Wang, Xiaoqun; Su, Yangfan; Yuan, Jiehao; Chen, Zhenxin; Yuan, Jie; Wu, Jueheng; Li, Jun; Zhu, Xun; Li, Mengfeng

2015-06-01

It has been well recognized that microRNA plays a role in the host-pathogen interaction network. The significance of microRNA in the regulation of dengue virus (DENV) replication, however, remains unknown. The objective of our study was to determine the biological function of miR-548g-3p in modulating the replication of dengue virus. Here we report that employment of a microRNA target search algorithm to analyze the 5' untranslated region (5'UTR) consensus sequences of DENV (DENV serotypes 1-4) led to a discovery that miR-548g-3p directly targets the stem loop A promoter element within the 5'UTR, a region essential for DENV replication. Real-time PCR was used to measure the expression levels of miR-548g-3p under DENV infection. We performed overexpression and inhibition assays to test the role of miR-548g-3p on DENV replication. The protein and mRNA levels of interferon were measured by ELISA and real-time PCR respectively. We found that overexpression of miR-548g-3p suppressed multiplication of DENV 1, 2, 3 and 4, and that miR-548g-3p was also found to interfere with DENV translation, thereby suppressing the expression of viral proteins. Our results suggest that miR-548g-3p directly regulates DENV replication and warrant further study to investigate the feasibility of microRNA-based anti-DENV approaches. Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

The Ethanolamine Permease EutH Promotes Vacuole Adaptation of Salmonella enterica and Listeria monocytogenes during Macrophage Infection.

PubMed

Anderson, Christopher J; Satkovich, John; Köseoğlu, Volkan K; Agaisse, Hervé; Kendall, Melissa M

2018-05-01

Ethanolamine is a ubiquitous and essential molecule within a host. Significantly, bacterial pathogens exploit ethanolamine during infection to promote growth and regulate virulence. The ethanolamine permease EutH is dispensable for growth in vitro under standard conditions, whereas EutH is required for ethanolamine utilization at low pH. These findings suggested a model in which EutH facilitates diffusion of ethanolamine into the bacterial cell in acidic environments. To date, the ecological significance of this model has not been thoroughly investigated, and the importance of EutH to bacterial growth under physiologically relevant conditions is not known. During infection, immune cells internalize invading bacteria within an acidic, nutrient-depleted vacuole called the phagosome. Here, we investigated the hypothesis that EutH promotes bacterial survival following phagocytosis. Our findings indicate that EutH is important for survival and replication of the facultative intracellular pathogens Salmonella enterica serovar Typhimurium and Listeria monocytogenes during prolonged or transient exposure to the phagosome, respectively. Furthermore, in agreement with EutH being important in the acidic environment, neutralization of the vacuole abolished the requirement for EutH. Significantly, consistent with a role for EutH in promoting intramacrophage survival, EutH was not required during S Typhimurium local intestinal infection but specifically conferred an advantage upon dissemination to peripheral organs. These findings reveal a physiologically relevant and conserved role for EutH in spatiotemporal niche adaptation during infection. Copyright © 2018 American Society for Microbiology.

Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor

PubMed Central

Richardson, Max W.; Ellebrecht, Christoph T.; Glover, Joshua A.; Secreto, Anthony J.; Kulikovskaya, Irina; Yi, Yanjie; Wang, Jianbin; Dufendach, Keith A.; Holmes, Michael C.; Collman, Ronald G.

2017-01-01

HIV is adept at avoiding naturally generated T cell responses; therefore, there is a need to develop HIV-specific T cells with greater potency for use in HIV cure strategies. Starting with a CD4-based chimeric antigen receptor (CAR) that was previously used without toxicity in clinical trials, we optimized the vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains to determine which components augmented the ability of T cells to control HIV replication. This re-engineered CAR was at least 50-fold more potent in vitro at controlling HIV replication than the original CD4 CAR, or a TCR-based approach, and substantially better than broadly neutralizing antibody-based CARs. A humanized mouse model of HIV infection demonstrated that T cells expressing optimized CARs were superior at expanding in response to antigen, protecting CD4 T cells from infection, and reducing viral loads compared to T cells expressing the original, clinical trial CAR. Moreover, in a humanized mouse model of HIV treatment, CD4 CAR T cells containing the 4-1BB costimulatory domain controlled HIV spread after ART removal better than analogous CAR T cells containing the CD28 costimulatory domain. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and that CAR T cells could be important components of an HIV cure strategy. PMID:29023549

A Study on Generic Representation of Skeletal Remains Replication of Prehistoric Burial

NASA Astrophysics Data System (ADS)

Shao, C.-W.; Chiu, H.-L.; Chang, S.-K.

2015-08-01

Generic representation of skeletal remains from burials consists of three dimensions which include physical anthropologists, replication technicians, and promotional educators. For the reason that archaeological excavation is irreversible and disruptive, detail documentation and replication technologies are surely needed for many purposes. Unearthed bones during the process of 3D digital scanning need to go through reverse procedure, 3D scanning, digital model superimposition, rapid prototyping, mould making, and the integrated errors generated from the presentation of colours and textures are important issues for the presentation of replicate skeleton remains among professional decisions conducted by physical anthropologists, subjective determination of makers, and the expectations of viewers. This study presents several cases and examines current issues on display and replication technologies for human skeletal remains of prehistoric burials. This study documented detail colour changes of human skeleton over time for the reference of reproduction. The tolerance errors of quantification and required technical qualification is acquired according to the precision of 3D scanning, the specification requirement of rapid prototyping machine, and the mould making process should following the professional requirement for physical anthropological study. Additionally, the colorimeter is adopted to record and analyse the "colour change" of the human skeletal remains from wet to dry condition. Then, the "colure change" is used to evaluate the "real" surface texture and colour presentation of human skeletal remains, and to limit the artistic presentation among the human skeletal remains reproduction. The"Lingdao man No.1", is a well preserved burial of early Neolithic period (8300 B.P.) excavated from Liangdao-Daowei site, Matsu, Taiwan , as the replicating object for this study. In this study, we examined the reproduction procedures step by step for ensuring the surface texture and colour of the replica matches the real human skeletal remains when discovered. The "colour change" of the skeleton documented and quantified in this study could be the reference for the future study and educational exhibition of human skeletal remain reproduction.

Condensin promotes the juxtaposition of DNA flanking its loading site in Bacillus subtilis

PubMed Central

Wang, Xindan; Le, Tung B.K.; Lajoie, Bryan R.; Dekker, Job; Laub, Michael T.; Rudner, David Z.

2015-01-01

SMC condensin complexes play a central role in compacting and resolving replicated chromosomes in virtually all organisms, yet how they accomplish this remains elusive. In Bacillus subtilis, condensin is loaded at centromeric parS sites, where it encircles DNA and individualizes newly replicated origins. Using chromosome conformation capture and cytological assays, we show that condensin recruitment to origin-proximal parS sites is required for the juxtaposition of the two chromosome arms. Recruitment to ectopic parS sites promotes alignment of large tracks of DNA flanking these sites. Importantly, insertion of parS sites on opposing arms indicates that these “zip-up” interactions only occur between adjacent DNA segments. Collectively, our data suggest that condensin resolves replicated origins by promoting the juxtaposition of DNA flanking parS sites, drawing sister origins in on themselves and away from each other. These results are consistent with a model in which condensin encircles the DNA flanking its loading site and then slides down, tethering the two arms together. Lengthwise condensation via loop extrusion could provide a generalizable mechanism by which condensin complexes act dynamically to individualize origins in B. subtilis and, when loaded along eukaryotic chromosomes, resolve them during mitosis. PMID:26253537

Stop codons in the hepatitis B surface proteins are enriched during antiviral therapy and are associated with host cell apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colledge, Danielle; Soppe, Sally; Yuen, Lilly

Premature stop codons in the hepatitis B virus (HBV) surface protein can be associated with nucleos(t)ide analogue resistance due to overlap of the HBV surface and polymerase genes. The aim of this study was to determine the effect of the replication of three common surface stop codon variants on the hepatocyte. Cell lines were transfected with infectious HBV clones encoding surface stop codons rtM204I/sW196*, rtA181T/sW172*, rtV191I/sW182*, and a panel of substitutions in the surface proteins. HBsAg was measured by Western blotting. Proliferation and apoptosis were measured using flow cytometry. All three surface stop codon variants were defective in HBsAg secretion.more » Cells transfected with these variants were less proliferative and had higher levels of apoptosis than those transfected with variants that did not encode surface stop codons. The most cytopathic variant was rtM204I/sW196*. Replication of HBV encoding surface stop codons was toxic to the cell and promoted apoptosis, exacerbating disease progression. - Highlights: •Under normal circumstances, HBV replication is not cytopathic. •Premature stop codons in the HBV surface protein can be selected and enriched during nucleos(t)ide analogue therapy. •Replication of these variants can be cytopathic to the cell and promote apoptosis. •Inadequate antiviral therapy may actually promote disease progression.« less

GC-Rich DNA Elements Enable Replication Origin Activity in the Methylotrophic Yeast Pichia pastoris

PubMed Central

Liachko, Ivan; Youngblood, Rachel A.; Tsui, Kyle; Bubb, Kerry L.; Queitsch, Christine; Raghuraman, M. K.; Nislow, Corey; Brewer, Bonita J.; Dunham, Maitreya J.

2014-01-01

The well-studied DNA replication origins of the model budding and fission yeasts are A/T-rich elements. However, unlike their yeast counterparts, both plant and metazoan origins are G/C-rich and are associated with transcription start sites. Here we show that an industrially important methylotrophic budding yeast, Pichia pastoris, simultaneously employs at least two types of replication origins—a G/C-rich type associated with transcription start sites and an A/T-rich type more reminiscent of typical budding and fission yeast origins. We used a suite of massively parallel sequencing tools to map and dissect P. pastoris origins comprehensively, to measure their replication dynamics, and to assay the global positioning of nucleosomes across the genome. Our results suggest that some functional overlap exists between promoter sequences and G/C-rich replication origins in P. pastoris and imply an evolutionary bifurcation of the modes of replication initiation. PMID:24603708

Rescue from replication stress during mitosis.

PubMed

Fragkos, Michalis; Naim, Valeria

2017-04-03

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

Initiation of DNA replication requires actin dynamics and formin activity.

PubMed

Parisis, Nikolaos; Krasinska, Liliana; Harker, Bethany; Urbach, Serge; Rossignol, Michel; Camasses, Alain; Dewar, James; Morin, Nathalie; Fisher, Daniel

2017-11-02

Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin- and redox-dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)-cargo release from RanGTP-importin complexes. Nuclear formin activity is further required to promote loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms. © 2017 The Authors.

Rescue from replication stress during mitosis

PubMed Central

Naim, Valeria

2017-01-01

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

GC-rich DNA elements enable replication origin activity in the methylotrophic yeast Pichia pastoris.

PubMed

Liachko, Ivan; Youngblood, Rachel A; Tsui, Kyle; Bubb, Kerry L; Queitsch, Christine; Raghuraman, M K; Nislow, Corey; Brewer, Bonita J; Dunham, Maitreya J

2014-03-01

The well-studied DNA replication origins of the model budding and fission yeasts are A/T-rich elements. However, unlike their yeast counterparts, both plant and metazoan origins are G/C-rich and are associated with transcription start sites. Here we show that an industrially important methylotrophic budding yeast, Pichia pastoris, simultaneously employs at least two types of replication origins--a G/C-rich type associated with transcription start sites and an A/T-rich type more reminiscent of typical budding and fission yeast origins. We used a suite of massively parallel sequencing tools to map and dissect P. pastoris origins comprehensively, to measure their replication dynamics, and to assay the global positioning of nucleosomes across the genome. Our results suggest that some functional overlap exists between promoter sequences and G/C-rich replication origins in P. pastoris and imply an evolutionary bifurcation of the modes of replication initiation.

Secure Base Priming Diminishes Conflict-Based Anger and Anxiety

PubMed Central

Koren, Tamara; Bartholomew, Kim

2016-01-01

This study examines the impact of a visual representation of a secure base (i.e. a secure base prime) on attenuating experimentally produced anger and anxiety. Specifically, we examined the assuaging of negative emotions through exposure to an image of a mother-infant embrace or a heterosexual couple embracing. Subjects seated at a computer terminal rated their affect (Pre Affect) using the Affect Adjective Checklist (AAC) then listened to two sets of intense two person conflicts. After the first conflict exposure they rated affect again (Post 1 AAC). Following the second exposure they saw a blank screen (control condition), pictures of everyday objects (distraction condition) or a photo of two people embracing (Secure Base Prime condition). They then reported emotions using the Post 2 AAC. Compared to either control or distraction subjects, Secure Base Prime (SBP) subjects reported significantly less anger and anxiety. These results were then replicated using an internet sample with control, SBP and two new controls: Smiling Man (to control for expression of positive affect) and Cold Mother (an unsmiling mother with infant). The SBP amelioration of anger and anxiety was replicated with the internet sample. No control groups produced this effect, which was generated only by a combination of positive affect in a physically embracing dyad. The results are discussed in terms of attachment theory and research on spreading activation. PMID:27606897

Forsythoside A Inhibits BVDV Replication via TRAF2-Dependent CD28-4-1BB Signaling in Bovine PBMCs.

PubMed

Song, Quan-Jiang; Weng, Xiao-Gang; Cai, Dong-Jie; Zhang, Wang; Wang, Jiu-Feng

2016-01-01

Bovine viral diarrhea virus (BVDV), the causative agent of bovine viral diarrhea/mucosal disease (BVD/MD), is an important pathogen of cattle and other wild animals throughout the world. BVDV infection typically leads to an impaired immune response in cattle. In the present study, we investigated the effect of Forsythoside A (FTA) on BVDV infection of bovine peripheral blood mononuclear cells (PBMCs). We found that Forsythoside A could not only promote proliferation of PBMCs and T cells activation but also inhibit the replication of BVDV as well as apoptosis induced by BVDV. FTA treatment could counteract the BVDV-induced overproduction of IFN-γ to maintain the immune homeostasis in bovine PBMCs. At same time, FTA can enhance the secretion of IL-2. What's more, BVDV promotes the expression of CD28, 4-1BB and TRAF-2, which can be modulated by FTA. Our data suggest that FTA protects PBMCs from BVDV infection possibly via TRAF2-dependent CD28-4-1BB signaling, which may activate PBMCs in response to BVDV infection. Therefore, this aids in the development of an effective adjuvant for vaccines against BVDV and other specific FTA-based therapies for preventing BVDV infection.

Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication

PubMed Central

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

Functional Incompatibility between the Generic NF-κB Motif and a Subtype-Specific Sp1III Element Drives the Formation of the HIV-1 Subtype C Viral Promoter

PubMed Central

Verma, Anjali; Rajagopalan, Pavithra; Lotke, Rishikesh; Varghese, Rebu; Selvam, Deepak; Kundu, Tapas K.

2016-01-01

ABSTRACT Of the various genetic subtypes of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), only in subtype C of HIV-1 is a genetically variant NF-κB binding site found at the core of the viral promoter in association with a subtype-specific Sp1III motif. How the subtype-associated variations in the core transcription factor binding sites (TFBS) influence gene expression from the viral promoter has not been examined previously. Using panels of infectious viral molecular clones, we demonstrate that subtype-specific NF-κB and Sp1III motifs have evolved for optimal gene expression, and neither of the motifs can be replaced by a corresponding TFBS variant. The variant NF-κB motif binds NF-κB with an affinity 2-fold higher than that of the generic NF-κB site. Importantly, in the context of an infectious virus, the subtype-specific Sp1III motif demonstrates a profound loss of function in association with the generic NF-κB motif. An additional substitution of the Sp1III motif fully restores viral replication, suggesting that the subtype C-specific Sp1III has evolved to function with the variant, but not generic, NF-κB motif. A change of only two base pairs in the central NF-κB motif completely suppresses viral transcription from the provirus and converts the promoter into heterochromatin refractory to tumor necrosis factor alpha (TNF-α) induction. The present work represents the first demonstration of functional incompatibility between an otherwise functional NF-κB motif and a unique Sp1 site in the context of an HIV-1 promoter. Our work provides important leads as to the evolution of the HIV-1 subtype C viral promoter with relevance for gene expression regulation and viral latency. IMPORTANCE Subtype-specific genetic variations provide a powerful tool to examine how these variations offer a replication advantage to specific viral subtypes, if any. Only in subtype C of HIV-1 are two genetically distinct transcription factor binding sites positioned at the most critical location of the viral promoter. Since a single promoter regulates viral gene expression, the promoter variations can play a critical role in determining the replication fitness of the viral strains. Our work for the first time provides a scientific explanation for the presence of a unique NF-κB binding motif in subtype C, a major HIV-1 genetic family responsible for half of the global HIV-1 infections. The results offer compelling evidence that the subtype C viral promoter not only is stronger but also is endowed with a qualitative gain-of-function advantage. The genetically variant NF-κB and the Sp1III motifs may be respond differently to specific cell signal pathways, and these mechanisms must be examined. PMID:27194770

TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells

PubMed Central

Pedersen, Rune Troelsgaard; Kruse, Thomas; Nilsson, Jakob

2015-01-01

Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis. PMID:26283799

Environmental stress speeds up DNA replication in Pseudomonas putida in chemostat cultivations.

PubMed

Lieder, Sarah; Jahn, Michael; Koepff, Joachim; Müller, Susann; Takors, Ralf

2016-01-01

Cellular response to different types of stress is the hallmark of the cell's strategy for survival. How organisms adjust their cell cycle dynamics to compensate for changes in environmental conditions is an important unanswered question in bacterial physiology. A cell using binary fission for reproduction passes through three stages during its cell cycle: a stage from cell birth to initiation of replication, a DNA replication phase and a period of cell division. We present a detailed analysis of durations of cell cycle phases, investigating their dynamics under environmental stress conditions. Applying continuous steady state cultivations (chemostats), the DNA content of a Pseudomonas putida KT2440 population was quantified with flow cytometry at distinct growth rates. Data-driven modeling revealed that under stress conditions, such as oxygen deprivation, solvent exposure and decreased iron availability, DNA replication was accelerated correlated to the severity of the imposed stress (up to 1.9-fold). Cells maintained constant growth rates by balancing the shortened replication phase with extended cell cycle phases before and after replication. Transcriptome data underpin the transcriptional upregulation of crucial genes of the replication machinery. Hence adaption of DNA replication speed appears to be an important strategy to withstand environmental stress. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Increased global transcription activity as a mechanism of replication stress in cancer

PubMed Central

Kotsantis, Panagiotis; Silva, Lara Marques; Irmscher, Sarah; Jones, Rebecca M.; Folkes, Lisa; Gromak, Natalia; Petermann, Eva

2016-01-01

Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRASV12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRASV12, elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer. PMID:27725641

Increased global transcription activity as a mechanism of replication stress in cancer.

PubMed

Kotsantis, Panagiotis; Silva, Lara Marques; Irmscher, Sarah; Jones, Rebecca M; Folkes, Lisa; Gromak, Natalia; Petermann, Eva

2016-10-11

Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRAS V12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRAS V12 , elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer.

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

PubMed

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.

Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses.

PubMed

Hamperl, Stephan; Bocek, Michael J; Saldivar, Joshua C; Swigut, Tomek; Cimprich, Karlene A

2017-08-10

Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states. Copyright © 2017 Elsevier Inc. All rights reserved.

Efficient Replication of over 180 Genetic Associations with Self-Reported Medical Data

PubMed Central

Tung, Joyce Y.; Do, Chuong B.; Hinds, David A.; Kiefer, Amy K.; Macpherson, J. Michael; Chowdry, Arnab B.; Francke, Uta; Naughton, Brian T.; Mountain, Joanna L.; Wojcicki, Anne; Eriksson, Nicholas

2011-01-01

While the cost and speed of generating genomic data have come down dramatically in recent years, the slow pace of collecting medical data for large cohorts continues to hamper genetic research. Here we evaluate a novel online framework for obtaining large amounts of medical information from a recontactable cohort by assessing our ability to replicate genetic associations using these data. Using web-based questionnaires, we gathered self-reported data on 50 medical phenotypes from a generally unselected cohort of over 20,000 genotyped individuals. Of a list of genetic associations curated by NHGRI, we successfully replicated about 75% of the associations that we expected to (based on the number of cases in our cohort and reported odds ratios, and excluding a set of associations with contradictory published evidence). Altogether we replicated over 180 previously reported associations, including many for type 2 diabetes, prostate cancer, cholesterol levels, and multiple sclerosis. We found significant variation across categories of conditions in the percentage of expected associations that we were able to replicate, which may reflect systematic inflation of the effects in some initial reports, or differences across diseases in the likelihood of misdiagnosis or misreport. We also demonstrated that we could improve replication success by taking advantage of our recontactable cohort, offering more in-depth questions to refine self-reported diagnoses. Our data suggest that online collection of self-reported data from a recontactable cohort may be a viable method for both broad and deep phenotyping in large populations. PMID:21858135

Efficient replication of over 180 genetic associations with self-reported medical data.

PubMed

Tung, Joyce Y; Do, Chuong B; Hinds, David A; Kiefer, Amy K; Macpherson, J Michael; Chowdry, Arnab B; Francke, Uta; Naughton, Brian T; Mountain, Joanna L; Wojcicki, Anne; Eriksson, Nicholas

2011-01-01

While the cost and speed of generating genomic data have come down dramatically in recent years, the slow pace of collecting medical data for large cohorts continues to hamper genetic research. Here we evaluate a novel online framework for obtaining large amounts of medical information from a recontactable cohort by assessing our ability to replicate genetic associations using these data. Using web-based questionnaires, we gathered self-reported data on 50 medical phenotypes from a generally unselected cohort of over 20,000 genotyped individuals. Of a list of genetic associations curated by NHGRI, we successfully replicated about 75% of the associations that we expected to (based on the number of cases in our cohort and reported odds ratios, and excluding a set of associations with contradictory published evidence). Altogether we replicated over 180 previously reported associations, including many for type 2 diabetes, prostate cancer, cholesterol levels, and multiple sclerosis. We found significant variation across categories of conditions in the percentage of expected associations that we were able to replicate, which may reflect systematic inflation of the effects in some initial reports, or differences across diseases in the likelihood of misdiagnosis or misreport. We also demonstrated that we could improve replication success by taking advantage of our recontactable cohort, offering more in-depth questions to refine self-reported diagnoses. Our data suggest that online collection of self-reported data from a recontactable cohort may be a viable method for both broad and deep phenotyping in large populations.

Regulated expression of the human cytomegalovirus pp65 gene: Octamer sequence in the promoter is required for activation by viral gene products

DOE Office of Scientific and Technical Information (OSTI.GOV)

Depto, A.S.; Stenberg, R.M.

1989-03-01

To better understand the regulation of late gene expression in human cytomegalovirus (CMV)-infected cells, the authors examined expression of the gene that codes for the 65-kilodalton lower-matrix phosphoprotein (pp65). Analysis of RNA isolated at 72 h from cells infected with CMV Towne or ts66, a DNA-negative temperature-sensitive mutant, supported the fact that pp65 is expressed at low levels prior to viral DNA replication but maximally expressed after the initiation of viral DNA replication. To investigate promoter activation in a transient expression assay, the pp65 promoter was cloned into the indicator plasmid containing the gene for chloramphenicol acetyltransferase (CAT). Transfection ofmore » the promoter-CAT construct and subsequent superinfection with CMV resulted in activation of the promoter at early times after infection. Cotransfection with plasmids capable of expressing immediate-early (IE) proteins demonstrated that the promoter was activated by IE proteins and that both IE regions 1 and 2 were necessary. These studies suggest that interactions between IE proteins and this octamer sequence may be important for the regulation and expression of this CMV gene.« less

Structural Basis of Mitochondrial Transcription Initiation.

PubMed

Hillen, Hauke S; Morozov, Yaroslav I; Sarfallah, Azadeh; Temiakov, Dmitry; Cramer, Patrick

2017-11-16

Transcription in human mitochondria is driven by a single-subunit, factor-dependent RNA polymerase (mtRNAP). Despite its critical role in both expression and replication of the mitochondrial genome, transcription initiation by mtRNAP remains poorly understood. Here, we report crystal structures of human mitochondrial transcription initiation complexes assembled on both light and heavy strand promoters. The structures reveal how transcription factors TFAM and TFB2M assist mtRNAP to achieve promoter-dependent initiation. TFAM tethers the N-terminal region of mtRNAP to recruit the polymerase to the promoter whereas TFB2M induces structural changes in mtRNAP to enable promoter opening and trapping of the DNA non-template strand. Structural comparisons demonstrate that the initiation mechanism in mitochondria is distinct from that in the well-studied nuclear, bacterial, or bacteriophage transcription systems but that similarities are found on the topological and conceptual level. These results provide a framework for studying the regulation of gene expression and DNA replication in mitochondria. Copyright © 2017 Elsevier Inc. All rights reserved.

Shining lights: studies that have most influenced the understanding of health promotion's financial impact.

PubMed

Golaszewski, T

2001-01-01

To examine the literature from the past 20 years and identify those studies that support the economic merit of health promotion. A panel of experts was used to identify the top studies supporting the purpose of this article. Studies were chosen based on the following criteria: the study (1) examined the relationship between health risks and financial outcomes, or health promotion programs and financial outcomes; (2) provided strong and compelling financial data supporting the worth of health promotion; (3) had a high-quality methodology; (4) answered an important question or replicated important findings with superior methodology; and (5) represented U.S.-based initiatives published since 1980. After initially nominating a group of studies for consideration, panelists rated each on a scale from 1 to 3 representing their opinion of importance. Studies rating the highest were included for this discussion. Studies were analyzed by population characteristics, design, statistical tests, limitations, and results. This information was summarized for each identified article. A relationship between modifiable health risk factors and health care costs is supported by research. Health promotion interventions appear to provide positive financial returns, most notably for health care costs and absenteeism reduction. Private sector initiatives seem to be driving economic-based research. Overall, health promotion shows promising results for providing financial advantages for its sponsors; however, if this discipline is to show its true worth, considerable funding is needed from government or philanthropic sources to cover the substantial costs of quality research.

DEIsoM: a hierarchical Bayesian model for identifying differentially expressed isoforms using biological replicates

PubMed Central

Peng, Hao; Yang, Yifan; Zhe, Shandian; Wang, Jian; Gribskov, Michael; Qi, Yuan

2017-01-01

Abstract Motivation High-throughput mRNA sequencing (RNA-Seq) is a powerful tool for quantifying gene expression. Identification of transcript isoforms that are differentially expressed in different conditions, such as in patients and healthy subjects, can provide insights into the molecular basis of diseases. Current transcript quantification approaches, however, do not take advantage of the shared information in the biological replicates, potentially decreasing sensitivity and accuracy. Results We present a novel hierarchical Bayesian model called Differentially Expressed Isoform detection from Multiple biological replicates (DEIsoM) for identifying differentially expressed (DE) isoforms from multiple biological replicates representing two conditions, e.g. multiple samples from healthy and diseased subjects. DEIsoM first estimates isoform expression within each condition by (1) capturing common patterns from sample replicates while allowing individual differences, and (2) modeling the uncertainty introduced by ambiguous read mapping in each replicate. Specifically, we introduce a Dirichlet prior distribution to capture the common expression pattern of replicates from the same condition, and treat the isoform expression of individual replicates as samples from this distribution. Ambiguous read mapping is modeled as a multinomial distribution, and ambiguous reads are assigned to the most probable isoform in each replicate. Additionally, DEIsoM couples an efficient variational inference and a post-analysis method to improve the accuracy and speed of identification of DE isoforms over alternative methods. Application of DEIsoM to an hepatocellular carcinoma (HCC) dataset identifies biologically relevant DE isoforms. The relevance of these genes/isoforms to HCC are supported by principal component analysis (PCA), read coverage visualization, and the biological literature. Availability and implementation The software is available at https://github.com/hao-peng/DEIsoM Contact pengh@alumni.purdue.edu Supplementary information Supplementary data are available at Bioinformatics online. PMID:28595376

Impact of Organic Carbon Electron Donors on Microbial Community Development under Iron- and Sulfate-Reducing Conditions

DOE PAGES

Kwon, Man Jae; O’Loughlin, Edward J.; Boyanov, Maxim I.; ...

2016-01-22

Although iron- and sulfate-reducing bacteria in subsurface environments have crucial roles in biogeochemical cycling of C, Fe, and S, how specific electron donors impact the compositional structure and activity of native iron- and/or sulfate-reducing communities is largely unknown. To understand this better, we created bicarbonate-buffered batch systems in duplicate with three different electron donors (acetate, lactate, or glucose) paired with ferrihydrite and sulfate as the electron acceptors and inoculated them with subsurface sediment as the microbial inoculum. Sulfate and ferrihydrite reduction occurred simultaneously and were faster with lactate than with acetate. 16S rRNA-based sequence analysis of the communities over timemore » revealed that Desulfotomaculum was the major driver for sulfate reduction coupled with propionate oxidation in lactate-amended incubations. The reduction of sulfate resulted in sulfide production and subsequent abiotic reduction of ferrihydrite. In contrast, glucose promoted faster reduction of ferrihydrite, but without reduction of sulfate. Interestingly, the glucose-amended incubations led to two different biogeochemical trajectories among replicate bottles that resulted in distinct coloration (white and brown). The two outcomes in geochemical evolution might be due to the stochastic evolution of the microbial communities or subtle differences in the initial composition of the fermenting microbial community and its development via the use of different glucose fermentation pathways available within the community. Synchrotron-based x-ray analysis indicated that siderite and amorphous Fe(II) were formed in the replicate bottles with glucose, while ferrous sulfide and vivianite were formed with lactate or acetate. As a result, these data sets reveal that use of different C utilization pathways projects significant changes in microbial community composition over time that uniquely impact both the geochemistry and mineralogy of subsurface environments.« less

Impact of Organic Carbon Electron Donors on Microbial Community Development under Iron- and Sulfate-Reducing Conditions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Man Jae; O’Loughlin, Edward J.; Boyanov, Maxim I.

Although iron- and sulfate-reducing bacteria in subsurface environments have crucial roles in biogeochemical cycling of C, Fe, and S, how specific electron donors impact the compositional structure and activity of native iron- and/or sulfate-reducing communities is largely unknown. To understand this better, we created bicarbonate-buffered batch systems in duplicate with three different electron donors (acetate, lactate, or glucose) paired with ferrihydrite and sulfate as the electron acceptors and inoculated them with subsurface sediment as the microbial inoculum. Sulfate and ferrihydrite reduction occurred simultaneously and were faster with lactate than with acetate. 16S rRNA-based sequence analysis of the communities over timemore » revealed that Desulfotomaculum was the major driver for sulfate reduction coupled with propionate oxidation in lactate-amended incubations. The reduction of sulfate resulted in sulfide production and subsequent abiotic reduction of ferrihydrite. In contrast, glucose promoted faster reduction of ferrihydrite, but without reduction of sulfate. Interestingly, the glucose-amended incubations led to two different biogeochemical trajectories among replicate bottles that resulted in distinct coloration (white and brown). The two outcomes in geochemical evolution might be due to the stochastic evolution of the microbial communities or subtle differences in the initial composition of the fermenting microbial community and its development via the use of different glucose fermentation pathways available within the community. Synchrotron-based x-ray analysis indicated that siderite and amorphous Fe(II) were formed in the replicate bottles with glucose, while ferrous sulfide and vivianite were formed with lactate or acetate. As a result, these data sets reveal that use of different C utilization pathways projects significant changes in microbial community composition over time that uniquely impact both the geochemistry and mineralogy of subsurface environments.« less

Impact of Organic Carbon Electron Donors on Microbial Community Development under Iron- and Sulfate-Reducing Conditions.

PubMed

Kwon, Man Jae; O'Loughlin, Edward J; Boyanov, Maxim I; Brulc, Jennifer M; Johnston, Eric R; Kemner, Kenneth M; Antonopoulos, Dionysios A

2016-01-01

Although iron- and sulfate-reducing bacteria in subsurface environments have crucial roles in biogeochemical cycling of C, Fe, and S, how specific electron donors impact the compositional structure and activity of native iron- and/or sulfate-reducing communities is largely unknown. To understand this better, we created bicarbonate-buffered batch systems in duplicate with three different electron donors (acetate, lactate, or glucose) paired with ferrihydrite and sulfate as the electron acceptors and inoculated them with subsurface sediment as the microbial inoculum. Sulfate and ferrihydrite reduction occurred simultaneously and were faster with lactate than with acetate. 16S rRNA-based sequence analysis of the communities over time revealed that Desulfotomaculum was the major driver for sulfate reduction coupled with propionate oxidation in lactate-amended incubations. The reduction of sulfate resulted in sulfide production and subsequent abiotic reduction of ferrihydrite. In contrast, glucose promoted faster reduction of ferrihydrite, but without reduction of sulfate. Interestingly, the glucose-amended incubations led to two different biogeochemical trajectories among replicate bottles that resulted in distinct coloration (white and brown). The two outcomes in geochemical evolution might be due to the stochastic evolution of the microbial communities or subtle differences in the initial composition of the fermenting microbial community and its development via the use of different glucose fermentation pathways available within the community. Synchrotron-based x-ray analysis indicated that siderite and amorphous Fe(II) were formed in the replicate bottles with glucose, while ferrous sulfide and vivianite were formed with lactate or acetate. These data sets reveal that use of different C utilization pathways projects significant changes in microbial community composition over time that uniquely impact both the geochemistry and mineralogy of subsurface environments.

Within and beyond the stringent response-RSH and (p)ppGpp in plants.

PubMed

Boniecka, Justyna; Prusińska, Justyna; Dąbrowska, Grażyna B; Goc, Anna

2017-11-01

Plant RSH proteins are able to synthetize and/or hydrolyze unusual nucleotides called (p)ppGpp or alarmones. These molecules regulate nuclear and chloroplast transcription, chloroplast translation and plant development and stress response. Homologs of bacterial RelA/SpoT proteins, designated RSH, and products of their activity, (p)ppGpp-guanosine tetra-and pentaphosphates, have been found in algae and higher plants. (p)ppGpp were first identified in bacteria as the effectors of the stringent response, a mechanism that orchestrates pleiotropic adaptations to nutritional deprivation and various stress conditions. (p)ppGpp accumulation in bacteria decreases transcription-with exception to genes that help to withstand or overcome current stressful situations, which are upregulated-and translation as well as DNA replication and eventually reduces metabolism and growth but promotes adaptive responses. In plants, RSH are nuclei-encoded and function in chloroplasts, where alarmones are produced and decrease transcription, translation, hormone, lipid and metabolites accumulation and affect photosynthetic efficiency and eventually plant growth and development. During senescence, alarmones coordinate nutrient remobilization and relocation from vegetative tissues into seeds. Despite the high conservancy of RSH protein domains among bacteria and plants as well as the bacterial origin of plant chloroplasts, in plants, unlike in bacteria, (p)ppGpp promote chloroplast DNA replication and division. Next, (p)ppGpp may also perform their functions in cytoplasm, where they would promote plant growth inhibition. Furthermore, (p)ppGpp accumulation also affects nuclear gene expression, i.a., decreases the level of Arabidopsis defense gene transcripts, and promotes plants susceptibility towards Turnip mosaic virus. In this review, we summarize recent findings that show the importance of RSH and (p)ppGpp in plant growth and development, and open an area of research aiming to understand the function of plant RSH in response to stress.

DNA synthesis by Pol η promotes fragile site stability by preventing under-replicated DNA in mitosis

PubMed Central

Bergoglio, Valérie; Boyer, Anne-Sophie; Walsh, Erin; Naim, Valeria; Legube, Gaëlle; Lee, Marietta Y.W.T.; Rey, Laurie; Rosselli, Filippo; Cazaux, Christophe; Eckert, Kristin A.

2013-01-01

Human DNA polymerase η (Pol η) is best known for its role in responding to UV irradiation–induced genome damage. We have recently observed that Pol η is also required for the stability of common fragile sites (CFSs), whose rearrangements are considered a driving force of oncogenesis. Here, we explored the molecular mechanisms underlying this newly identified role. We demonstrated that Pol η accumulated at CFSs upon partial replication stress and could efficiently replicate non-B DNA sequences within CFSs. Pol η deficiency led to persistence of checkpoint-blind under-replicated CFS regions in mitosis, detectable as FANCD2-associated chromosomal sites that were transmitted to daughter cells in 53BP1-shielded nuclear bodies. Expression of a catalytically inactive mutant of Pol η increased replication fork stalling and activated the replication checkpoint. These data are consistent with the requirement of Pol η–dependent DNA synthesis during S phase at replication forks stalled in CFS regions to suppress CFS instability by preventing checkpoint-blind under-replicated DNA in mitosis. PMID:23609533

HMGB1 Protein Binds to Influenza Virus Nucleoprotein and Promotes Viral Replication

PubMed Central

Moisy, Dorothée; Avilov, Sergiy V.; Jacob, Yves; Laoide, Brid M.; Ge, Xingyi; Baudin, Florence; Jestin, Jean-Luc

2012-01-01

Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs). High-mobility-group box (HMGB) proteins were found to bind to the nucleoprotein (NP) component of vRNPs. HMGB1 and HMGB2 bind directly to the purified NP in the absence of viral RNA, and the HMG box A domain is sufficient to bind the NP. We show that HMGB1 associates with the viral NP in the nuclei of infected cells, promotes viral growth, and enhances the activity of the viral polymerase. The presence of a functional HMGB1 DNA-binding site is required to enhance influenza virus replication. Glycyrrhizin, which reduces HMGB1 binding to DNA, inhibits influenza virus polymerase activity. Our data show that the HMGB1 protein can play a significant role in intranuclear replication of influenza viruses, thus extending previous findings on the bornavirus and on a number of DNA viruses. PMID:22696656

Timely binding of IHF and Fis to DARS2 regulates ATP–DnaA production and replication initiation

PubMed Central

Kasho, Kazutoshi; Fujimitsu, Kazuyuki; Matoba, Toshihiro; Oshima, Taku; Katayama, Tsutomu

2014-01-01

In Escherichia coli, the ATP-bound form of DnaA (ATP–DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP–DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP–DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP–DnaA was fully active in replication initiation and underwent DnaA–ATP hydrolysis. ADP–DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP–DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP–DnaA production, thereby promoting timely initiation. Moreover, we show that IHF–DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP–DnaA and replication initiation in coordination with the cell cycle and growth phase. PMID:25378325

Stronger enhancer II/core promoter activities of hepatitis B virus isolates of B2 subgenotype than those of C2 subgenotype

PubMed Central

Qin, Yanli; Zhou, Xueshi; Jia, Haodi; Chen, Chaoyang; Zhao, Weifeng; Zhang, Jiming; Tong, Shuping

2016-01-01

Hepatitis B virus (HBV) genotype C causes prolonged chronic infection and increased risk for liver cancer than genotype B. Our previous work revealed lower replication capacity of wild-type genotype C2 than B2 isolates. HBV DNA replication is driven by pregenomic RNA, which is controlled by core promoter (CP) and further augmented by enhancer I (ENI) and enhancer II (ENII). DNA fragments covering these regulatory elements were amplified from B2 and C2 isolates to generate luciferase reporter constructs. As ENII is fully embedded in CP, we inserted HBV DNA fragments in the sense orientation to determine their combined activities, and in the antisense orientation to measure enhancer activities alone. Genotype B2 isolates displayed higher ENI+ENII+CP, ENII+CP, and ENII activities, but not ENI or ENI+ENII activity, than C2 isolates. The higher ENII+CP activity was partly attributable to 4 positions displaying genotype-specific variability. Exchanging CP region was sufficient to revert the replication phenotypes of several B2 and C2 clones tested. These results suggest that a weaker ENII and/or CP at least partly accounts for the lower replication capacities of wild-type C2 isolates, which could drive the subsequent acquisition of CP mutations. Such mutations increase genome replication and are implicated in liver cancer development. PMID:27461034

Stronger enhancer II/core promoter activities of hepatitis B virus isolates of B2 subgenotype than those of C2 subgenotype.

PubMed

Qin, Yanli; Zhou, Xueshi; Jia, Haodi; Chen, Chaoyang; Zhao, Weifeng; Zhang, Jiming; Tong, Shuping

2016-07-27

Hepatitis B virus (HBV) genotype C causes prolonged chronic infection and increased risk for liver cancer than genotype B. Our previous work revealed lower replication capacity of wild-type genotype C2 than B2 isolates. HBV DNA replication is driven by pregenomic RNA, which is controlled by core promoter (CP) and further augmented by enhancer I (ENI) and enhancer II (ENII). DNA fragments covering these regulatory elements were amplified from B2 and C2 isolates to generate luciferase reporter constructs. As ENII is fully embedded in CP, we inserted HBV DNA fragments in the sense orientation to determine their combined activities, and in the antisense orientation to measure enhancer activities alone. Genotype B2 isolates displayed higher ENI+ENII+CP, ENII+CP, and ENII activities, but not ENI or ENI+ENII activity, than C2 isolates. The higher ENII+CP activity was partly attributable to 4 positions displaying genotype-specific variability. Exchanging CP region was sufficient to revert the replication phenotypes of several B2 and C2 clones tested. These results suggest that a weaker ENII and/or CP at least partly accounts for the lower replication capacities of wild-type C2 isolates, which could drive the subsequent acquisition of CP mutations. Such mutations increase genome replication and are implicated in liver cancer development.

MCM5: a new actor in the link between DNA replication and Meier-Gorlin syndrome.

PubMed

Vetro, Annalisa; Savasta, Salvatore; Russo Raucci, Annalisa; Cerqua, Cristina; Sartori, Geppo; Limongelli, Ivan; Forlino, Antonella; Maruelli, Silvia; Perucca, Paola; Vergani, Debora; Mazzini, Giuliano; Mattevi, Andrea; Stivala, Lucia Anna; Salviati, Leonardo; Zuffardi, Orsetta

2017-05-01

Meier-Gorlin syndrome (MGORS) is a rare disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recessive mutations in ORC1, ORC4, ORC6, CDT1, CDC6, and CDC45, encoding members of the pre-replication (pre-RC) and pre-initiation (pre-IC) complexes, and heterozygous mutations in GMNN, a regulator of cell-cycle progression and DNA replication, have already been associated with this condition. We performed whole-exome sequencing (WES) in a patient with a clinical diagnosis of MGORS and identified biallelic variants in MCM5. This gene encodes a subunit of the replicative helicase complex, which represents a component of the pre-RC. Both variants, a missense substitution within a conserved domain critical for the helicase activity, and a single base deletion causing a frameshift and a premature stop codon, were predicted to be detrimental for the MCM5 function. Although variants of MCM5 have never been reported in specific human diseases, defect of this gene in zebrafish causes a phenotype of growth restriction overlapping the one associated with orc1 depletion. Complementation experiments in yeast showed that the plasmid carrying the missense variant was unable to rescue the lethal phenotype caused by mcm5 deletion. Moreover cell-cycle progression was delayed in patient's cells, as already shown for mutations in the ORC1 gene. Altogether our findings support the role of MCM5 as a novel gene involved in MGORS, further emphasizing that this condition is caused by impaired DNA replication.

Statistical models for RNA-seq data derived from a two-condition 48-replicate experiment.

PubMed

Gierliński, Marek; Cole, Christian; Schofield, Pietà; Schurch, Nicholas J; Sherstnev, Alexander; Singh, Vijender; Wrobel, Nicola; Gharbi, Karim; Simpson, Gordon; Owen-Hughes, Tom; Blaxter, Mark; Barton, Geoffrey J

2015-11-15

High-throughput RNA sequencing (RNA-seq) is now the standard method to determine differential gene expression. Identifying differentially expressed genes crucially depends on estimates of read-count variability. These estimates are typically based on statistical models such as the negative binomial distribution, which is employed by the tools edgeR, DESeq and cuffdiff. Until now, the validity of these models has usually been tested on either low-replicate RNA-seq data or simulations. A 48-replicate RNA-seq experiment in yeast was performed and data tested against theoretical models. The observed gene read counts were consistent with both log-normal and negative binomial distributions, while the mean-variance relation followed the line of constant dispersion parameter of ∼0.01. The high-replicate data also allowed for strict quality control and screening of 'bad' replicates, which can drastically affect the gene read-count distribution. RNA-seq data have been submitted to ENA archive with project ID PRJEB5348. g.j.barton@dundee.ac.uk. © The Author 2015. Published by Oxford University Press.

Statistical models for RNA-seq data derived from a two-condition 48-replicate experiment

PubMed Central

Cole, Christian; Schofield, Pietà; Schurch, Nicholas J.; Sherstnev, Alexander; Singh, Vijender; Wrobel, Nicola; Gharbi, Karim; Simpson, Gordon; Owen-Hughes, Tom; Blaxter, Mark; Barton, Geoffrey J.

2015-01-01

Motivation: High-throughput RNA sequencing (RNA-seq) is now the standard method to determine differential gene expression. Identifying differentially expressed genes crucially depends on estimates of read-count variability. These estimates are typically based on statistical models such as the negative binomial distribution, which is employed by the tools edgeR, DESeq and cuffdiff. Until now, the validity of these models has usually been tested on either low-replicate RNA-seq data or simulations. Results: A 48-replicate RNA-seq experiment in yeast was performed and data tested against theoretical models. The observed gene read counts were consistent with both log-normal and negative binomial distributions, while the mean-variance relation followed the line of constant dispersion parameter of ∼0.01. The high-replicate data also allowed for strict quality control and screening of ‘bad’ replicates, which can drastically affect the gene read-count distribution. Availability and implementation: RNA-seq data have been submitted to ENA archive with project ID PRJEB5348. Contact: g.j.barton@dundee.ac.uk PMID:26206307

Identification of the subgenomic promoter of the coat protein gene of cucumber fruit mottle mosaic virus and development of a heterologous expression vector.

PubMed

Rhee, Sun-Ju; Jang, Yoon Jeong; Lee, Gung Pyo

2016-06-01

Heterologous gene expression using plant virus vectors enables research on host-virus interactions and the production of useful proteins, but the host range of plant viruses limits the practical applications of such vectors. Here, we aimed to develop a viral vector based on cucumber fruit mottle mosaic virus (CFMMV), a member of the genus Tobamovirus, whose members infect cucurbits. The subgenomic promoter (SGP) in the coat protein (CP) gene, which was used to drive heterologous expression, was mapped by analyzing deletion mutants from a CaMV 35S promoter-driven infectious CFMMV clone. The region from nucleotides (nt) -55 to +160 relative to the start codon of the open reading frame (ORF) of CP was found to be a fully active promoter, and the region from nt -55 to +100 was identified as the active core promoter. Based on these SGPs, we constructed a cloning site in the CFMMV vector and successfully expressed enhanced green fluorescent protein (EGFP) in Nicotiana benthamiana and watermelon (Citrullus lanatus). Co-inoculation with the P19 suppressor increased EGFP expression and viral replication by blocking degradation of the viral genome. Our CFMMV vector will be useful as an expression vector in cucurbits.

Ectromelia virus upregulates the expression of heat shock protein 70 to promote viral replication.

PubMed

Cheng, Wenyu; Jia, Huaijie; Wang, Xiaoxia; He, Xiaobing; Jin, Qiwang; Cao, Jingxin; Jing, Zhizhong

2018-08-01

The ectromelia virus (ECTV) is a mouse specific Orthopoxvirus that causes lethal infection in some mouse strains. ECTV infection of these mouse strains has been used as a valuable model for understanding the interplay between Orthopoxvirus species and their hosts, including variola virus in humans. Although poxviruses encode numerous proteins required for DNA and RNA synthesis, and are less dependent on host functions than other DNA viruses, a detailed understanding of the host factors required for the replication of poxviruses is lacking. Heat shock protein 70 (Hsp70) isoforms have been reported to serve various roles in the replication cycle of numerous viruses. In the present study, microarray and reverse transcription‑quantitative polymerase chain reaction analysis were conducted to investigate the host gene expression profiles following ECTV infection in mice and cell cultures. The results indicated that one Hsp70 isoform, Hsp70 member 1B (Hspa1b), was highly upregulated during ECTV infection in vitro and in vivo. Subsequently, overexpression of Hspa1b protein and small interfering RNA‑mediated gene silencing of Hspa1b revealed that Hspa1b is required for efficient replication of ECTV. Furthermore, the results demonstrated that ECTV replication may be significantly suppressed by two chemical Hspa1b inhibitors: Quercetin and VER155008. In conclusion, the present study clearly demonstrated that ECTV infection upregulates the expression of Hspa1b in order to promote its replication. The dependence on Hsp70 may be used as a novel therapeutic target for the treatment of Orthopoxvirus infection.

Family options for parents with mental illnesses: a developmental, mixed methods pilot study.

PubMed

Nicholson, Joanne; Albert, Karen; Gershenson, Bernice; Williams, Valerie; Biebel, Kathleen

2009-01-01

The objective of this paper is to provide a description of Family Options, a rehabilitation intervention for parents with serious mental illnesses and their children focusing on recovery and resilience, and to report the findings from a pilot study at 6-months post-enrollment for participating mothers. A developmental design, and mixed quantitative and qualitative methods facilitate an in-depth understanding of Family Options and its impact on parents early in the implementation process. Participating families faced significant challenges, including long-term mental health conditions in adults, and emotional and behavioral difficulties in children. Data from mothers (n = 22) demonstrate significant improvements in well-being, functioning, and supports and resources at 6 months post-enrollment in Family Options. Mothers report help from Family Options staff consistent with the intervention as conceptualized, and high levels of satisfaction with the intervention as delivered. Innovative study design and analytic strategies are required to build the evidence base and promote rapid dissemination of effective interventions. Findings from this study will assist purveyors in refining the intervention, and will lay the groundwork for further replication and testing to build the evidence base for parents with serious mental illnesses and their families.

Expression of the Caulobacter heat shock gene dnaK is developmentally controlled during growth at normal temperatures.

PubMed Central

Gomes, S L; Gober, J W; Shapiro, L

1990-01-01

Caulobacter crescentus has a single dnaK gene that is highly homologous to the hsp70 family of heat shock genes. Analysis of the cloned and sequenced dnaK gene has shown that the deduced amino acid sequence could encode a protein of 67.6 kilodaltons that is 68% identical to the DnaK protein of Escherichia coli and 49% identical to the Drosophila and human hsp70 protein family. A partial open reading frame 165 base pairs 3' to the end of dnaK encodes a peptide of 190 amino acids that is 59% identical to DnaJ of E. coli. Northern blot analysis revealed a single 4.0-kilobase mRNA homologous to the cloned fragment. Since the dnaK coding region is 1.89 kilobases, dnaK and dnaJ may be transcribed as a polycistronic message. S1 mapping and primer extension experiments showed that transcription initiated at two sites 5' to the dnaK coding sequence. A single start site of transcription was identified during heat shock at 42 degrees C, and the predicted promoter sequence conformed to the consensus heat shock promoters of E. coli. At normal growth temperature (30 degrees C), a different start site was identified 3' to the heat shock start site that conformed to the E. coli sigma 70 promoter consensus sequence. S1 protection assays and analysis of expression of the dnaK gene fused to the lux transcription reporter gene showed that expression of dnaK is temporally controlled under normal physiological conditions and that transcription occurs just before the initiation of DNA replication. Thus, in both human cells (I. K. L. Milarski and R. I. Morimoto, Proc. Natl. Acad. Sci. USA 83:9517-9521, 1986) and in a simple bacterium, the transcription of a hsp70 gene is temporally controlled as a function of the cell cycle under normal growth conditions. Images PMID:2345134

Phage transposon mutagenesis.

PubMed

Siegrist, M Sloan; Rubin, Eric J

2009-01-01

Phage transduction is an attractive method of genetic manipulation in mycobacteria. PhiMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an Escherichia coli oriR6 K origin of replication. Mycobacterial transposon mutant libraries produced by PhiMycoMarT7 transduction are amenable to both forward and reverse genetic studies. In this protocol, we detail the preparation of PhiMycoMarT7, including a description of the phage, reconstitution of the phage, purification of plaques, preparation of phage stock, and titering of phage stock. We then describe the transduction procedure and finally outline the isolation of individual transposon mutants.

Human Cytomegalovirus Strategies to Maintain and Promote mRNA Translation

PubMed Central

Vincent, Heather A.; Ziehr, Benjamin; Moorman, Nathaniel J.

2016-01-01

mRNA translation requires the ordered assembly of translation initiation factors and ribosomal subunits on a transcript. Host signaling pathways regulate each step in this process to match levels of protein synthesis to environmental cues. In response to infection, cells activate multiple defenses that limit viral protein synthesis, which viruses must counteract to successfully replicate. Human cytomegalovirus (HCMV) inhibits host defenses that limit viral protein expression and manipulates host signaling pathways to promote the expression of both host and viral proteins necessary for virus replication. Here we review key regulatory steps in mRNA translation, and the strategies used by HCMV to maintain protein synthesis in infected cells. PMID:27089357

Nitric oxide prevents a pathogen permissive granulocytic inflammation during tuberculosis

PubMed Central

Mishra, Bibhuti B.; Lovewell, Rustin R.; Olive, Andrew J; Zhang, Guoliang; Wang, Wenfei; Eugenin, Eliseo; Smith, Clare M; Yao, Jia Phuah; Long, Jarukit E; Dubuke, Michelle L; Palace, Samantha G.; Goguen, Jon D.; Baker, Richard E.; Nambi, Subhalaxmi; Mishra, Rabinarayan; Booty, Matthew G; Baer, Christina E.; Shaffer, Scott A; Dartois, Veronique; McCormick, Beth; Chen, Xinchun; Sassetti, Christopher M.

2017-01-01

Nitric oxide (NO) contributes to protection from tuberculosis (TB). It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis (Mtb) growth, which prevents subsequent pathological inflammation. In contrast, we report NO primarily protects mice by repressing an interleukin-1 and 12/15-lipoxygenase dependent neutrophil recruitment cascade that promotes bacterial replication. Using Mtb mutants as indicators of the pathogen's environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports Mtb growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes TB in patients. The human 12/15 lipoxygenase ortholog, ALOX12, is expressed in cavitary TB lesions, the abundance of its products correlate with the number of airway neutrophils and bacterial burden, and a genetic polymorphism that increases ALOX12 expression is associated with TB risk. These data suggest that Mtb exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion. PMID:28504669

Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis.

PubMed

Mishra, Bibhuti B; Lovewell, Rustin R; Olive, Andrew J; Zhang, Guoliang; Wang, Wenfei; Eugenin, Eliseo; Smith, Clare M; Phuah, Jia Yao; Long, Jarukit E; Dubuke, Michelle L; Palace, Samantha G; Goguen, Jon D; Baker, Richard E; Nambi, Subhalaxmi; Mishra, Rabinarayan; Booty, Matthew G; Baer, Christina E; Shaffer, Scott A; Dartois, Veronique; McCormick, Beth A; Chen, Xinchun; Sassetti, Christopher M

2017-05-15

Nitric oxide contributes to protection from tuberculosis. It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis growth, which prevents subsequent pathological inflammation. In contrast, we report that nitric oxide primarily protects mice by repressing an interleukin-1- and 12/15-lipoxygenase-dependent neutrophil recruitment cascade that promotes bacterial replication. Using M. tuberculosis mutants as indicators of the pathogen's environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports M. tuberculosis growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes tuberculosis in patients. The human 12/15-lipoxygenase orthologue, ALOX12, is expressed in cavitary tuberculosis lesions; the abundance of its products correlates with the number of airway neutrophils and bacterial burden and a genetic polymorphism that increases ALOX12 expression is associated with tuberculosis risk. These data suggest that M. tuberculosis exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion.

Re-wiring of energy metabolism promotes viability during hyperreplication stress in E. coli

PubMed Central

Campion, Christopher; Weimann, Allan

2017-01-01

Chromosome replication in Escherichia coli is initiated by DnaA. DnaA binds ATP which is essential for formation of a DnaA-oriC nucleoprotein complex that promotes strand opening, helicase loading and replisome assembly. Following initiation, DnaAATP is converted to DnaAADP primarily by the Regulatory Inactivation of DnaA process (RIDA). In RIDA deficient cells, DnaAATP accumulates leading to uncontrolled initiation of replication and cell death by accumulation of DNA strand breaks. Mutations that suppress RIDA deficiency either dampen overinitiation or permit growth despite overinitiation. We characterize mutations of the last group that have in common that distinct metabolic routes are rewired resulting in the redirection of electron flow towards the cytochrome bd-1. We propose a model where cytochrome bd-1 lowers the formation of reactive oxygen species and hence oxidative damage to the DNA in general. This increases the processivity of replication forks generated by overinitiation to a level that sustains viability. PMID:28129339

Promoting Community Language Learning in the United Kingdom

ERIC Educational Resources Information Center

Handley, Sharon

2011-01-01

The COLT (Community and Lesser Taught Languages) project is a consortium of five UK universities, working with various other regional organisations to set up replicable projects and structures to promote languages in North West England. It received funding under the UK's national "Routes into Languages" initiative. One objective was to…

Myxoma Virus Protein M029 Is a Dual Function Immunomodulator that Inhibits PKR and Also Conscripts RHA/DHX9 to Promote Expanded Host Tropism and Viral Replication

PubMed Central

Rahman, Masmudur M.; Liu, Jia; Chan, Winnie M.; Rothenburg, Stefan; McFadden, Grant

2013-01-01

Myxoma virus (MYXV)-encoded protein M029 is a member of the poxvirus E3 family of dsRNA-binding proteins that antagonize the cellular interferon signaling pathways. In order to investigate additional functions of M029, we have constructed a series of targeted M029-minus (vMyx-M029KO and vMyx-M029ID) and V5-tagged M029 MYXV. We found that M029 plays a pivotal role in determining the cellular tropism of MYXV in all mammalian cells tested. The M029-minus viruses were able to replicate only in engineered cell lines that stably express a complementing protein, such as vaccinia E3, but underwent abortive or abated infection in all other tested mammalian cell lines. The M029-minus viruses were dramatically attenuated in susceptible host European rabbits and caused no observable signs of myxomatosis. Using V5-tagged M029 virus, we observed that M029 expressed as an early viral protein is localized in both the nuclear and cytosolic compartments in virus-infected cells, and is also incorporated into virions. Using proteomic approaches, we have identified Protein Kinase R (PKR) and RNA helicase A (RHA)/DHX9 as two cellular binding partners of M029 protein. In virus-infected cells, M029 interacts with PKR in a dsRNA-dependent manner, while binding with DHX9 was not dependent on dsRNA. Significantly, PKR knockdown in human cells rescued the replication defect of the M029-knockout viruses. Unexpectedly, this rescue of M029-minus virus replication by PKR depletion could then be reversed by RHA/DHX9 knockdown in human monocytic THP1 cells. This indicates that M029 not only inhibits generic PKR anti-viral pathways, but also binds and conscripts RHA/DHX9 as a pro-viral effector to promote virus replication in THP1 cells. Thus, M029 is a critical host range and virulence factor for MYXV that is required for replication in all mammalian cells by antagonizing PKR-mediated anti-viral functions, and also conscripts pro-viral RHA/DHX9 to promote viral replication specifically in myeloid cells. PMID:23853588

The Impact of the Mode of Thought in Complex Decisions: Intuitive Decisions are Better

PubMed Central

Usher, Marius; Russo, Zohar; Weyers, Mark; Brauner, Ran; Zakay, Dan

2011-01-01

A number of recent studies have reported that decision quality is enhanced under conditions of inattention or distraction (unconscious thought; Dijksterhuis, 2004; Dijksterhuis and Nordgren, 2006; Dijksterhuis et al., 2006). These reports have generated considerable controversy, for both experimental (problems of replication) and theoretical reasons (interpretation). Here we report the results of four experiments. The first experiment replicates the unconscious thought effect, under conditions that validate and control the subjective criterion of decision quality. The second and third experiments examine the impact of a mode of thought manipulation (without distraction) on decision quality in immediate decisions. Here we find that intuitive or affective manipulations improve decision quality compared to analytic/deliberation manipulations. The fourth experiment combines the two methods (distraction and mode of thought manipulations) and demonstrates enhanced decision quality, in a situation that attempts to preserve ecological validity. The results are interpreted within a framework that is based on two interacting subsystems of decision-making: an affective/intuition based system and an analytic/deliberation system. PMID:21716605

Potential roles of DNA methylation in the initiation and establishment of replicative senescence revealed by array-based methylome and transcriptome analyses

PubMed Central

Sakaki, Mizuho; Ebihara, Yukiko; Okamura, Kohji; Nakabayashi, Kazuhiko; Igarashi, Arisa; Matsumoto, Kenji; Hata, Kenichiro; Kobayashi, Yoshiro

2017-01-01

Cellular senescence is classified into two groups: replicative and premature senescence. Gene expression and epigenetic changes are reported to differ between these two groups and cell types. Normal human diploid fibroblast TIG-3 cells have often been used in cellular senescence research; however, their epigenetic profiles are still not fully understood. To elucidate how cellular senescence is epigenetically regulated in TIG-3 cells, we analyzed the gene expression and DNA methylation profiles of three types of senescent cells, namely, replicatively senescent, ras-induced senescent (RIS), and non-permissive temperature-induced senescent SVts8 cells, using gene expression and DNA methylation microarrays. The expression of genes involved in the cell cycle and immune response was commonly either down- or up-regulated in the three types of senescent cells, respectively. The altered DNA methylation patterns were observed in replicatively senescent cells, but not in prematurely senescent cells. Interestingly, hypomethylated CpG sites detected on non-CpG island regions (“open sea”) were enriched in immune response-related genes that had non-CpG island promoters. The integrated analysis of gene expression and methylation in replicatively senescent cells demonstrated that differentially expressed 867 genes, including cell cycle- and immune response-related genes, were associated with DNA methylation changes in CpG sites close to the transcription start sites (TSSs). Furthermore, several miRNAs regulated in part through DNA methylation were found to affect the expression of their targeted genes. Taken together, these results indicate that the epigenetic changes of DNA methylation regulate the expression of a certain portion of genes and partly contribute to the introduction and establishment of replicative senescence. PMID:28158250

Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly.

PubMed

Romero-Brey, Inés; Bartenschlager, Ralf

2016-06-07

The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles.

Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly

PubMed Central

Romero-Brey, Inés; Bartenschlager, Ralf

2016-01-01

The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles. PMID:27338443

The Effectiveness of Prompts to Promote Engagement With Digital Interventions: A Systematic Review.

PubMed

Alkhaldi, Ghadah; Hamilton, Fiona L; Lau, Rosa; Webster, Rosie; Michie, Susan; Murray, Elizabeth

2016-01-08

Digital interventions have been effective in improving numerous health outcomes and health behaviors; furthermore, they are increasingly being used in different health care areas, including self-management of long-term conditions, mental health, and health promotion. The full potential of digital interventions is hindered by a lack of user engagement. There is an urgent need to develop effective strategies that can promote users' engagement with digital interventions. One potential method is the use of technology-based reminders or prompts. To evaluate the effectiveness of technology-based strategies for promoting engagement with digital interventions. Cochrane Collaboration guidelines on systematic review methodology were followed. The search strategy was executed across 7 electronic databases: the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, Web of Science, the Education Resources Information Center (ERIC), PsycINFO, and the Cumulative Index to Nursing and Allied Health Literature (CINAHL). Databases were searched from inception to September 13, 2013, with no language or publication type restrictions, using three concepts: randomized controlled trials, digital interventions, and engagement. Gray literature and reference lists of included studies were also searched. Titles and abstracts were independently screened by 2 authors, then the full texts of potentially eligible papers were obtained and double-screened. Data from eligible papers were extracted by one author and checked for accuracy by another author. Bias was assessed using the Cochrane risk of bias assessment tool. Narrative synthesis was performed on all included studies and, where appropriate, data were pooled using meta-analysis. All findings were reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A total of 14 studies were included in the review with 8774 participants. Of the 14 studies, 9 had sufficient data to be included in the meta-analyses. The meta-analyses suggested that technology-based strategies can potentially promote engagement compared to no strategy for dichotomous outcomes (relative risk [RR] 1.27, 95% CI 1.01-1.60, I(2)=71%), but due to considerable heterogeneity and the small sample sizes in most studies, this result should be treated with caution. No studies reported adverse or economic outcomes. Only one study with a small sample size compared different characteristics; the study found that strategies promoting new digital intervention content and those sent to users shortly after they started using the digital intervention were more likely to engage users. Overall, studies reported borderline positive effects of technology-based strategies on engagement compared to no strategy. However, the results have to be interpreted with caution. More research is needed to replicate findings and understand which characteristics of the strategies are effective in promoting engagement and how cost-effective they are.

A 4-study replication of the moderating effects of greed on socioeconomic status and unethical behaviour.

PubMed

Balakrishnan, Anjana; Palma, Paolo A; Patenaude, Joshua; Campbell, Lorne

2017-01-31

Four replications of Piff and colleagues' study examined the moderating effects of greed attitudes on the relationship between socio-economic status (SES) and unethical behaviour (Study 7). In the original study, the researchers found that both greed and SES predicted increased propensity to engage in unethical behavior. Furthermore, this association was moderated such that the effects of SES on unethical behaviour were no longer present in the greed prime condition versus the neutral condition. In replication 1 of the original study main effects of greed attitudes and SES were found, but no interaction was found. Main effects for greed emerged in replications 3 and 4. However no main effects for SES or interactions emerged for replications 2-4. A meta-analysis was conducted with all replications and the original study, and found no moderating effect of greed on the relationship between SES and unethical behavior.

A 4-study replication of the moderating effects of greed on socioeconomic status and unethical behaviour

PubMed Central

Balakrishnan, Anjana; Palma, Paolo A.; Patenaude, Joshua; Campbell, Lorne

2017-01-01

Four replications of Piff and colleagues’ study examined the moderating effects of greed attitudes on the relationship between socio-economic status (SES) and unethical behaviour (Study 7). In the original study, the researchers found that both greed and SES predicted increased propensity to engage in unethical behavior. Furthermore, this association was moderated such that the effects of SES on unethical behaviour were no longer present in the greed prime condition versus the neutral condition. In replication 1 of the original study main effects of greed attitudes and SES were found, but no interaction was found. Main effects for greed emerged in replications 3 and 4. However no main effects for SES or interactions emerged for replications 2–4. A meta-analysis was conducted with all replications and the original study, and found no moderating effect of greed on the relationship between SES and unethical behavior. PMID:28140396

Nuclear Mitochondrial DNA Activates Replication in Saccharomyces cerevisiae

PubMed Central

Chatre, Laurent; Ricchetti, Miria

2011-01-01

The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in. PMID:21408151

Nuclear mitochondrial DNA activates replication in Saccharomyces cerevisiae.

PubMed

Chatre, Laurent; Ricchetti, Miria

2011-03-08

The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in.

Calcein represses human papillomavirus 16 E1-E2 mediated DNA replication via blocking their binding to the viral origin of replication.

PubMed

Das, Dipon; Smith, Nathan W; Wang, Xu; Richardson, Stacie L; Hartman, Matthew C T; Morgan, Iain M

2017-08-01

Human papillomaviruses are causative agents in several human diseases ranging from genital warts to ano-genital and oropharyngeal cancers. Currently only symptoms of HPV induced disease are treated; there are no antivirals available that directly target the viral life cycle. Previously, we determined that the cellular protein TopBP1 interacts with the HPV16 replication/transcription factor E2. This E2-TopBP1 interaction is essential for optimal E1-E2 DNA replication and for the viral life cycle. The drug calcein disrupts the interaction of TopBP1 with itself and other host proteins to promote cell death. Here we demonstrate that calcein blocks HPV16 E1-E2 DNA replication via blocking the viral replication complex forming at the origin of replication. This occurs at non-toxic levels of calcein and demonstrates specificity as it does not block the ability of E2 to regulate transcription. We propose that calcein or derivatives could be developed as an anti-HPV therapeutic. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of proteins that may directly interact with human RPA.

PubMed

Nakaya, Ryou; Takaya, Junichiro; Onuki, Takeshi; Moritani, Mariko; Nozaki, Naohito; Ishimi, Yukio

2010-11-01

RPA, which consisted of three subunits (RPA1, 2 and 3), plays essential roles in DNA transactions. At the DNA replication forks, RPA binds to single-stranded DNA region to stabilize the structure and to assemble other replication proteins. Interactions between RPA and several replication proteins have been reported but the analysis is not comprehensive. We systematically performed the qualitative analysis to identify RPA interaction partners to understand the protein-protein interaction at the replication forks. We expressed in insect cells the three subunits of human RPA, together with one replication protein, which is present at the forks under normal conditions and/or under the replication stress conditions, to examine the interaction. Among 30 proteins examined in total, it was found that at least 14 proteins interacted with RPA. RPA interacted with MCM3-7, MCM-BP and CDC45 proteins among the proteins that play roles in the initiation and the elongation of the DNA replication. RPA bound with TIPIN, CLASPIN and RAD17, which are involved in the DNA replication checkpoint functions. RPA also bound with cyclin-dependent kinases and an amino-terminal fragment of Rb protein that negatively regulates DNA replication. These results suggest that RPA interacts with the specific proteins among those that play roles in the regulation of the replication fork progression.

Antiretroviral therapy for adults infected with HIV: Guidelines for health care professionals from the Quebec HIV care committee.

PubMed

Rouleau, Danielle; Fortin, Claude; Trottier, Benoît; Lalonde, Richard; Lapointe, Normand; Côté, Pierre; Routy, Jean-Pierre; Matte, Marie-France; Tsarevsky, Irina; Baril, Jean-Guy

2011-01-01

The appropriate use of antiretrovirals reduces morbidity and mortality caused by HIV infection. The present article provides health care professionals with a practical guide for the use of antiretrovirals. Therapy should be initiated based predominantly on clinical presentation and CD4 count, and should consist of three active drugs or at least two active drugs when this is not possible, as in cases of some treatment-experienced patients. This is the most effective way to achieve long-term suppression of viral replication. Selection of individual drugs in the regimen should consider the weight of the evidence supporting these choices, as well as their tolerability profiles and ease of use, the patients' comorbidities and treatment history. Treatment interruption is not recommended, either in aviremic patients or in those who have experienced virological failure. Instead, the therapeutic regimen should be adjusted to minimize side effects, promote adherence and suppress viral replication.

RPA Interacts with HIRA and Regulates H3.3 Deposition at Gene Regulatory Elements in Mammalian Cells.

PubMed

Zhang, Honglian; Gan, Haiyun; Wang, Zhiquan; Lee, Jeong-Heon; Zhou, Hui; Ordog, Tamas; Wold, Marc S; Ljungman, Mats; Zhang, Zhiguo

2017-01-19

The histone chaperone HIRA is involved in depositing histone variant H3.3 into distinct genic regions, including promoters, enhancers, and gene bodies. However, how HIRA deposits H3.3 to these regions remains elusive. Through a short hairpin RNA (shRNA) screening, we identified single-stranded DNA binding protein replication protein A (RPA) as a regulator of the deposition of newly synthesized H3.3 into chromatin. We show that RPA physically interacts with HIRA to form RPA-HIRA-H3.3 complexes, and it co-localizes with HIRA and H3.3 at gene promoters and enhancers. Depletion of RPA1, the largest subunit of the RPA complex, dramatically reduces both HIRA association with chromatin and the deposition of newly synthesized H3.3 at promoters and enhancers and leads to altered transcription at gene promoters. These results support a model whereby RPA, best known for its role in DNA replication and repair, recruits HIRA to promoters and enhancers and regulates deposition of newly synthesized H3.3 to these regulatory elements for gene regulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Error-free replicative bypass of (6–4) photoproducts by DNA polymerase ζ in mouse and human cells

PubMed Central

Yoon, Jung-Hoon; Prakash, Louise; Prakash, Satya

2010-01-01

The ultraviolet (UV)-induced (6–4) pyrimidine–pyrimidone photoproduct [(6–4) PP] confers a large structural distortion in DNA. Here we examine in human cells the roles of translesion synthesis (TLS) DNA polymerases (Pols) in promoting replication through a (6–4) TT photoproduct carried on a duplex plasmid where bidirectional replication initiates from an origin of replication. We show that TLS contributes to a large fraction of lesion bypass and that it is mostly error-free. We find that, whereas Pol η and Pol ι provide alternate pathways for mutagenic TLS, surprisingly, Pol ζ functions independently of these Pols and in a predominantly error-free manner. We verify and extend these observations in mouse cells and conclude that, in human cells, TLS during replication can be markedly error-free even opposite a highly distorting DNA lesion. PMID:20080950

Acetyl-coenzyme A synthetase 2 is a nuclear protein required for replicative longevity in Saccharomyces cerevisiae

PubMed Central

Falcón, Alaric A.; Chen, Shaoping; Wood, Michael S.

2013-01-01

Acs2p is one of two acetyl-coenzyme A synthetases in Saccharomyces cerevisiae. We have prepared and characterized a monoclonal antibody specific for Acs2p and find that Acs2p is localized primarily to the nucleus, including the nucleolus, with a minor amount in the cytosol. We find that Acs2p is required for replicative longevity: an acs2Δ strain has a reduced replicative life span compared to wild-type and acs1Δ strains. Furthermore, replicatively aged acs2Δ cells contain elevated levels of extrachromosomal rDNA circles, and silencing at the rDNA locus is impaired in an acs2Δ strain. These findings indicate that Acs2p-mediated synthesis of acetyl-CoA in the nucleus functions to promote rDNA silencing and replicative longevity in yeast. PMID:19618123

RTEL1 is a replisome-associated helicase that promotes telomere and genome-wide replication.

PubMed

Vannier, Jean-Baptiste; Sandhu, Sumit; Petalcorin, Mark I R; Wu, Xiaoli; Nabi, Zinnatun; Ding, Hao; Boulton, Simon J

2013-10-11

Regulator of telomere length 1 (RTEL1) is an essential DNA helicase that disassembles telomere loops (T loops) and suppresses telomere fragility to maintain the integrity of chromosome ends. We established that RTEL1 also associates with the replisome through binding to proliferating cell nuclear antigen (PCNA). Mouse cells disrupted for the RTEL1-PCNA interaction (PIP mutant) exhibited accelerated senescence, replication fork instability, reduced replication fork extension rates, and increased origin usage. Although T-loop disassembly at telomeres was unaffected in the mutant cells, telomere replication was compromised, leading to fragile sites at telomeres. RTEL1-PIP mutant mice were viable, but loss of the RTEL1-PCNA interaction accelerated the onset of tumorigenesis in p53-deficient mice. We propose that RTEL1 plays a critical role in both telomere and genome-wide replication, which is crucial for genetic stability and tumor avoidance.

Association Study of Genes Controlling IL-12-dependent IFN-γ Immunity: STAT4 Alleles Increase Risk of Pulmonary Tuberculosis in Morocco

PubMed Central

Sabri, Ayoub; Grant, Audrey V.; Cosker, Kristel; El Azbaoui, Safa; Abid, Ahmed; Abderrahmani Rhorfi, Ismail; Souhi, Hicham; Janah, Hicham; Alaoui-Tahiri, Kebir; Gharbaoui, Yasser; Benkirane, Majid; Orlova, Marianna; Boland, Anne; Deswarte, Caroline; Migaud, Melanie; Bustamante, Jacinta; Schurr, Erwin; Boisson-Dupuis, Stephanie; Casanova, Jean-Laurent; Abel, Laurent; El Baghdadi, Jamila

2014-01-01

Background. Only a minority of individuals infected with Mycobacterium tuberculosis develop clinical tuberculosis. Genetic epidemiological evidence suggests that pulmonary tuberculosis has a strong human genetic component. Previous genetic findings in Mendelian predisposition to more severe mycobacterial infections, including by M. tuberculosis, underlined the importance of the interleukin 12 (IL-12)/interferon γ (IFN-γ) circuit in antimycobacterial immunity. Methods. We conducted an association study in Morocco between pulmonary tuberculosis and a panel of single-nucleotide polymorphisms (SNPs) covering 14 core IL-12/IFN-γ circuit genes. The analyses were performed in a discovery family-based sample followed by replication in a case-control population. Results. Out of 228 SNPs tested in the family-based sample, 6 STAT4 SNPs were associated with pulmonary tuberculosis (P = .0013–.01). We replicated the same direction of association for 1 cluster of 3 SNPs encompassing the promoter region of STAT4. In the combined sample, the association was stronger among younger subjects (pulmonary tuberculosis onset <25 years) with an odds ratio of developing pulmonary tuberculosis at rs897200 for GG vs AG/AA subjects of 1.47 (1.06–2.04). Previous functional experiments showed that the G allele of rs897200 was associated with lower STAT4 expression. Conclusions. Our present findings in a Moroccan population support an association of pulmonary tuberculosis with STAT4 promoter-region polymorphisms that may impact STAT4 expression. PMID:24610875

In-Game Health Communication: Delivering Low-Fear Health Messages in a Low-Fear Videogame.

PubMed

Burrows, Christopher N; Blanton, Hart

2018-06-01

Prior research has demonstrated that psychological immersion (or "transportation") into virtual gaming worlds can heighten influence from health-promotion messages embedded in the backgrounds of gaming scenes. However, research to date has only studied the effectiveness of embedding graphic, fear-based messages in the background of violent, first-person videogames. This study sought to examine whether transportation into a nonviolent videogame can heighten persuasion from low-fear, nongraphic health messages. Willingness to drive under the influence of alcohol (DUI) was measured in an undergraduate sample (M AGE  = 19) at the start of the semester, and n = 220 returned 0-3 months later to play a car-racing videogame. All were randomly assigned to play either a version of the game containing background billboards that delivered simple, text-based anti-DUI messages or consumer advertising. Self-reported levels of transportation were assessed, as well as postgame willingness to DUI. Exposure to anti-DUI messages from within the game predicted lower postgame willingness to DUI. Moreover, replicating prior research, the effect of message exposure was largest among those who reported higher levels of pregame willingness to DUI, and higher levels of in-game transportation. This study replicates prior research by suggesting that immersive videogames that promote transportation can be used to effectively deliver health communications. Results also expand prior research by providing the first evidence that such effects can generalize when health messaging orients around nongraphic, lower fear messages embedded within immersive but lower fear videogames.

Timely binding of IHF and Fis to DARS2 regulates ATP-DnaA production and replication initiation.

PubMed

Kasho, Kazutoshi; Fujimitsu, Kazuyuki; Matoba, Toshihiro; Oshima, Taku; Katayama, Tsutomu

2014-12-01

In Escherichia coli, the ATP-bound form of DnaA (ATP-DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP-DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP-DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP-DnaA was fully active in replication initiation and underwent DnaA-ATP hydrolysis. ADP-DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP-DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP-DnaA production, thereby promoting timely initiation. Moreover, we show that IHF-DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP-DnaA and replication initiation in coordination with the cell cycle and growth phase. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118▿ †

PubMed Central

Fang, Fang; Flynn, Sarah; Li, Yin; Claesson, Marcus J.; van Pijkeren, Jan-Peter; Collins, J. Kevin; van Sinderen, Douwe; O'Toole, Paul W.

2008-01-01

The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK homologs, and this plasmid could be cured when PemI was produced in trans. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector derivatives of pSF118-20 were generated that included the replication region (pLS203) and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in Lactobacillus plantarum, Lactobacillus fermentum, and the pSF118-20-cured derivative strain of L. salivarius UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and green fluorescent protein, and expression from a constitutive L. salivarius promoter, demonstrated the utility of this vector for the expression of heterologous genes in Lactobacillus. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli. PMID:18390685

Managing conflict between large carnivores and livestock.

PubMed

van Eeden, Lily M; Crowther, Mathew S; Dickman, Chris R; Macdonald, David W; Ripple, William J; Ritchie, Euan G; Newsome, Thomas M

2018-02-01

Large carnivores are persecuted globally because they threaten human industries and livelihoods. How this conflict is managed has consequences for the conservation of large carnivores and biodiversity more broadly. Mitigating human-predator conflict should be evidence-based and accommodate people's values while protecting carnivores. Despite much research into human and large-carnivore coexistence strategies, there have been few attempts to document the success of conflict-mitigation strategies on a global scale. We conducted a meta-analysis of global research on conflict mitigation related to large carnivores and humans. We focused on conflicts that arise from the threat large carnivores pose to livestock. We first used structured and unstructured searching to identify replicated studies that used before-after or control-impact design to measure change in livestock loss as a result of implementing a management intervention. We then extracted relevant data from these studies to calculate an overall effect size for each intervention type. Research effort and focus varied among continents and aligned with the histories and cultures that shaped livestock production and attitudes toward carnivores. Livestock guardian animals most effectively reduced livestock losses. Lethal control was the second most effective control, although its success varied the most, and guardian animals and lethal control did not differ significantly. Financial incentives have promoted tolerance of large carnivores in some settings and reduced retaliatory killings. We suggest coexistence strategies be location-specific, incorporate cultural values and environmental conditions, and be designed such that return on financial investment can be evaluated. Improved monitoring of mitigation measures is urgently required to promote effective evidence-based policy. © 2017 Society for Conservation Biology.

Drawing ability in typical and atypical development; colour cues and the effect of oblique lines.

PubMed

Farran, E K; Dodd, G F

2015-06-01

Individuals with Williams syndrome (WS) have poor drawing ability. Here, we investigated whether colour could be used as a facilitation cue during a drawing task. Participants with WS and non-verbal ability matched typically developing (TD) children were shown line figures presented on a 3 by 3 dot matrix, and asked to replicate the figures by drawing on an empty dot matrix. The dots of the matrix were either all black (control condition), or nine different coloured dots (colour condition). In a third condition, which also used coloured dots, participants were additionally asked to verbalise the colours of the dots prior to replicating the line drawings (colour-verbal condition). Performance was stronger in both WS and TD groups on the two coloured conditions, compared with the control condition. However, the facilitation effect of colour was significantly weaker in the WS group than in the TD group. Replication of oblique line segments was less successful than replication of non-oblique line segments for both groups; this effect was reduced by colour facilitation in the TD group only. Verbalising the colours had no additional impact on performance in either group. We suggest that colour acted as a cue to individuate the dots, thus enabling participants to better ascertain the spatial relationships between the parts of each figure, to determine the start and end points of component lines, and to determine the correspondence between the model and their replication. The reduced facilitation in the WS group is discussed in relation to the effect of oblique versus non-oblique lines, the use of atypical drawing strategies, and reduced attention to the model when drawing the replication. © 2014 MENCAP and International Association of the Scientific Study of Intellectual and Developmental Disabilities and John Wiley & Sons Ltd.

Lexical Coverage of TED Talks: Implications for Vocabulary Instruction

ERIC Educational Resources Information Center

Nurmukhamedov, Ulugbek

2017-01-01

Teachers of English are often in search of authentic audio and video materials that promote learners' listening comprehension and vocabulary development. TED Talks, a set of freely available web presentations, could be a useful resource to promote vocabulary instruction. The present replication study examines the lexical coverage of TED Talks by…

Promoting the Development of Mentor Teachers: Theory and Research Programs Using Guided Reflection.

ERIC Educational Resources Information Center

Reiman, Alan J.; Thies-Sprinthall, Lois

1993-01-01

Describes theory and a research program that uses guided reflection to promote the development of mentor teachers. Significant findings from a quasi-experimental study and a replication study are presented. The paper can assist teacher educators, policymakers, and school personnel who are searching for a teacher induction model. (GLR)

Purification of full-length VP22 from cells infected with HSV-1: A two-pronged approach for the solubilization and purification of viral proteins for use in biochemical studies

PubMed Central

Dewberry, Ebony J.; Dunkerley, Eric; Duffy, Carol

2012-01-01

Summary VP22, encoded by the UL49 gene, is one of the most abundant proteins of the herpes simplex virus type 1 (HSV-1) tegument and has been shown to be important for virus replication and spread. However, the exact role(s) played by VP22 in the HSV-1 replication cycle have yet to be delineated. The lack of a procedure to purify full-length VP22 has limited molecular studies on VP22 function. A procedure was developed for the purification of soluble, full-length VP22 from cells infected with HSV-1. A recombinant virus encoding His-tagged VP22 was generated and found to express VP22 at levels comparable to the wild type virus upon infection of Vero cells. By experimenting with a wide variety of cell lysis buffer conditions, several buffers that promote the solubility of full-length VP22 were identified. Buffers that gave the highest levels of solubility were then used in immobilized metal ion affinity chromatography experiments to identify conditions that provided the greatest level of VP22 binding and recovery from cobalt and nickel affinity resins. Using this strategy soluble, full-length VP22 was purified from cells infected with HSV-1. PMID:22569534

DNA Damage and Genomic Instability Induced by Inappropriate DNA Re-replication

DTIC Science & Technology

2006-04-01

replication in yeast cells. In the prior reporting period we demonstrated that re-replication induces a rapid and significant decrease in cell viability...repair, DNA replication, checkpoint, cell cycle, yeast , RAD9 16. SECURITY CLASSIFICATION OF: 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON...initiation, our laboratory has been able to conditionally induce varying amounts of re- replication in yeast cells. Effectively, cells enter, but do not

Forsythoside A Inhibits BVDV Replication via TRAF2-Dependent CD28–4-1BB Signaling in Bovine PBMCs

PubMed Central

Song, Quan-Jiang; Weng, Xiao-Gang; Cai, Dong-Jie; Zhang, Wang; Wang, Jiu-Feng

2016-01-01

Bovine viral diarrhea virus (BVDV), the causative agent of bovine viral diarrhea/mucosal disease (BVD/MD), is an important pathogen of cattle and other wild animals throughout the world. BVDV infection typically leads to an impaired immune response in cattle. In the present study, we investigated the effect of Forsythoside A (FTA) on BVDV infection of bovine peripheral blood mononuclear cells (PBMCs). We found that Forsythoside A could not only promote proliferation of PBMCs and T cells activation but also inhibit the replication of BVDV as well as apoptosis induced by BVDV. FTA treatment could counteract the BVDV-induced overproduction of IFN-γ to maintain the immune homeostasis in bovine PBMCs. At same time, FTA can enhance the secretion of IL-2. What’s more, BVDV promotes the expression of CD28, 4-1BB and TRAF-2, which can be modulated by FTA. Our data suggest that FTA protects PBMCs from BVDV infection possibly via TRAF2-dependent CD28–4-1BB signaling, which may activate PBMCs in response to BVDV infection. Therefore, this aids in the development of an effective adjuvant for vaccines against BVDV and other specific FTA-based therapies for preventing BVDV infection. PMID:27617959

Nuclear scaffold attachment stimulates, but is not essential for ARS activity in Saccharomyces cerevisiae: analysis of the Drosophila ftz SAR.

PubMed Central

Amati, B; Pick, L; Laroche, T; Gasser, S M

1990-01-01

Nuclei isolated from eukaryotic cells can be depleted of histones and most soluble nuclear proteins to isolate a structural framework called the nuclear scaffold. This structure maintains specific interactions with genomic DNA at sites known as scaffold attached regions (SARs), which are thought to be the bases of DNA loops. In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, genomic ARS elements are recovered as SARs. In addition, SARs from Drosophila melanogaster bind to yeast nuclear scaffolds in vitro and a subclass of these promotes autonomous replication of plasmids in yeast. In the present report, we present fine mapping studies of the Drosophila ftz SAR, which has both SAR and ARS activities in yeast. The data establish a close relationship between the sequences involved in ARS activity and scaffold binding: ARS elements that can bind the nuclear scaffold in vitro promote more efficient plasmid replication in vivo, but scaffold association is not a strict prerequisite for ARS function. Efficient interaction with nuclear scaffolds from both yeast and Drosophila requires a minimal length of SAR DNA that contains reiteration of a narrow minor groove structure of the double helix. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2123454

Variability in group size and the evolution of collective action.

PubMed

Peña, Jorge; Nöldeke, Georg

2016-01-21

Models of the evolution of collective action typically assume that interactions occur in groups of identical size. In contrast, social interactions between animals occur in groups of widely dispersed size. This paper models collective action problems as two-strategy multiplayer games and studies the effect of variability in group size on the evolution of cooperative behavior under the replicator dynamics. The analysis identifies elementary conditions on the payoff structure of the game implying that the evolution of cooperative behavior is promoted or inhibited when the group size experienced by a focal player is more or less variable. Similar but more stringent conditions are applicable when the confounding effect of size-biased sampling, which causes the group-size distribution experienced by a focal player to differ from the statistical distribution of group sizes, is taken into account. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines

PubMed Central

Gasper, David J.; Neldner, Brandon; Plisch, Erin H.; Rustom, Hani; Imai, Hirotaka; Kawaoka, Yoshihiro; Suresh, M.

2016-01-01

CD8+ cytotoxic T lymphocytes (CTLs) are critical for clearing many viral infections, and protective CTL memory can be induced by vaccination with attenuated viruses and vectors. Non-replicating vaccines are typically potentiated by the addition of adjuvants that enhance humoral responses, however few are capable of generating CTL responses. Adjuplex is a carbomer-lecithin-based adjuvant demonstrated to elicit robust humoral immunity to non-replicating antigens. We report that mice immunized with non-replicating Adjuplex-adjuvanted vaccines generated robust antigen-specific CTL responses. Vaccination by the subcutaneous or the intranasal route stimulated systemic and mucosal CTL memory respectively. However, only CTL memory induced by intranasal vaccination was protective against influenza viral challenge, and correlated with an enhancement of memory CTLs in the airways and CD103+ CD69+ CXCR3+ resident memory-like CTLs in the lungs. Mechanistically, Myd88-deficient mice mounted primary CTL responses to Adjuplex vaccines that were similar in magnitude to wild-type mice, but exhibited altered differentiation of effector cell subsets. Immune potentiating effects of Adjuplex entailed alterations in the frequency of antigen-presenting-cell subsets in vaccine draining lymph nodes, and in the lungs and airways following intranasal vaccination. Further, Adjuplex enhanced the ability of dendritic cells to promote antigen-induced proliferation of naïve CD8 T cells by modulating antigen uptake, its intracellular localization, and rate of processing. Taken together, we have identified an adjuvant that elicits both systemic and mucosal CTL memory to non-replicating antigens, and engenders protective CTL-based heterosubtypic immunity to influenza A virus in the respiratory tract. Further, findings presented in this manuscript have provided key insights into the mechanisms and factors that govern the induction and programming of systemic and protective memory CTLs in the respiratory tract. PMID:27997610

Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines.

PubMed

Gasper, David J; Neldner, Brandon; Plisch, Erin H; Rustom, Hani; Carrow, Emily; Imai, Hirotaka; Kawaoka, Yoshihiro; Suresh, M

2016-12-01

CD8+ cytotoxic T lymphocytes (CTLs) are critical for clearing many viral infections, and protective CTL memory can be induced by vaccination with attenuated viruses and vectors. Non-replicating vaccines are typically potentiated by the addition of adjuvants that enhance humoral responses, however few are capable of generating CTL responses. Adjuplex is a carbomer-lecithin-based adjuvant demonstrated to elicit robust humoral immunity to non-replicating antigens. We report that mice immunized with non-replicating Adjuplex-adjuvanted vaccines generated robust antigen-specific CTL responses. Vaccination by the subcutaneous or the intranasal route stimulated systemic and mucosal CTL memory respectively. However, only CTL memory induced by intranasal vaccination was protective against influenza viral challenge, and correlated with an enhancement of memory CTLs in the airways and CD103+ CD69+ CXCR3+ resident memory-like CTLs in the lungs. Mechanistically, Myd88-deficient mice mounted primary CTL responses to Adjuplex vaccines that were similar in magnitude to wild-type mice, but exhibited altered differentiation of effector cell subsets. Immune potentiating effects of Adjuplex entailed alterations in the frequency of antigen-presenting-cell subsets in vaccine draining lymph nodes, and in the lungs and airways following intranasal vaccination. Further, Adjuplex enhanced the ability of dendritic cells to promote antigen-induced proliferation of naïve CD8 T cells by modulating antigen uptake, its intracellular localization, and rate of processing. Taken together, we have identified an adjuvant that elicits both systemic and mucosal CTL memory to non-replicating antigens, and engenders protective CTL-based heterosubtypic immunity to influenza A virus in the respiratory tract. Further, findings presented in this manuscript have provided key insights into the mechanisms and factors that govern the induction and programming of systemic and protective memory CTLs in the respiratory tract.

Getting away and Getting by: The Experiences of Self-Employed Homemakers.

ERIC Educational Resources Information Center

Jurik, Nancy C.

1998-01-01

Home-based self-employed workers (n=46, including 35 women) viewed home work as a valuable nontraditional option, especially mothers who combined work and child care. Family-work conflicts and economic issues sometimes replicated negative, exploitative conditions of traditional workplaces. Gender, family status, resources, race/ethnicity, and…

The Proteasomal Rpn11 Metalloprotease Suppresses Tombusvirus RNA Recombination and Promotes Viral Replication via Facilitating Assembly of the Viral Replicase Complex

PubMed Central

Prasanth, K. Reddisiva; Barajas, Daniel

2014-01-01

ABSTRACT 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 p92pol 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. IMPORTANCE 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 of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, and in vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination. PMID:25540361

Atlantic salmon eggs favour sperm in competition that have similar major histocompatibility alleles.

PubMed

Yeates, Sarah E; Einum, Sigurd; Fleming, Ian A; Megens, Hendrik-Jan; Stet, René J M; Hindar, Kjetil; Holt, William V; Van Look, Katrien J W; Gage, Matthew J G

2009-02-07

Polyandry and post-copulatory sexual selection provide opportunities for the evolution of female differential sperm selection. Here, we examined the influence of variation in major histocompatibility (MH) class I allelic composition upon sperm competition dynamics in Atlantic salmon. We ran in vitro fertilization competitions that mimicked the gametic microenvironment, and replicated a paired-male experimental design that allowed us to compare differences in sperm competition success among males when their sperm compete for eggs from females that were genetically either similar or dissimilar at the MH class I locus. Concurrently, we measured variation in spermatozoal traits that are known to influence relative fertilization success under these conditions. Contrary to the findings demonstrating mechanisms that promote MH complex heterozygosity, our results showed that males won significantly greater relative fertilization success when competing for eggs from genetically similar females at the MH class I. This result also showed covariation with the known influences of sperm velocity on relative fertilization success. We discuss these unexpected findings in relation to sperm-egg recognition and hybridization avoidance mechanisms based upon immunogenetic variation.

Management of fluorescent lamps in controlled environment chambers

NASA Technical Reports Server (NTRS)

Romer, Mark

1994-01-01

Management of fluorescent lights is recommended to (1) maintain uniformity of light intensity over time and (2) permit reproducibility of lighting conditions during experimental replications. At the McGill Phytotron, the lighting intensity can be controlled to desired level because any individual pair of the 40 lamps in each chamber can be set to be 'on' at any particular time. A lamp canopy service history is maintained for each experiment permitting accurate replication of lighting conditions for subsequent replicate trials.

A plasmid-based reverse genetics system for influenza A virus.

PubMed Central

Pleschka, S; Jaskunas, R; Engelhardt, O G; Zürcher, T; Palese, P; García-Sastre, A

1996-01-01

A reverse genetics system for negative-strand RNA viruses was first successfully developed for influenza viruses. This technology involved the transfection of in vitro-reconstituted ribonucleoprotein (RNP) complexes into influenza virus-infected cells. We have now developed a method that allows intracellular reconstitution of RNP complexes from plasmid-based expression vectors. Expression of a viral RNA-like transcript is achieved from a plasmid containing a truncated human polymerase I (polI) promoter and a ribozyme sequence that generates the desired 3' end by autocatalytic cleavage. The polI-driven plasmid is cotransfected into human 293 cells with polII-responsive plasmids that express the viral PB1, PB2, PA, and NP proteins. This exclusively plasmid-driven system results in the efficient transcription and replication of the viral RNA-like reporter and allows the study of cis- and trans-acting signals involved in the transcription and replication of influenza virus RNAs. Using this system, we have also been able to rescue a synthetic neuraminidase gene into a recombinant influenza virus. This method represents a convenient alternative to the previously established RNP transfection system. PMID:8648766

Parvovirus Minute Virus of Mice Induces a DNA Damage Response That Facilitates Viral Replication

PubMed Central

Adeyemi, Richard O.; Landry, Sebastien; Davis, Meredith E.; Weitzman, Matthew D.; Pintel, David J.

2010-01-01

Infection by DNA viruses can elicit DNA damage responses (DDRs) in host cells. In some cases the DDR presents a block to viral replication that must be overcome, and in other cases the infecting agent exploits the DDR to facilitate replication. We find that low multiplicity infection with the autonomous parvovirus minute virus of mice (MVM) results in the activation of a DDR, characterized by the phosphorylation of H2AX, Nbs1, RPA32, Chk2 and p53. These proteins are recruited to MVM replication centers, where they co-localize with the main viral replication protein, NS1. The response is seen in both human and murine cell lines following infection with either the MVMp or MVMi strains. Replication of the virus is required for DNA damage signaling. Damage response proteins, including the ATM kinase, accumulate in viral-induced replication centers. Using mutant cell lines and specific kinase inhibitors, we show that ATM is the main transducer of the signaling events in the normal murine host. ATM inhibitors restrict MVM replication and ameliorate virus-induced cell cycle arrest, suggesting that DNA damage signaling facilitates virus replication, perhaps in part by promoting cell cycle arrest. Thus it appears that MVM exploits the cellular DNA damage response machinery early in infection to enhance its replication in host cells. PMID:20949077

Tombusviruses upregulate phospholipid biosynthesis via interaction between p33 replication protein and yeast lipid sensor proteins during virus replication in yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barajas, Daniel; Xu, Kai; Sharma, Monika

Positive-stranded RNA viruses induce new membranous structures and promote membrane proliferation in infected cells to facilitate viral replication. In this paper, the authors show that a plant-infecting tombusvirus upregulates transcription of phospholipid biosynthesis genes, such as INO1, OPI3 and CHO1, and increases phospholipid levels in yeast model host. This is accomplished by the viral p33 replication protein, which interacts with Opi1p FFAT domain protein and Scs2p VAP protein. Opi1p and Scs2p are phospholipid sensor proteins and they repress the expression of phospholipid genes. Accordingly, deletion of OPI1 transcription repressor in yeast has a stimulatory effect on TBSV RNA accumulation andmore » enhanced tombusvirus replicase activity in an in vitro assay. Altogether, the presented data convincingly demonstrate that de novo lipid biosynthesis is required for optimal TBSV replication. Overall, this work reveals that a (+)RNA virus reprograms the phospholipid biosynthesis pathway in a unique way to facilitate its replication in yeast cells. - Highlights: • Tombusvirus p33 replication protein interacts with FFAT-domain host protein. • Tombusvirus replication leads to upregulation of phospholipids. • Tombusvirus replication depends on de novo lipid synthesis. • Deletion of FFAT-domain host protein enhances TBSV replication. • TBSV rewires host phospholipid synthesis.« less

MMS Exposure Promotes Increased MtDNA Mutagenesis in the Presence of Replication-Defective Disease-Associated DNA Polymerase γ Variants

PubMed Central

Stumpf, Jeffrey D.; Copeland, William C.

2014-01-01

Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but suppresses MMS-induced mutagenesis. These results suggest a novel mechanism wherein mutations that lead to hypermutation by DNA base-damaging agents and associate with mitochondrial disease may contribute to previously unexplained phenomena, such as the wide variation of age of disease onset and acquired mitochondrial toxicities. PMID:25340760

MMS exposure promotes increased MtDNA mutagenesis in the presence of replication-defective disease-associated DNA polymerase γ variants.

PubMed

Stumpf, Jeffrey D; Copeland, William C

2014-10-01

Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but suppresses MMS-induced mutagenesis. These results suggest a novel mechanism wherein mutations that lead to hypermutation by DNA base-damaging agents and associate with mitochondrial disease may contribute to previously unexplained phenomena, such as the wide variation of age of disease onset and acquired mitochondrial toxicities.

Novel Nonreplicating Vaccinia Virus Vector Enhances Expression of Heterologous Genes and Suppresses Synthesis of Endogenous Viral Proteins.

PubMed

Wyatt, Linda S; Xiao, Wei; Americo, Jeffrey L; Earl, Patricia L; Moss, Bernard

2017-06-06

Viruses are used as expression vectors for protein synthesis, immunology research, vaccines, and therapeutics. Advantages of poxvirus vectors include the accommodation of large amounts of heterologous DNA, the presence of a cytoplasmic site of transcription, and high expression levels. On the other hand, competition of approximately 200 viral genes with the target gene for expression and immune recognition may be disadvantageous. We describe a vaccinia virus (VACV) vector that uses an early promoter to express the bacteriophage T7 RNA polymerase; has the A23R intermediate transcription factor gene deleted, thereby restricting virus replication to complementing cells; and has a heterologous gene regulated by a T7 promoter. In noncomplementing cells, viral early gene expression and DNA replication occurred normally but synthesis of intermediate and late proteins was prevented. Nevertheless, the progeny viral DNA provided templates for abundant expression of heterologous genes regulated by a T7 promoter. Selective expression of the Escherichia coli lac repressor gene from an intermediate promoter reduced transcription of the heterologous gene specifically in complementing cells, where large amounts might adversely impact VACV replication. Expression of heterologous proteins mediated by the A23R deletion vector equaled that of a replicating VACV, was higher than that of a nonreplicating modified vaccinia virus Ankara (MVA) vector used for candidate vaccines in vitro and in vivo , and was similarly immunogenic in mice. Unlike the MVA vector, the A23R deletion vector still expresses numerous early genes that can restrict immunogenicity as demonstrated here by the failure of the prototype vector to induce interferon alpha. By deleting immunomodulatory genes, we anticipate further improvements in the system. IMPORTANCE Vaccines provide an efficient and effective way of preventing infectious diseases. Nevertheless, new and better vaccines are needed. Vaccinia virus, which was used successfully as a live vaccine to eradicate smallpox, has been further attenuated and adapted as a recombinant vector for immunization against other pathogens. However, since the initial description of this vector system, only incremental improvements largely related to safety have been implemented. Here we described novel modifications of the platform that increased expression of the heterologous target gene and decreased expression of endogenous vaccinia virus genes while providing safety by preventing replication of the candidate vaccine except in complementing cells used for vector propagation. Copyright © 2017 Wyatt et al.

Identification of Novel Kaposi's Sarcoma-Associated Herpesvirus Orf50 Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential

PubMed Central

Wakeman, Brian S.; Izumiya, Yoshihiro

2016-01-01

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma and multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50. RTA is a known transactivator of multiple viral genes, allowing it to control the switch between latency and virus replication. We report here the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial), 3T12 (mouse fibroblast), and RAW 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 to 10 residues at the amino terminus of the protein. We show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation. IMPORTANCE Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma and multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV-infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication and is conserved among all known gammaherpesviruses. We show here for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of several isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression, leading to the efficient production of mature virions. PMID:27795414

Immune System Dysregulation and Latent Herpesvirus Reactivation During Winterover at Concordia Station, Dome C, Antarctica

NASA Technical Reports Server (NTRS)

Crucian, B. E.; Feuerecker, M.; Salam, A. P.; Rybka, A.; Stowe, R. P.; Morrels, M.; Meta, S. K.; Quiriarte, H.; Quintens, Roel; Thieme, U.;

Enhancing creativity: Proper body posture meets proper emotion.

PubMed

Hao, Ning; Xue, Hua; Yuan, Huan; Wang, Qing; Runco, Mark A

2017-02-01

This study tested whether compatibility or incompatibility between body posture and emotion was beneficial for creativity. In Study 1, participants were asked to solve the Alternative Uses Task (AUT) problems when performing open or closed body posture in positive or negative emotional state respectively. The results showed that originality of AUT performance was higher in the compatible conditions (i.e., open-positive and closed-negative) than in the incompatible conditions (i.e., closed-positive and open-negative). In Study 2, the compatibility effect was replicated in both the AUT and the Realistic Presented Problem test (i.e., RPP). Moreover, it was revealed that participants exhibited the highest associative flexibility in the open-positive condition, and the highest persistence in the closed-negative condition. These findings indicate that compatibility between body posture and emotion is beneficial for creativity. This may be because when the implicit emotions elicited by body posture match explicit emotions, the effects of emotions on creativity are enhanced, therefore promoting creativity through the flexibility or the persistence pathway respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Sequence Analysis of the Cryptic Plasmid pMG101 from Rhodopseudomonas palustris and Construction of Stable Cloning Vectors

PubMed Central

Inui, Masayuki; Roh, Jung Hyeob; Zahn, Kenneth; Yukawa, Hideaki

2000-01-01

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of α-proteobacteria. PMID:10618203

Accelerated Self-Replication under Non-Equilibrium, Periodic Energy Delivery

NASA Astrophysics Data System (ADS)

Zhang, Rui; Olvera de La Cruz, Monica

2014-03-01

Self-replication is a remarkable phenomenon in nature that has fascinated scientists for decades. In a self-replicating system, the original units are attracted to a template, which induce their binding. In equilibrium, the energy required to disassemble the newly assembled copy from the mother template is supplied by thermal energy. The possibility of optimizing self-replication is explored by controlling the frequency at which energy is supplied to the system. A model system inspired by a class of light switchable colloids is considered where light is used to control the interactions. Conditions under which self-replication can be significantly more effective under non-equilibrium, cyclic energy delivery than under equilibrium constant energy conditions are identified. Optimal self-replication does not require constant energy expenditure. Instead, the proper timing at which energy is delivered to the system is an essential controllable parameter to induce high replication rates. This work was supported by the Non-Equilibrium Energy Research Center (NERC), which is an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0000989.

Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis.

PubMed

Castro-Cerritos, Karla Viridiana; Yasbin, Ronald E; Robleto, Eduardo A; Pedraza-Reyes, Mario

2017-02-15

The Gram-positive microorganism Bacillus subtilis relies on a single class Ib ribonucleotide reductase (RNR) to generate 2'-deoxyribonucleotides (dNDPs) for DNA replication and repair. In this work, we investigated the influence of RNR levels on B. subtilis stationary-phase-associated mutagenesis (SPM). Since RNR is essential in this bacterium, we engineered a conditional mutant of strain B. subtilis YB955 (hisC952 metB5 leu427) in which expression of the nrdEF operon was modulated by isopropyl-β-d-thiogalactopyranoside (IPTG). Moreover, genetic inactivation of ytcG, predicted to encode a repressor (NrdR) of nrdEF in this strain, dramatically increased the expression levels of a transcriptional nrdE-lacZ fusion. The frequencies of mutations conferring amino acid prototrophy in three genes were measured in cultures under conditions that repressed or induced RNR-encoding genes. The results revealed that RNR was necessary for SPM and overexpression of nrdEF promoted growth-dependent mutagenesis and SPM. We also found that nrdEF expression was induced by H 2 O 2 and such induction was dependent on the master regulator PerR. These observations strongly suggest that the metabolic conditions operating in starved B. subtilis cells increase the levels of RNR, which have a direct impact on SPM. Results presented in this study support the concept that the adverse metabolic conditions prevailing in nutritionally stressed bacteria activate an oxidative stress response that disturbs ribonucleotide reductase (RNR) levels. Such an alteration of RNR levels promotes mutagenic events that allow Bacillus subtilis to escape from growth-limited conditions. Copyright © 2017 American Society for Microbiology.

Transition-state destabilization reveals how human DNA polymerase β proceeds across the chemically unstable lesion N7-methylguanine

PubMed Central

Ouzon-Shubeita, Hala; Lee, Seongmin

2014-01-01

N7-Methyl-2′-deoxyguanosine (m7dG) is the predominant lesion formed by methylating agents. A systematic investigation on the effect of m7dG on DNA replication has been difficult due to the chemical instability of m7dG. To gain insights into the m7dG effect, we employed a 2′-fluorine-mediated transition-state destabilzation strategy. Specifically, we determined kinetic parameters for dCTP insertion opposite a chemically stable m7dG analogue, 2′-fluoro-m7dG (Fm7dG), by human DNA polymerase β (polβ) and solved three X-ray structures of polβ in complex with the templating Fm7dG paired with incoming dCTP or dTTP analogues. The kinetic studies reveal that the templating Fm7dG slows polβ catalysis ∼300-fold, suggesting that m7dG in genomic DNA may impede replication by some DNA polymerases. The structural analysis reveals that Fm7dG forms a canonical Watson–Crick base pair with dCTP, but metal ion coordination is suboptimal for catalysis in the polβ-Fm7dG:dCTP complex, which partially explains the slow insertion of dCTP opposite Fm7dG by polβ. In addition, the polβ-Fm7dG:dTTP structure shows open protein conformations and staggered base pair conformations, indicating that N7-methylation of dG does not promote a promutagenic replication. Overall, the first systematic studies on the effect of m7dG on DNA replication reveal that polβ catalysis across m7dG is slow, yet highly accurate. PMID:24966350

Identification of acidic and aromatic residues in the Zta activation domain essential for Epstein-Barr virus reactivation.

PubMed

Deng, Z; Chen, C J; Zerby, D; Delecluse, H J; Lieberman, P M

2001-11-01

Epstein-Barr virus (EBV) lytic cycle transcription and DNA replication require the transcriptional activation function of the viral immediate-early protein Zta. We describe a series of alanine substitution mutations in the Zta activation domain that reveal two functional motifs based on amino acid composition. Alanine substitution of single or paired hydrophobic aromatic amino acid residues resulted in modest transcription activation defects, while combining four substitutions of aromatic residues (F22/F26/W74/F75) led to more severe transcription defects. Substitution of acidic amino acid residue E27, D35, or E54 caused severe transcription defects on most viral promoters. Promoter- and cell-specific defects were observed for some substitution mutants. Aromatic residues were required for Zta interaction with TFIIA-TFIID and the CREB-binding protein (CBP) and for stimulation of CBP histone acetyltransferase activity in vitro. In contrast, acidic amino acid substitution mutants interacted with TFIIA-TFIID and CBP indistinguishably from the wild type. The nuclear domain 10 (ND10) protein SP100 was dispersed by most Zta mutants, but acidic residue mutations led to reduced, while aromatic substitution mutants led to increased SP100 nuclear staining. Acidic residue substitution mutants had more pronounced defects in transcription activation of endogenous viral genes in latently infected cells and for viral replication, as measured by the production of infectious virus. One mutant, K12/F13, was incapable of stimulating EBV lytic replication but had only modest transcription defects. These results indicate that Zta stimulates viral reactivation through two nonredundant structural motifs, one of which interacts with general transcription factors and coactivators, and the other has an essential but as yet not understood function in lytic transcription.

Requirement of the cyclic adenosine monophosphate response element-binding protein for hepatitis B virus replication.

PubMed

Kim, Bo Kyung; Lim, Seoung Ok; Park, Yun Gyu

2008-08-01

The cyclic adenosine monophosphate-response element (CRE)-transcription factor complex participates in the regulation of viral gene expression and pathologic processes caused by various viruses. The hepatitis B virus (HBV) enhancer I directs liver-specific transcription of viral genes and contains a CRE sequence (HBV-CRE); however, whether the HBV-CRE and CRE-binding protein (CREB) are required for the HBV life cycle remains to be determined. This study was designed to investigate the role of CREB in HBV replication and gene expression. Sequence-comparison analysis of 984 HBVs reported worldwide showed that the HBV-CRE sequence is highly conserved, indicating the possibility that it plays an important role in the HBV life cycle. The binding of CREB to the HBV-CRE site was markedly inhibited by oligonucleotides containing HBV-CRE and consensus CRE sequences in vitro and in vivo. The HBV promoter activity was demonstrated to be dependent upon the transactivation activity of CREB. Treatment with CRE decoy oligonucleotides reduced HBV promoter activity, and this was reversed by CREB overexpression. The levels of viral transcripts, DNA, and antigens were remarkably decreased in response to the overexpression of CREB mutants or treatment with the CRE decoy oligonucleotides, whereas enhancing CREB activity increased the levels of viral transcripts. In addition, introduction of a three-base mutation into the HBV-CRE led to a marked reduction in HBV messenger RNA synthesis. Taken together, our results demonstrate that both replication and gene expression of HBV require a functional CREB and HBV-CRE. We have also demonstrated that CRE decoy oligonucleotides and the overexpression of CREB mutants can effectively block the HBV life cycle, suggesting that interventions against CREB activity could provide a new avenue to treat HBV infection.

Filovirus pathogenesis and immune evasion: insights from Ebola virus and Marburg virus

PubMed Central

Messaoudi, Ilhem; Amarasinghe, Gaya K.; Basler, Christopher F.

2016-01-01

Ebola viruses and Marburg viruses, members of the filovirus family, are zoonotic pathogens that cause severe disease in people. The Ebola virus epidemic in West Africa, which was first recognized in early 2014, highlights the threat posed by these deadly viruses. Filovirus disease is characterized by uncontrolled virus replication and the activation of damaging host pathways. Underlying these phenomena is the potent suppression of host innate antiviral responses, particularly the type I interferon (IFN) response, which allows high levels of replication. Here we review the mechanisms deployed by filoviruses to block host innate immunity and discuss aspects of virus replication that promote disease. PMID:26439085

Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B

PubMed Central

Kong, Lingbao; Fujimoto, Akira; Nakamura, Mariko; Aoyagi, Haruyo; Matsuda, Mami; Watashi, Koichi; Suzuki, Ryosuke; Arita, Minetaro; Yamagoe, Satoshi; Dohmae, Naoshi; Suzuki, Takehiro; Sakamaki, Yuriko; Ichinose, Shizuko; Suzuki, Tetsuro; Wakita, Takaji

2016-01-01

ABSTRACT It has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors. IMPORTANCE The hepatitis C virus (HCV) protein NS4B can induce alteration of the endoplasmic reticulum and the formation of a membranous web structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces the membranous HCV replication compartment is not understood. We screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis, followed by screening with small interfering RNA. We identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. PREB is induced by HCV infection and recruited into the replication complex by interaction with NS4B. Recruited PREB promotes HCV RNA replication by participating in the formation of the membranous HCV replication compartment. To our knowledge, the effect of NS4B-binding protein on the formation of the membranous HCV replication compartment is newly described in this report. Our findings are expected to provide new insights into HCV host cofactors. PMID:26739056

Mycobacterium tuberculosis Ser/Thr protein kinase B mediates an oxygen-dependent replication switch

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ortega, Corrie; Liao, Reiling; Anderson, Lindsey N.

In the majority of cases, Mycobacterium tuberculosis (Mtb) infections are clinically latent, characterized by little or no bacterial replication and drug tolerance. Low oxygen tension is a major host factor inducing bacteriostasis, but the molecular mechanisms driving oxygen-dependent replication are poorly understood. Mtb encodes eleven serine/threonine protein kinases, a family of signaling molecules known to regulate similar replicative adaptations in other bacteria. Here, we tested the role of serine/threonine phosphorylation in the Mtb response to altered oxygen status, using an in vitro model of latency (hypoxia) and reactivation (reaeration). Broad kinase inhibition compromised survival of Mtb in hypoxia. Activity-based proteinmore » profiling and genetic mutation identified PknB as the kinase critical for surviving hypoxia. Mtb replication was highly sensitive to changes in PknB levels in aerated culture, and even more so in hypoxia. A mutant overexpressing PknB specifically in hypoxia showed a 10-fold loss in viability in low oxygen conditions. In contrast, chemically reducing PknB activity during hypoxia specifically compromised resumption of growth during reaeration. These data support a model in which PknB activity is reduced to achieve bacteriostasis, and elevated when replication resumes. Together, these data show that phosphosignaling controls replicative transitions associated with latency and reactivation, that PknB is a major regulator of these transitions, and that PknB could provide a highly vulnerable therapeutic target at every step of the Mtb life cycle - active disease, latency, and reactivation.« less

Matrix Conditions and KLF2-Dependent Induction of Heme Oxygenase-1 Modulate Inhibition of HCV Replication by Fluvastatin

PubMed Central

Singethan, Katrin; Sirma, Hüseyin; Keller, Amelie Dorothea; Rosal, Sergio René Perez; Schrader, Jörg; Loscher, Christine; Volz, Tassilo; Bartenschlager, Ralf; Lohmann, Volker; Protzer, Ulrike; Dandri, Maura; Lohse, Ansgar W.; Tiegs, Gisa; Sass, Gabriele

2014-01-01

Background & Aims HMG-CoA-reductase-inhibitors (statins) have been shown to interfere with HCV replication in vitro. We investigated the mechanism, requirements and contribution of heme oxygenase-1(HO-1)-induction by statins to interference with HCV replication. Methods HO-1-induction by fluva-, simva-, rosuva-, atorva- or pravastatin was correlated to HCV replication, using non-infectious replicon systems as well as the infectious cell culture system. The mechanism of HO-1-induction by statins as well as its relevance for interference with HCV replication was investigated using transient or permanent knockdown cell lines. Polyacrylamide(PAA) gels of different density degrees or the Rho-kinase-inhibitor Hydroxyfasudil were used in order to mimic matrix conditions corresponding to normal versus fibrotic liver tissue. Results All statins used, except pravastatin, decreased HCV replication and induced HO-1 expression, as well as interferon response in vitro. HO-1-induction was mediated by reduction of Bach1 expression and induction of the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) cofactor Krueppel-like factor 2 (KLF2). Knockdown of KLF2 or HO-1 abrogated effects of statins on HCV replication. HO-1-induction and anti-viral effects of statins were more pronounced under cell culture conditions mimicking advanced stages of liver disease. Conclusions Statin-mediated effects on HCV replication seem to require HO-1-induction, which is more pronounced in a microenvironment resembling fibrotic liver tissue. This implicates that certain statins might be especially useful to support HCV therapy of patients at advanced stages of liver disease. PMID:24801208

Stronger activation of SREBP-1a by nucleus-localized HBx

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Qi; Qiao, Ling; Yang, Jian

2015-05-08

We previously showed that hepatitis B virus (HBV) X protein activates the sterol regulatory element-binding protein-1a (SREBP-1a). Here we examined the role of nuclear localization of HBx in this process. In comparison to the wild-type and cytoplasmic HBx, nuclear HBx had stronger effects on SREBP-1a and fatty acid synthase transcription activation, intracellular lipid accumulation and cell proliferation. Furthermore, nuclear HBx could activate HBV enhancer I/X promoter and was more effective on up-regulating HBV mRNA level in the context of HBV replication than the wild-type HBx, while the cytoplasmic HBx had no effect. Our results demonstrate the functional significance of themore » nucleus-localized HBx in regulating host lipogenic pathway and HBV replication. - Highlights: • Nuclear HBx is more effective on activating SREBP-1a and FASN transcription. • Nuclear HBx is more effective on enhancing intracellular lipid accumulation. • Nuclear HBx is more effective on enhancing cell proliferation. • Nuclear HBx up-regulates HBV enhancer I/X promoter activity. • Nuclear HBx increases HBV mRNA level in the context of HBV replication.« less

Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells

PubMed Central

Mansilla, Sabrina F; Bertolin, Agustina P; Bergoglio, Valérie; Pillaire, Marie-Jeanne; González Besteiro, Marina A; Luzzani, Carlos; Miriuka, Santiago G; Hoffmann, Jean-Sébastien; Gottifredi, Vanesa

2016-01-01

The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21’s PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis. DOI: http://dx.doi.org/10.7554/eLife.18020.001 PMID:27740454

Ethylene production with engineered Synechocystis sp PCC 6803 strains.

PubMed

Veetil, Vinod Puthan; Angermayr, S Andreas; Hellingwerf, Klaas J

2017-02-23

Metabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae pv. phaseolicola PK2 (the Kudzu strain), which catalyzes the conversion of the ubiquitous tricarboxylic acid cycle intermediate 2-oxoglutarate into ethylene. This study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine-Dalgarno sequence (5'-AGGAGG-3') as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background. Making use of different RBS sequences, production of ethylene ranging over a 20-fold difference has been achieved. However, a further increase of production through multiple tandem promoters and a broad-host plasmid was not achieved speculating that the transcription strength and the gene copy number are not the limiting factors in our system.

APOBEC4 Enhances the Replication of HIV-1

PubMed Central

Hofmann, Henning; Hanschmann, Kay-Martin; Mühlebach, Michael D.; Schumann, Gerald G.; König, Renate; Cichutek, Klaus; Häussinger, Dieter; Münk, Carsten

2016-01-01

APOBEC4 (A4) is a member of the AID/APOBEC family of cytidine deaminases. In this study we found a high mRNA expression of A4 in human testis. In contrast, there were only low levels of A4 mRNA detectable in 293T, HeLa, Jurkat or A3.01 cells. Ectopic expression of A4 in HeLa cells resulted in mostly cytoplasmic localization of the protein. To test whether A4 has antiviral activity similar to that of proteins of the APOBEC3 (A3) subfamily, A4 was co-expressed in 293T cells with wild type HIV-1 and HIV-1 luciferase reporter viruses. We found that A4 did not inhibit the replication of HIV-1 but instead enhanced the production of HIV-1 in a dose-dependent manner and seemed to act on the viral LTR. A4 did not show detectable cytidine deamination activity in vitro and weakly interacted with single-stranded DNA. The presence of A4 in virus producer cells enhanced HIV-1 replication by transiently transfected A4 or stably expressed A4 in HIV-susceptible cells. APOBEC4 was capable of similarly enhancing transcription from a broad spectrum of promoters, regardless of whether they were viral or mammalian. We hypothesize that A4 may have a natural role in modulating host promoters or endogenous LTR promoters. PMID:27249646

Human Adenovirus Core Protein V Is Targeted by the Host SUMOylation Machinery To Limit Essential Viral Functions.

PubMed

Freudenberger, Nora; Meyer, Tina; Groitl, Peter; Dobner, Thomas; Schreiner, Sabrina

2018-02-15

Human adenoviruses (HAdV) are nonenveloped viruses containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear pore complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood. The core protein V is speculated to bridge the core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus, protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultaneously, the altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than that observed during wild-type infection. Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies. IMPORTANCE Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this concept needs expansion as the functions are more diverse. We showed that capsid protein VI regulates the antiviral response by modulation of the transcription factor Daxx during infection. Moreover, core protein VII interacts with SPOC1 restriction factor, which is beneficial for efficient viral gene expression. Here, we were able to show that core protein V also represents a novel substrate of the host SUMOylation machinery and contains several conserved SCMs; mutation of these consensus motifs reduced SUMOylation of the protein. Unexpectedly, we observed that introducing these mutations into HAdV promotes adenoviral replication. In conclusion, we offer novel insights into adenovirus core proteins and provide evidence that SUMOylation of HAdV factors regulates replication efficiency. Copyright © 2018 American Society for Microbiology.

Development of a Reverse Genetic System for Infectious Salmon Anemia Virus: Rescue of Recombinant Fluorescent Virus by Using Salmon Internal Transcribed Spacer Region 1 as a Novel Promoter

PubMed Central

Toro-Ascuy, Daniela; Tambley, Carolina; Beltran, Carolina; Mascayano, Carolina; Sandoval, Nicolas; Olivares, Eduardo; Medina, Rafael A.; Spencer, Eugenio

2014-01-01

Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV901_09 and rISAVrS6-NotI-HPR containing a NotI restriction site and rISAVS6/EGFP-HPR harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5 × 105 PFU/ml, similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry. PMID:25480750

Bacterial lipopolysaccharide binding enhances virion stability and promotes environmental fitness of an enteric virus

PubMed Central

Robinson, Christopher M.; Jesudhasan, Palmy R.; Pfeiffer, Julie K.

2014-01-01

Summary Enteric viruses, including poliovirus and reovirus, encounter a vast microbial community in the mammalian gastrointestinal tract, which has been shown to promote virus replication and pathogenesis. Investigating the underlying mechanisms, we find that poliovirus binds bacterial surface polysaccharides, which enhances virion stability and cell attachment by increasing binding to the viral receptor. Additionally, we identified a poliovirus mutant, VP1-T99K, with reduced lipopolysaccharide (LPS) binding. Although T99K and WT poliovirus cell attachment, replication and pathogenesis in mice are equivalent, following peroral inoculation of mice, VP1-T99K poliovirus was unstable in feces. Consequently, the ratio of mutant virus in feces is reduced following additional cycles of infection in mice. Thus, the mutant virus incurs a fitness cost when environmental stability is a factor. These data suggest that poliovirus binds bacterial surface polysaccharides, enhancing cell attachment and environmental stability, potentially promoting transmission to a new host. PMID:24439896

Rif1 Binding and Control of Chromosome-Internal DNA Replication Origins Is Limited by Telomere Sequestration.

PubMed

Hafner, Lukas; Lezaja, Aleksandra; Zhang, Xu; Lemmens, Laure; Shyian, Maksym; Albert, Benjamin; Follonier, Cindy; Nunes, Jose Manuel; Lopes, Massimo; Shore, David; Mattarocci, Stefano

2018-04-24

The Saccharomyces cerevisiae telomere-binding protein Rif1 plays an evolutionarily conserved role in control of DNA replication timing by promoting PP1-dependent dephosphorylation of replication initiation factors. However, ScRif1 binding outside of telomeres has never been detected, and it has thus been unclear whether Rif1 acts directly on the replication origins that it controls. Here, we show that, in unperturbed yeast cells, Rif1 primarily regulates late-replicating origins within 100 kb of a telomere. Using the chromatin endogenous cleavage ChEC-seq technique, we robustly detect Rif1 at late-replicating origins that we show are targets of its inhibitory action. Interestingly, abrogation of Rif1 telomere association by mutation of its Rap1-binding module increases Rif1 binding and origin inhibition elsewhere in the genome. Our results indicate that Rif1 inhibits replication initiation by interacting directly with origins and suggest that Rap1-dependent sequestration of Rif1 increases its effective concentration near telomeres, while limiting its action at chromosome-internal sites. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

FANCJ promotes DNA synthesis through G-quadruplex structures

PubMed Central

Castillo Bosch, Pau; Segura-Bayona, Sandra; Koole, Wouter; van Heteren, Jane T; Dewar, James M; Tijsterman, Marcel; Knipscheer, Puck

2014-01-01

Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ−/− cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes. PMID:25193968

Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes

PubMed Central

Min, Jaewon; Wright, Woodring E.

2017-01-01

ABSTRACT Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Saccharomyces cerevisiae. Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer. PMID:28760773

Quantum information and the problem of mechanisms of biological evolution.

PubMed

Melkikh, Alexey V

2014-01-01

One of the most important conditions for replication in early evolution is the de facto elimination of the conformational degrees of freedom of the replicators, the mechanisms of which remain unclear. In addition, realistic evolutionary timescales can be established based only on partially directed evolution, further complicating this issue. A division of the various evolutionary theories into two classes has been proposed based on the presence or absence of a priori information about the evolving system. A priori information plays a key role in solving problems in evolution. Here, a model of partially directed evolution, based on the learning automata theory, which includes a priori information about the fitness space, is proposed. A potential repository of such prior information is the states of biologically important molecules. Thus, the need for extended evolutionary synthesis is discussed. Experiments to test the hypothesis of partially directed evolution are proposed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Functional centromeres determine the activation time of pericentric origins of DNA replication in Saccharomyces cerevisiae.

PubMed

Pohl, Thomas J; Brewer, Bonita J; Raghuraman, M K

2012-01-01

The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation.

Functional Centromeres Determine the Activation Time of Pericentric Origins of DNA Replication in Saccharomyces cerevisiae

PubMed Central

Pohl, Thomas J.; Brewer, Bonita J.; Raghuraman, M. K.

2012-01-01

The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation. PMID:22589733

Activation of human herpesvirus replication by apoptosis.

PubMed

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.

Activation of Human Herpesvirus Replication by Apoptosis

PubMed Central

Prasad, Alka; Remick, Jill

2013-01-01

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. PMID:23885073

[The prevalence and clinical significance of precore and core promoter mutations in Korean patients with chronic hepatitis B virus infection].

PubMed

Kim, Hyung Joon; Yoo, Byung Chul

2002-06-01

Precore and core promoter mutations of hepatitis B virus (HBV) have been reported in Korea but their prevalence and clinical significance have not been determined. The aims of this study were to determine the prevalence of precore and core promoter mutations and their relationships to hepatitis B e antigen (HBeAg) status, viral replication level, and severity of liver disease in Korea. Among the patients who visited the Liver Diseases Clinics (Chung Ang University Hospital) between December 1998 and August 1999, 150 patients were randomly selected: 50 HBeAg-positive HBV-DNA positive patients by a branched DNA (bDNA) assay, 50 HBeAg-negative bDNA-positive patients, and 50 HBeAg-negative bDNA-negative patients. Serum HBV-DNA was amplified by a polymerase chain reaction (PCR) in these patients and the core promoter/precore HBV sequence was determined in 135 of the patients whose sera were positive for HBV-DNA by PCR. All of the 135 determined HBV-DNA sequences had HBV genotype with T at nucleotide 1858. Precore mutation (A1896) was detected in 95.7% of HBeAg-negative bDNA-positive patients and 94.9% of HBeAg-negative bDNA-negative patients. In HBeAg-positive patients 88% had wild type and 12% had mixture of wild type and A1896 mutant. Core promoter TA mutation (T1762/A1764) was detected in 93.5% of HBeAg-negative bDNA-positive patients, 94.9% of HBeAg-negative bDNA-negative patients and 74% of HBeAg-positive patients. No correlation was found between the presence of precore/core promoter mutations and liver disease severity or HBV-DNA levels. Precore stop codon mutation occurred almost invariably, along with HBeAg seroconversion, irrespective of subsequent viral replication levels or disease severity. Core promoter TA mutation was frequent both in the HBeAg-positive patients and HBeAg-negative patients irrespective of viral replication levels or disease severity.

Claspin Promotes Normal Replication Fork Rates in Human Cells

PubMed Central

Helleday, Thomas; Caldecott, Keith W.

2008-01-01

The S phase-specific adaptor protein Claspin mediates the checkpoint response to replication stress by facilitating phosphorylation of Chk1 by ataxia-telangiectasia and Rad3-related (ATR). Evidence suggests that these components of the ATR pathway also play a critical role during physiological S phase. Chk1 is required for high rates of global replication fork progression, and Claspin interacts with the replication machinery and might therefore monitor normal DNA replication. Here, we have used DNA fiber labeling to investigate, for the first time, whether human Claspin is required for high rates of replication fork progression during normal S phase. We report that Claspin-depleted HeLa and HCT116 cells display levels of replication fork slowing similar to those observed in Chk1-depleted cells. This was also true in primary human 1BR3 fibroblasts, albeit to a lesser extent, suggesting that Claspin is a universal requirement for high replication fork rates in human cells. Interestingly, Claspin-depleted cells retained significant levels of Chk1 phosphorylation at both Ser317 and Ser345, raising the possibility that Claspin function during normal fork progression may extend beyond facilitating phosphorylation of either individual residue. Consistent with this possibility, depletion of Chk1 and Claspin together doubled the percentage of very slow forks, compared with depletion of either protein alone. PMID:18353973

Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen

PubMed Central

2013-01-01

Background HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex. Results Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades. Conclusions Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I. PMID:24229420

Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression.

PubMed

Franz, André; Pirson, Paul A; Pilger, Domenic; Halder, Swagata; Achuthankutty, Divya; Kashkar, Hamid; Ramadan, Kristijan; Hoppe, Thorsten

2016-02-04

The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging.

Knockdown of RMI1 impairs DNA repair under DNA replication stress.

PubMed

Xu, Chang; Fang, Lianying; Kong, Yangyang; Xiao, Changyan; Yang, Mengmeng; Du, Li-Qing; Liu, Qiang

2017-12-09

RMI1 (RecQ-mediated genome instability protein 1) forms a conserved BTR complex with BLM, Topo IIIα, and RMI2, and its absence causes genome instability. It has been revealed that RMI1 localizes to nuclear foci with BLM and Topo IIIα in response to replication stress, and that RMI1 functions downstream of BLM in promoting replication elongation. However, the precise functions of RMI1 during replication stress are not completely understood. Here we report that RMI1 knockdown cells are hypersensitive to hydroxyurea (HU). Using comet assay, we show that RMI1 knockdown cells exhibit accumulation of broken DNAs after being released from HU treatment. Moreover, we demonstrate that RMI1 facilitates the recovery from activated checkpoint and resuming the cell cycle after replicative stress. Surprisingly, loss of RMI1 results in a failure of RAD51 loading onto DNA damage sites. These findings reveal the importance of RMI1 in response to replication stress, which could explain the molecular basis for its function in maintaining genome integrity. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression

PubMed Central

Franz, André; Pirson, Paul A.; Pilger, Domenic; Halder, Swagata; Achuthankutty, Divya; Kashkar, Hamid; Ramadan, Kristijan; Hoppe, Thorsten

2016-01-01

The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging. PMID:26842564

A baculovirus (Bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells.

PubMed

Lu, M; Farrell, P J; Johnson, R; Iatrou, K

1997-12-05

It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant proteins and the capability of expression of proteins from genomic as well as cDNA sequences.

Identification and Characterization of Alternative Promoters, Transcripts and Protein Isoforms of Zebrafish R2 Gene

PubMed Central

Shang, Hanqiao; Li, Qing; Feng, Guohui; Cui, Zongbin

2011-01-01

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5′ termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress. PMID:21887375

Identification and characterization of alternative promoters, transcripts and protein isoforms of zebrafish R2 gene.

PubMed

Shang, Hanqiao; Li, Qing; Feng, Guohui; Cui, Zongbin

2011-01-01

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5' termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress.

Phosphatidic Acid Produced by Phospholipase D Promotes RNA Replication of a Plant RNA Virus

PubMed Central

Hyodo, Kiwamu; Taniguchi, Takako; Manabe, Yuki; Kaido, Masanori; Mise, Kazuyuki; Sugawara, Tatsuya; Taniguchi, Hisaaki; Okuno, Tetsuro

2015-01-01

Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate. PMID:26020241

A Case for Developing a Ground Based Replication of the Earth, Moon and Mars Spaceflight Infrastructure

NASA Technical Reports Server (NTRS)

Bradford, Robert N.; Best, Susan L.

2006-01-01

When the systems are developed and in place to provide the services needed to operate en route and on the Lunar and Martian surfaces, an Earth based replication will need to be in place for the safety and protection of mission success. The replication will entail all aspects of the flight configuration end to end but will not include any closed loop systems. This would replicate the infrastructure from Lunar and Martian robots, manned surface excursions, through man and unmanned terrestrial bases, through the various types of communication systems and technologies, manned and un-manned space vehicles (large and small), to Earth based systems and control centers. An Earth based replicated infrastructure will enable checkout and test of new technologies, hardware, software updates and upgrades and procedures without putting humans and missions at risk. Analysis of events, what ifs and trouble resolution could be played out on the ground to remove as much risk as possible from any type of proposed change to flight operational systems. With adequate detail, it is possible that failures could be predicted with a high probability and action taken to eliminate failures. A major factor in any mission to the Moon and to Mars is the complexity of systems, interfaces, processes, their limitations, associated risks and the factor of the unknown including the development by many contractors and NASA centers. The need to be able to introduce new technologies over the life of the program requires an end to end test bed to analyze and evaluate these technologies and what will happen when they are introduced into the flight system. The ability to analyze system behaviors end to end under varying conditions would enhance safety e.g. fault tolerances. This analysis along with the ability to mine data from the development environment (e.g. test data), flight ops and modeling/simulations data would provide a level of information not currently available to operations and astronauts. In this paper we will analyze the beginnings of such a replication and what it could do in terms of reducing risk in the near term for development. We will analyze the Space Shuttle Main Engine (SSME) test lab which has to a large extent accomplished this replication for the SSME and has been highly successful in analyzing hardware and software problems and changes. The cost of replicating the flight system as proposed here could be very high if attempted as an afterthought. We will describe the initial steps for the development of a replication of this infrastructure starting with the communication infrastructure. The Constellation of Labs (CofL) under the Command, Control, Communication and Information (C3I) project for the NASA Exploration Initiative will provide the initial foundation upon which to base this replication. Simply put, there is very little margin for error in high latency situations e.g. en-route to/from Mars or in an autonomous process on the Lunar far side. Any thought out approach to reduce risk and increase safety needs to be accomplished end to end with the actual systems configuration.

Switch from translation to RNA replication in a positive-stranded RNA virus

PubMed Central

Gamarnik, Andrea V.; Andino, Raul

1998-01-01

In positive-stranded viruses, the genomic RNA serves as a template for both translation and RNA replication. Using poliovirus as a model, we examined the interaction between these two processes. We show that the RNA polymerase is unable to replicate RNA templates undergoing translation. We discovered that an RNA structure at the 5′ end of the viral genome, next to the internal ribosomal entry site, carries signals that control both viral translation and RNA synthesis. The interaction of this RNA structure with the cellular factor PCBP up-regulates viral translation, while the binding of the viral protein 3CD represses translation and promotes negative-strand RNA synthesis. We propose that the interaction of 3CD with this RNA structure controls whether the genomic RNA is used for translation or RNA replication. PMID:9694795

Viral and cellular subnuclear structures in human cytomegalovirus-infected cells.

PubMed

Strang, Blair L

2015-02-01

In human cytomegalovirus (HCMV)-infected cells, a dramatic remodelling of the nuclear architecture is linked to the creation, utilization and manipulation of subnuclear structures. This review outlines the involvement of several viral and cellular subnuclear structures in areas of HCMV replication and virus-host interaction that include viral transcription, viral DNA synthesis and the production of DNA-filled viral capsids. The structures discussed include those that promote or impede HCMV replication (such as viral replication compartments and promyelocytic leukaemia nuclear bodies, respectively) and those whose role in the infected cell is unclear (for example, nucleoli and nuclear speckles). Viral and cellular proteins associated with subnuclear structures are also discussed. The data reviewed here highlight advances in our understanding of HCMV biology and emphasize the complexity of HCMV replication and virus-host interactions in the nucleus. © 2015 The Authors.

Nanobacteria: Fact or Fiction? Characteristics, Detection and Medical Importance of Novel Self-Replicating, Calcifying Nanoparticles

NASA Technical Reports Server (NTRS)

Ciftcioglu, Neva; Mckay, David S.; Mathew, Grace; Kajander, E. Olavi

2006-01-01

There is some debate in microbiology as to whether Nanobacteria (NB) are alive. This paper reviews some aspects of NB. In summary, Nanobacteria is a perfect model for studying biogenic mineralization/calcification because NB a) are self-replicating particles and have less complicated metabolic pathways b) accumulate calcium and phosphate under physiological conditions, c)produce a calcium phosphate mineral similar to bone, d) exist in physical conditions (pH, gravity, temperature, etc) that are easy to manipulate, and which can be replicated for the physiological model.

Probe-level linear model fitting and mixture modeling results in high accuracy detection of differential gene expression.

PubMed

Lemieux, Sébastien

2006-08-25

The identification of differentially expressed genes (DEGs) from Affymetrix GeneChips arrays is currently done by first computing expression levels from the low-level probe intensities, then deriving significance by comparing these expression levels between conditions. The proposed PL-LM (Probe-Level Linear Model) method implements a linear model applied on the probe-level data to directly estimate the treatment effect. A finite mixture of Gaussian components is then used to identify DEGs using the coefficients estimated by the linear model. This approach can readily be applied to experimental design with or without replication. On a wholly defined dataset, the PL-LM method was able to identify 75% of the differentially expressed genes within 10% of false positives. This accuracy was achieved both using the three replicates per conditions available in the dataset and using only one replicate per condition. The method achieves, on this dataset, a higher accuracy than the best set of tools identified by the authors of the dataset, and does so using only one replicate per condition.

A Drosophila Toolkit for the Visualization and Quantification of Viral Replication Launched from Transgenic Genomes

PubMed Central

Wernet, Mathias F.; Klovstad, Martha; Clandinin, Thomas R.

2014-01-01

Arthropod RNA viruses pose a serious threat to human health, yet many aspects of their replication cycle remain incompletely understood. Here we describe a versatile Drosophila toolkit of transgenic, self-replicating genomes (‘replicons’) from Sindbis virus that allow rapid visualization and quantification of viral replication in vivo. We generated replicons expressing Luciferase for the quantification of viral replication, serving as useful new tools for large-scale genetic screens for identifying cellular pathways that influence viral replication. We also present a new binary system in which replication-deficient viral genomes can be activated ‘in trans’, through co-expression of an intact replicon contributing an RNA-dependent RNA polymerase. The utility of this toolkit for studying virus biology is demonstrated by the observation of stochastic exclusion between replicons expressing different fluorescent proteins, when co-expressed under control of the same cellular promoter. This process is analogous to ‘superinfection exclusion’ between virus particles in cell culture, a process that is incompletely understood. We show that viral polymerases strongly prefer to replicate the genome that encoded them, and that almost invariably only a single virus genome is stochastically chosen for replication in each cell. Our in vivo system now makes this process amenable to detailed genetic dissection. Thus, this toolkit allows the cell-type specific, quantitative study of viral replication in a genetic model organism, opening new avenues for molecular, genetic and pharmacological dissection of virus biology and tool development. PMID:25386852

Novel cis-acting replication element in the adeno-associated virus type 2 genome is involved in amplification of integrated rep-cap sequences.

PubMed

Nony, P; Tessier, J; Chadeuf, G; Ward, P; Giraud, A; Dugast, M; Linden, R M; Moullier, P; Salvetti, A

2001-10-01

This study identifies a region of the adeno-associated virus type 2 (AAV-2) rep gene (nucleotides 190 to 540 of wild-type AAV-2) as a cis-acting Rep-dependent element able to promote the replication of transiently transfected plasmids. This viral element is also shown to be involved in the amplification of integrated sequences in the presence of adenovirus and Rep proteins.

Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins

PubMed Central

Hegde, Muralidhar L.; Hegde, Pavana M.; Bellot, Larry J.; Mandal, Santi M.; Hazra, Tapas K.; Li, Guo-Min; Boldogh, Istvan; Tomkinson, Alan E.; Mitra, Sankar

2013-01-01

Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a “cowcatcher” and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function. PMID:23898192

MAP kinase dependent cyclinE/cdk2 activity promotes DNA replication in early sea urchin embryos

PubMed Central

Kisielewska, J.; Philipova, R.; Huang, J.-Y.; Whitaker, M.

2009-01-01

Sea urchins provide an excellent model for studying cell cycle control mechanisms governing DNA replication in vivo. Fertilization and cell cycle progression are tightly coordinated by Ca2+ signals, but the mechanisms underlying the onset of DNA replication after fertilization remain less clear. In this study we demonstrate that calcium-dependent activation of ERK1 promotes accumulation of cyclinE/cdk2 into the male and female pronucleus and entry into first S-phase. We show that cdk2 activity rises quickly after fertilization to a maximum at 4 min, corresponding in timing to the early ERK1 activity peak. Abolishing MAP kinase activity after fertilization with MEK inhibitor, U0126, substantially reduces the early peak of cdk2 activity and prevents cyclinE and cdk2 accumulation in both sperm pronucleus and zygote nucleus in vivo. Both p27kip1 and roscovitine, cdk2 inhibitors, prevented DNA replication suggesting cdk2 involvement in this process in sea urchin. Inhibition of cdk2 activity using p27kip1 had no effect on the phosphorylation of MBP by ERK, but completely abolished phosphorylation of retinoblastoma protein, a cdk2 substrate, indicating that cdk2 activity is downstream of ERK1 activation. This pattern of regulation of DNA synthesis conforms to the pattern observed in mammalian somatic cells. PMID:19665013

The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ

PubMed Central

Hirota, Kouji; Yoshikiyo, Kazunori; Guilbaud, Guillaume; Tsurimoto, Toshiki; Murai, Junko; Tsuda, Masataka; Phillips, Lara G.; Narita, Takeo; Nishihara, Kana; Kobayashi, Kaori; Yamada, Kouich; Nakamura, Jun; Pommier, Yves; Lehmann, Alan; Sale, Julian E.; Takeda, Shunichi

2015-01-01

The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases. PMID:25628356

Analysis of model replication origins in Drosophila reveals new aspects of the chromatin landscape and its relationship to origin activity and the prereplicative complex

PubMed Central

Liu, Jun; McConnell, Kristopher; Dixon, Michael; Calvi, Brian R.

2012-01-01

Epigenetic regulation exerts a major influence on origins of DNA replication during development. The mechanisms for this regulation, however, are poorly defined. We showed previously that acetylation of nucleosomes regulates the origins that mediate developmental gene amplification during Drosophila oogenesis. Here we show that developmental activation of these origins is associated with acetylation of multiple histone lysines. Although these modifications are not unique to origin loci, we find that the level of acetylation is higher at the active origins and quantitatively correlated with the number of times these origins initiate replication. All of these acetylation marks were developmentally dynamic, rapidly increasing with origin activation and rapidly declining when the origins shut off and neighboring promoters turn on. Fine-scale analysis of the origins revealed that both hyperacetylation of nucleosomes and binding of the origin recognition complex (ORC) occur in a broad domain and that acetylation is highest on nucleosomes adjacent to one side of the major site of replication initiation. It was surprising to find that acetylation of some lysines depends on binding of ORC to the origin, suggesting that multiple histone acetyltransferases may be recruited during origin licensing. Our results reveal new insights into the origin epigenetic landscape and lead us to propose a chromatin switch model to explain the coordination of origin and promoter activity during development. PMID:22049023

Self-replication of chemical systems based on recognition within a double or a triple helix - A realistic hypothesis

NASA Technical Reports Server (NTRS)

Kanavarioti, Anastassia

1992-01-01

A scenario is proposed for the non-enzymatic self-replication of short RNA molecules. The self-replication of an oligopyrimidine strand is considered and the process of template-directed synthesis based on recognition within a double helix is discussed. Replication mechanisms are suggested for selected oligonucleotides. The mechanisms are based on Watson-Crick base pairing between complementary nucleotides as well as Hoogsteen base pairing between a duplex and the complementary third strand. It is suggested that self-replication based on these mechanisms may be accomplished but may result in a substantial amount of misinformation transfer when mixed oligonucleotides are used.

T135I substitution in the nonstructural protein 2C enhances foot-and-mouth disease virus replication.

PubMed

Yuan, Tiangang; Wang, Haiwei; Li, Chen; Yang, Decheng; Zhou, Guohui; Yu, Li

2017-12-01

The foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays an important role in viral replication, virulence, and host range. It has been shown that deletions of 10 or 19-20 amino acids in the C-terminal half of 3A attenuate serotype O and C FMDVs, which replicate poorly in bovine cells but normally in porcine-derived cells, and the C-terminal half of 3A is not essential for serotype Asia1 FMDV replication in BHK-21 cells. In this study, we constructed a 3A deletion FMDV mutant based on a serotype O FMDV, the wild-type virus O/YS/CHA/05, with a 60-amino acid deletion in the 3A protein sequence, between residues 84 and 143. The rescued virus O/YS/CHA/05-Δ3A exhibited slower growth kinetics and formed smaller plaques compared to O/YS/CHA/05 in both BHK-21 and IBRS-2 cells, indicating that the 60-amino acid deletion in the 3A protein impaired FMDV replication. After 14 passages in BHK-21 cells, the replication capacity of the passaged virus O/YS/CHA/05-Δ3A-P14 returned to a level similar to the wild-type virus, suggesting that amino acid substitutions responsible for the enhanced replication capacity occurred in the genome of O/YS/CHA/05-Δ3A-P14. By sequence analysis, two amino acid substitutions, P153L in VP1 and T135I in 2C, were found in the O/YS/CHA/05-Δ3A-P14 genome compared to the O/YS/CHA/05-Δ3A genome. Subsequently, the amino acid substitutions VP1 P153L and 2C T135I were separately introduced into O/YS/CHA/05-Δ3A to rescue mutant viruses for examining their growth kinetics. Results showed that the 2C T135I instead of the VP1 P153L enhanced the virus replication capacity. The 2C T135I substitution also improved the replication of the wild-type virus, indicating that the effect of 2C T135I substitution on FMDV replication is not associated with the 3A deletion. Furthermore, our results showed that the T135I substitution in the nonstructural protein 2C enhanced O/YS/CHA/05 replication through promoting viral RNA synthesis.

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages

PubMed Central

Lathrop, Stephanie K.; Binder, Kelsey A.; Starr, Tregei; Cooper, Kendal G.; Chong, Audrey; Carmody, Aaron B.

2015-01-01

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages. PMID:25895967

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages.

PubMed

Lathrop, Stephanie K; Binder, Kelsey A; Starr, Tregei; Cooper, Kendal G; Chong, Audrey; Carmody, Aaron B; Steele-Mortimer, Olivia

2015-07-01

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

76 FR 3638 - Nominations Requested for the 2011 Healthy Living Innovation Awards

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-20

... health promotion areas: (1) Healthy weight; (2) Physical activity; and (3) Nutrition. The Department... consideration upon review: Creativity and Innovation Leadership Sustainability Replicability Results/Outcomes...

The 5'-poly(A) leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation

PubMed Central

Dhungel, Pragyesh; Cao, Shuai

2017-01-01

The poly(A) leader at the 5’-untranslated region (5’-UTR) is an unusually striking feature of all poxvirus mRNAs transcribed after viral DNA replication (post-replicative mRNAs). These poly(A) leaders are non-templated and of heterogeneous lengths; and their function during poxvirus infection remains a long-standing question. Here, we discovered that a 5’-poly(A) leader conferred a selective translational advantage to mRNA in poxvirus-infected cells. A constitutive and uninterrupted 5’-poly(A) leader with 12 residues was optimal. Because the most frequent lengths of the 5’-poly(A) leaders are 8–12 residues, the result suggests that the poly(A) leader has been evolutionarily optimized to boost poxvirus protein production. A 5’-poly(A) leader also could increase protein production in the bacteriophage T7 promoter-based expression system of vaccinia virus, the prototypic member of poxviruses. Interestingly, although vaccinia virus post-replicative mRNAs do have 5’- methylated guanosine caps and can use cap-dependent translation, in vaccinia virus-infected cells, mRNA with a 5’-poly(A) leader could also be efficiently translated in cells with impaired cap-dependent translation. However, the translation was not mediated through an internal ribosome entry site (IRES). These results point to a fundamental mechanism poxvirus uses to efficiently translate its post-replicative mRNAs. PMID:28854224

Paucity and preferential suppression of transgenes in late replication domains of the D. melanogaster genome.

PubMed

Babenko, Vladimir N; Makunin, Igor V; Brusentsova, Irina V; Belyaeva, Elena S; Maksimov, Daniil A; Belyakin, Stepan N; Maroy, Peter; Vasil'eva, Lyubov A; Zhimulev, Igor F

2010-05-21

Eukaryotic genomes are organized in extended domains with distinct features intimately linking genome structure, replication pattern and chromatin state. Recently we identified a set of long late replicating euchromatic regions that are underreplicated in salivary gland polytene chromosomes of D. melanogaster. Here we demonstrate that these underreplicated regions (URs) have a low density of P-element and piggyBac insertions compared to the genome average or neighboring regions. In contrast, Minos-based transposons show no paucity in URs but have a strong bias to testis-specific genes. We estimated the suppression level in 2,852 stocks carrying a single P-element by analysis of eye color determined by the mini-white marker gene and demonstrate that the proportion of suppressed transgenes in URs is more than three times higher than in the flanking regions or the genomic average. The suppressed transgenes reside in intergenic, genic or promoter regions of the annotated genes. We speculate that the low insertion frequency of P-elements and piggyBacs in URs partially results from suppression of transgenes that potentially could prevent identification of transgenes due to complete suppression of the marker gene. In a similar manner, the proportion of suppressed transgenes is higher in loci replicating late or very late in Kc cells and these loci have a lower density of P-elements and piggyBac insertions. In transgenes with two marker genes suppression of mini-white gene in eye coincides with suppression of yellow gene in bristles. Our results suggest that the late replication domains have a high inactivation potential apparently linked to the silenced or closed chromatin state in these regions, and that such inactivation potential is largely maintained in different tissues.

Association study of genes controlling IL-12-dependent IFN-γ immunity: STAT4 alleles increase risk of pulmonary tuberculosis in Morocco.

PubMed

Sabri, Ayoub; Grant, Audrey V; Cosker, Kristel; El Azbaoui, Safa; Abid, Ahmed; Abderrahmani Rhorfi, Ismail; Souhi, Hicham; Janah, Hicham; Alaoui-Tahiri, Kebir; Gharbaoui, Yasser; Benkirane, Majid; Orlova, Marianna; Boland, Anne; Deswarte, Caroline; Migaud, Melanie; Bustamante, Jacinta; Schurr, Erwin; Boisson-Dupuis, Stephanie; Casanova, Jean-Laurent; Abel, Laurent; El Baghdadi, Jamila

2014-08-15

Only a minority of individuals infected with Mycobacterium tuberculosis develop clinical tuberculosis. Genetic epidemiological evidence suggests that pulmonary tuberculosis has a strong human genetic component. Previous genetic findings in Mendelian predisposition to more severe mycobacterial infections, including by M. tuberculosis, underlined the importance of the interleukin 12 (IL-12)/interferon γ (IFN-γ) circuit in antimycobacterial immunity. We conducted an association study in Morocco between pulmonary tuberculosis and a panel of single-nucleotide polymorphisms (SNPs) covering 14 core IL-12/IFN-γ circuit genes. The analyses were performed in a discovery family-based sample followed by replication in a case-control population. Out of 228 SNPs tested in the family-based sample, 6 STAT4 SNPs were associated with pulmonary tuberculosis (P = .0013-.01). We replicated the same direction of association for 1 cluster of 3 SNPs encompassing the promoter region of STAT4. In the combined sample, the association was stronger among younger subjects (pulmonary tuberculosis onset <25 years) with an odds ratio of developing pulmonary tuberculosis at rs897200 for GG vs AG/AA subjects of 1.47 (1.06-2.04). Previous functional experiments showed that the G allele of rs897200 was associated with lower STAT4 expression. Our present findings in a Moroccan population support an association of pulmonary tuberculosis with STAT4 promoter-region polymorphisms that may impact STAT4 expression. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.

Interferon alpha inhibits viral replication of a live-attenuated porcine reproductive and respiratory syndrome virus vaccine preventing development of an adaptive immune response in swine

USDA-ARS?s Scientific Manuscript database

Type I interferons, such as interferon alpha (IFNa), contribute to innate antiviral immunity by promoting production of antiviral mediators and are also involved in promoting an adaptive immune response. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating and c...

Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

PubMed Central

Joseph, Joan; Fernández-Lloris, Raquel; Pezzat, Elías; Saubi, Narcís; Cardona, Pere-Joan; Mothe, Beatriz; Gatell, Josep Maria

2010-01-01

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261) and Mycobacteria spp. α-antigen promoter (in plasmid pJH222). Among 14 rBCG:HIV-1gp120 (pMV261) colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222) colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors. PMID:20617151

Role of the Adenovirus DNA-Binding Protein in In Vitro Adeno-Associated Virus DNA Replication

PubMed Central

Ward, Peter; Dean, Frank B.; O’Donnell, Michael E.; Berns, Kenneth I.

1998-01-01

A basic question in adeno-associated virus (AAV) biology has been whether adenovirus (Ad) infection provided any function which directly promoted replication of AAV DNA. Previously in vitro assays for AAV DNA replication, using linear duplex AAV DNA as the template, uninfected or Ad-infected HeLa cell extracts, and exogenous AAV Rep protein, demonstrated that Ad infection provides a direct helper effect for AAV DNA replication. It was shown that the nature of this helper effect was to increase the processivity of AAV DNA replication. Left unanswered was the question of whether this effect was the result of cellular factors whose activity was enhanced by Ad infection or was the result of direct participation of Ad proteins in AAV DNA replication. In this report, we show that in the in vitro assay, enhancement of processivity occurs with the addition of either the Ad DNA-binding protein (Ad-DBP) or the human single-stranded DNA-binding protein (replication protein A [RPA]). Clearly Ad-DBP is present after Ad infection but not before, whereas the cellular level of RPA is not apparently affected by Ad infection. However, we have not measured possible modifications of RPA which might occur after Ad infection and affect AAV DNA replication. When the substrate for replication was an AAV genome inserted into a plasmid vector, RPA was not an effective substitute for Ad-DBP. Extracts supplemented with Ad-DBP preferentially replicated AAV sequences rather than adjacent vector sequences; in contrast, extracts supplemented with RPA preferentially replicated vector sequences. PMID:9420241

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Perturbs the G1-S Cell Cycle Progression via Deregulation of the cyclin D1 Gene.

PubMed

Lee, Hye-Ra; Mitra, Jaba; Lee, Stacy; Gao, Shou-Jiang; Oh, Tae-Kwang; Kim, Myung Hee; Ha, Taekjip; Jung, Jae U

2016-01-15

Kaposi's sarcoma-associated herpesvirus (KSHV) infection modulates the host cell cycle to create an environment optimal for its viral-DNA replication during the lytic life cycle. We report here that KSHV vIRF4 targets the β-catenin/CBP cofactor and blocks its occupancy on the cyclin D1 promoter, suppressing the G1-S cell cycle progression and enhancing KSHV replication. This shows that KSHV vIRF4 suppresses host G1-S transition, possibly providing an intracellular milieu favorable for its replication. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Forging Ahead through Darkness: PCNA, Still the Principal Conductor at the Replication Fork.

PubMed

Choe, Katherine N; Moldovan, George-Lucian

2017-02-02

Proliferating cell nuclear antigen (PCNA) lies at the center of the faithful duplication of eukaryotic genomes. With its distinctive doughnut-shaped molecular structure, PCNA was originally studied for its role in stimulating DNA polymerases. However, we now know that PCNA does much more than promote processive DNA synthesis. Because of the complexity of the events involved, cellular DNA replication poses major threats to genomic integrity. Whatever predicament lies ahead for the replication fork, PCNA is there to orchestrate the events necessary to handle it. Through its many protein interactions and various post-translational modifications, PCNA has far-reaching impacts on a myriad of cellular functions. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanisms of bacterial DNA replication restart

PubMed Central

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

The role of MatP, ZapA and ZapB in chromosomal organization and dynamics in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mannik, Jaana; Castillo, Daniel E.; Yang, Da

Despite extensive research over several decades, a comprehensive view of how the Escherichia coli chromosome is organized within the nucleoid, and how two daughter chromosomes segregate has yet to emerge. Here we investigate the role of the MatP, ZapA and ZapB proteins in organizing the replication terminus (Ter) region and in the chromosomal segregation process. Quantitative image analysis of the fluorescently labeled Ter region shows that the replication terminus attaches to the divisome in a single segment along the perimeter of the cell in a MatP, ZapA and ZapB-dependent manner. The attachment does not significantly affect the bulk chromosome segregationmore » in slow growth conditions. With or without the attachment, two chromosomal masses separate from each other at a speed comparable to the cell growth. The separation starts even before the replication terminus region positions itself at the center of the nucleoid. Modeling of the segregation based on conformational entropy correctly predicts the positioning of the replication terminus region within the nucleoid. Furthermore, the model produces a distinctly different chromosomal density distribution than the experiment, indicating that the conformational entropy plays a limited role in segregating the chromosomes in the late stages of replication.« less

The role of MatP, ZapA and ZapB in chromosomal organization and dynamics in Escherichia coli

DOE PAGES

Mannik, Jaana; Castillo, Daniel E.; Yang, Da; ...

2016-01-13

Despite extensive research over several decades, a comprehensive view of how the Escherichia coli chromosome is organized within the nucleoid, and how two daughter chromosomes segregate has yet to emerge. Here we investigate the role of the MatP, ZapA and ZapB proteins in organizing the replication terminus (Ter) region and in the chromosomal segregation process. Quantitative image analysis of the fluorescently labeled Ter region shows that the replication terminus attaches to the divisome in a single segment along the perimeter of the cell in a MatP, ZapA and ZapB-dependent manner. The attachment does not significantly affect the bulk chromosome segregationmore » in slow growth conditions. With or without the attachment, two chromosomal masses separate from each other at a speed comparable to the cell growth. The separation starts even before the replication terminus region positions itself at the center of the nucleoid. Modeling of the segregation based on conformational entropy correctly predicts the positioning of the replication terminus region within the nucleoid. Furthermore, the model produces a distinctly different chromosomal density distribution than the experiment, indicating that the conformational entropy plays a limited role in segregating the chromosomes in the late stages of replication.« less

A functional analysis of social reinforcement in vicarious verbal conditioning.

PubMed

Donohue, G B; Tryon, W W

1985-01-01

This article reports the results of 629 subjects in three experiments designed to replicate and extend the phenomenon of vicarious verbal conditioning. Experiment I replicated the finding that subjects who responded most to vicarious verbal conditioning were aware of the contingency involved. Experiment II attempted to examine the effects of prior history with the verbal reinforcer on vicarious verbal conditioning by providing seven groups of subjects with varying classic conditioning histories prior to vicarious verbal conditioning. The null results associated with this experiment were hypothesized to be due to the fact that the vicarious verbal conditioning took place in a language laboratory where the subjects could hear but not see the model. Experiment III replicated Experiment II in a live group context as was done in Experiment I. The results showed that vicarious verbal conditioning was again found to take place, that associating the verbal reinforcer with a tone or tone plus money via forward classic conditioning potentiated the effects of the verbal reinforcer, that backward classic conditioning did not potentiate the reinforcer, d) nor did either of two sensitization procedures potentiate the effects of the verbal reinforcer. Both aware and unaware subjects evidenced vicarious verbal conditioning.

PUTATIVE GENE PROMOTER SEQUENCES IN THE CHLORELLA VIRUSES

PubMed Central

Fitzgerald, Lisa A.; Boucher, Philip T.; Yanai-Balser, Giane; Suhre, Karsten; Graves, Michael V.; Van Etten, James L.

2008-01-01

Three short (7 to 9 nucleotides) highly conserved nucleotide sequences were identified in the putative promoter regions (150 bp upstream and 50 bp downstream of the ATG translation start site) of three members of the genus Chlorovirus, family Phycodnaviridae. Most of these sequences occurred in similar locations within the defined promoter regions. The sequence and location of the motifs were often conserved among homologous ORFs within the Chlorovirus family. One of these conserved sequences (AATGACA) is predominately associated with genes expressed early in virus replication. PMID:18768195

Oxidative peptide /and amide/ formation from Schiff base complexes

NASA Technical Reports Server (NTRS)

Strehler, B. L.; Li, M. P.; Martin, K.; Fliss, H.; Schmid, P.

1982-01-01

One hypothesis of the origin of pre-modern forms of life is that the original replicating molecules were specific polypeptides which acted as templates for the assembly of poly-Schiff bases complementary to the template, and that these polymers were then oxidized to peptide linkages, probably by photo-produced oxidants. A double cycle of such anti-parallel complementary replication would yield the original peptide polymer. If this model were valid, the Schiff base between an N-acyl alpha mino aldehyde and an amino acid should yield a dipeptide in aqueous solution in the presence of an appropriate oxidant. In the present study it is shown that the substituted dipeptide, N-acetyl-tyrosyl-tyrosine, is produced in high yield in aqueous solution at pH 9 through the action of H2O2 on the Schiff-base complex between N-acetyl-tyrosinal and tyrosine and that a great variety of N-acyl amino acids are formed from amino acids and aliphatic aldehydes under similar conditions.

A new paradigm for transcription factor TFIIB functionality

PubMed Central

Gelev, Vladimir; Zabolotny, Janice M.; Lange, Martin; Hiromura, Makoto; Yoo, Sang Wook; Orlando, Joseph S.; Kushnir, Anna; Horikoshi, Nobuo; Paquet, Eric; Bachvarov, Dimcho; Schaffer, Priscilla A.; Usheva, Anny

2014-01-01

Experimental and bioinformatic studies of transcription initiation by RNA polymerase II (RNAP2) have revealed a mechanism of RNAP2 transcription initiation less uniform across gene promoters than initially thought. However, the general transcription factor TFIIB is presumed to be universally required for RNAP2 transcription initiation. Based on bioinformatic analysis of data and effects of TFIIB knockdown in primary and transformed cell lines on cellular functionality and global gene expression, we report that TFIIB is dispensable for transcription of many human promoters, but is essential for herpes simplex virus-1 (HSV-1) gene transcription and replication. We report a novel cell cycle TFIIB regulation and localization of the acetylated TFIIB variant on the transcriptionally silent mitotic chromatids. Taken together, these results establish a new paradigm for TFIIB functionality in human gene expression, which when downregulated has potent anti-viral effects. PMID:24441171

An intervention to promote physical activity in Mexican elementary school students: building public policy to prevent noncommunicable diseases.

PubMed

Polo-Oteyza, Ernestina; Ancira-Moreno, Mónica; Rosel-Pech, Cecilia; Sánchez-Mendoza, María Teresa; Salinas-Martínez, Vicente; Vadillo-Ortega, Felipe

2017-01-01

Physical activity is an important component of strategies for health promotion and prevention of noncommunicable diseases. It is also associated with decreased risk for cardiovascular disease in overweight and obese adults and children. This article addresses the initial description of a physical activity intervention for children attending public elementary schools in Mexico. The objective was to develop a replicable model based on a strategic public, private, academic, and social partnership that would have a short-term impact on the metabolic health of children and be useful for building effective public policy. Forty-nine schools (20 000 students) participated, and 5 schools were selected for evaluation. The intervention included a 30-minute supervised middle-effort interchangeable routine, 5 days a week for a complete school year, adapted for different school conditions and students of different ages. Evaluation included anthropometric measurements and biochemical markers. Actual prevalence of combined overweight and obesity in these children was 31.9%. The intervention was successfully implemented in all schools. No change in body mass index, waist circumference, or other anthropometric indicators was found. However, changes in biochemical markers showed a significant decrease in blood glucose, total cholesterol, and cholesterol-low-density lipoproteins, reflecting a positive effect on cardiovascular health indicators. © The Author(s) 2016. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Mobilizing the private sector. Indonesia.

PubMed

Ferraz-tabor, L

1993-12-01

The national diarrheal disease control program has made progress against diarrheal illness in Indonesia, but diarrhea still accounts for 130,000 deaths of under-5 year olds annually. The potential of the private sector had been virtually untapped until the PRITECH Project designed and implemented a private sector based program to complement government efforts to reduce childhood morbidity and mortality related to diarrhea. PRITECH wanted to motivate commercial firms to invest the capital necessary to commercialize oral rehydration salts (ORS) so that program self-sufficiency would be maximized over the long term without depending upon donor support. Sections describe the lack of commercial sector ORS promotion, supply and demand factors, raising interest in ORS, conditions for change, the potential market for ORS, demand, production capacity, previous collaborative efforts, areas for improvement, PRITECH's role as catalyst, coordination with commercial companies, marketing workshops, collaboration with the Indonesian Medical Association, the handwashing campaign, and program impact. Although the program has been in place for just 1 year, sales of ORS have increased along with the degree of sustainable collaboration among local institutions. The author stresses that for the model to be successfully replicated elsewhere, the government, medical and pediatric associations, the World Health Organization, UNICEF, groups which can promote oral rehydration therapy, and opinion leaders in the medical community must be involved. Finally, note is made that the image of ORS must be improved and that the work of the public and commercial sectors can be complementary.

Mobile phone-based asthma self-management aid for adolescents (mASMAA): a feasibility study.

PubMed

Rhee, Hyekyun; Allen, James; Mammen, Jennifer; Swift, Mary

2014-01-01

Adolescents report high asthma-related morbidity that can be prevented by adequate self-management of the disease. Therefore, there is a need for a developmentally appropriate strategy to promote effective asthma self-management. Mobile phone-based technology is portable, commonly accessible, and well received by adolescents. The purpose of this study was to develop and evaluate the feasibility and acceptability of a comprehensive mobile phone-based asthma self-management aid for adolescents (mASMAA) that was designed to facilitate symptom monitoring, treatment adherence, and adolescent-parent partnership. The system used state-of-the-art natural language-understanding technology that allowed teens to use unconstrained English in their texts, and to self-initiate interactions with the system. mASMAA was developed based on an existing natural dialogue system that supports broad coverage of everyday natural conversation in English. Fifteen adolescent-parent dyads participated in a 2-week trial that involved adolescents' daily scheduled and unscheduled interactions with mASMAA and parents responding to daily reports on adolescents' asthma condition automatically generated by mASMAA. Subsequently, four focus groups were conducted to systematically obtain user feedback on the system. Frequency data on the daily usage of mASMAA over the 2-week period were tabulated, and content analysis was conducted for focus group interview data. Response rates for daily text messages were 81%-97% in adolescents. The average number of self-initiated messages to mASMAA was 19 per adolescent. Symptoms were the most common topic of teen-initiated messages. Participants concurred that use of mASMAA improved awareness of symptoms and triggers, promoted treatment adherence and sense of control, and facilitated adolescent-parent partnerships. This study demonstrates the utility and user acceptability of mASMAA as a potential asthma self-management tool in a selective group of adolescents. Further research is needed to replicate the findings in a large group of adolescents from sociodemographically diverse backgrounds to validate the findings.

Prior task experience and comparable stimulus exposure nullify focal and nonfocal prospective memory retrieval differences.

PubMed

Hicks, Jason L; Franks, Bryan A; Spitler, Samantha N

2017-10-01

We explored the nature of focal versus nonfocal event-based prospective memory retrieval. In the context of a lexical decision task, people received an intention to respond to a single word (focal) in one condition and to a category label (nonfocal) for the other condition. Participants experienced both conditions, and their order was manipulated. The focal instruction condition was a single word presented multiple times. In Experiment 1, the stimuli in the nonfocal condition were different exemplars from a category, each presented once. In the nonfocal condition retrieval was poorer and reaction times were slower during the ongoing task as compared to the focal condition, replicating prior findings. In Experiment 2, the stimulus in the nonfocal condition was a single category exemplar repeated multiple times. When this single-exemplar nonfocal condition followed in time the single-item focal condition, focal versus nonfocal performance was virtually indistinguishable. These results demonstrate that people can modify their stimulus processing and expectations in event-based prospective memory tasks based on experience with the nature of prospective cues and with the ongoing task.

An Inadvertent Concurrent Replication: Same Roadmap, Different Journey

ERIC Educational Resources Information Center

Lemons, Christopher J.; King, Seth A.; Davidson, Kimberly A.; Berryessa, Teresa L.; Gajjar, Shimul A.; Sacks, Lia H.

2016-01-01

Replication is a critical aspect of scientific inquiry that presents a variety of challenges to researchers, even under the best of conditions. We conducted a review of replication rates in special education journals similar to the review conducted by Makel et al. in this issue. Unknowingly conducting independent reviews allowed for an unexpected…

Replication of associations between LRP5 and ESRRA variants and bone density in premenopausal women.

PubMed

Giroux, S; Elfassihi, L; Cole, D E C; Rousseau, F

2008-12-01

Replication is a critical step to validate positive genetic associations. In this study, we tested two previously reported positive associations. The low density lipoprotein receptor-related protein 5 (LRP5) Val667Met and lumbar spine bone density are replicated. This result is in line with results from large consortiums such as Genomos. However, the estrogen-related receptor alpha (ESRRA) repeat in the promoter is not replicated although the polymorphism studied was functional and could have been a causative variant. We sought to validate associations previously reported between LRP5 V667M polymorphism and lumbar spine (LS, p = 0.013) and femoral neck (FN, p = 0.0002) bone mineral density (BMD), and between ESRRA 23 base pair repeat polymorphism and LS BMD (p = 0.0036) in a sample of premenopausal Caucasian women using an independent sample. For the replication sample, we recruited 673 premenopausal women from the Toronto metropolitan area. All women were Caucasian and had BMD measured. LRP5 V667M was genotyped by allele-specific PCR and ESRRA repeats by sizing of PCR products on agarose gels. We reproduced the same association as we reported previously between LRP5 V667M and LS BMD (p = 0.015) but not with FN BMD (p = 0.254). The combined data from the two populations indicate an effect size of 0.28SD for LS BMD (p = 0.00048) and an effect size of 0.26 SD for FN BMD (p = 0.00037). In contrast, the association we reported earlier between ESRRA repeats and LS BMD was not replicated in the sample from Toronto (p = 0.645). The association between LRP5 V667M and LS BMD is confirmed but not that between ESRRA repeats and LS BMD. This result indicates that it is imperative to validate any positive association in an independent sample.

The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating assembly of the viral replicase complex.

PubMed

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 of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, and in vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The evolution of replicators.

PubMed Central

Szathmáry, E

2000-01-01

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

The evolution of replicators.

PubMed

Szathmáry, E

2000-11-29

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

Bridging from Replication to Translation with a Thermal, Autonomous Replicator Made from Transfer RNA

NASA Astrophysics Data System (ADS)

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

2013-03-01

Central to the understanding of living systems is the interplay between DNA/RNA and proteins. Known as Eigen paradox, proteins require genetic information while proteins are needed for the replication of genes. RNA world scenarios focus on a base by base replication disconnected from translation. Here we used strategies from DNA machines to demonstrate a tight connection between a basic replication mechanism and translation. A pool of hairpin molecules replicate a two-letter code. The replication is thermally driven: the energy and negative entropy to drive replication is initially stored in metastable hairpins by kinetic cooling. Both are released by a highly specific and exponential replication reaction that is solely implemented by base hybridization. The duplication time is 30s. The reaction is monitored by fluorescence and described by a detailed kinetic model. The RNA hairpins usetransfer RNA sequences and the replication is driven by the simple disequilibrium setting of a thermal gradient The experiments propose a physical rather than a chemical scenario for the autonomous replication of protein encoding information. Supported by the NanoSystems Initiative Munich and ERC.

Imperfect Symmetry of Sp1 and Core Promoter Sequences Regulates Early and Late Virus Gene Expression of the Bidirectional BK Polyomavirus Noncoding Control Region.

PubMed

Bethge, Tobias; Ajuh, Elvis; Hirsch, Hans H

2016-11-15

Rearrangements or point mutations in the noncoding control region (NCCR) of BK polyomavirus (BKPyV) have been associated with higher viral loads and more pronounced organ pathology in immunocompromised patients. The respective alterations affect a multitude of transcription factor binding sites (TFBS) but consistently cause increased expression of the early viral gene region (EVGR) at the expense of late viral gene region (LVGR) expression. By mutating TFBS, we identified three phenotypic groups leading to strong, intermediate, or impaired EVGR expression and corresponding BKPyV replication. Unexpectedly, Sp1 TFBS mutants either activated or inhibited EVGR expression when located proximal to the LVGR (sp1-4) or the EVGR (sp1-2), respectively. We now demonstrate that the bidirectional balance of EVGR and LVGR expression is dependent on affinity, strand orientation, and the number of Sp1 sites. Swapping the LVGR-proximal high-affinity SP1-4 with the EVGR-proximal low-affinity SP1-2 in site strand flipping or inserting an additional SP1-2 site caused a rearranged NCCR phenotype of increased EVGR expression and faster BKPyV replication. The 5' rapid amplification of cDNA ends revealed an imperfect symmetry between the EVGR- and LVGR-proximal parts of the NCCR, consisting of TATA and TATA-like elements, initiator elements, and downstream promoter elements. Mutation or deletion of the archetypal LVGR promoter, which is found in activated NCCR variants, abrogated LVGR expression, which could be restored by providing large T antigen (LTag) in trans Thus, whereas Sp1 sites control the initial EVGR-LVGR expression balance, LTag expression can override inactivation of the LVGR promoter and acts as a key driver of LVGR expression independently of the Sp1 sites and core promoter elements. Polyomaviridae currently comprise more than 70 members, including 13 human polyomaviruses (PyVs), all of which share a bidirectional genome organization mediated by the NCCR, which determines species and host cell specificity, persistence, replication, and virulence. Here, we demonstrate that the BKPyV NCCR is fine-tuned by an imperfect symmetry of core promoter elements centered around TATA and TATA-like sequences close to the EVGR and LVGR, respectively, which are governed by the directionality and affinity of two Sp1 sites. The data indicated that the BKPyV NCCR is poised toward EVGR expression, which can be readily unlatched by a simple switch affecting Sp1 binding. The resulting LTag, which is the major EVGR protein, drives viral genome replication, renders subsequent LVGR expression independently of archetypal promoter elements, and can overcome enhancer/promoter mutations and deletions. The data are pivotal for understanding how human PyV NCCRs mediate secondary host cell specificity, reactivation, and virulence in their natural hosts. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Temperature and Nutrient Effects on Periphyton Associated Bacterial Communities in Continuous Flow-Through Estuarine Mesocosms

NASA Astrophysics Data System (ADS)

Houghton, K.; James, J. B.; Devereux, R.; Friedman, S. D.

2016-02-01

Nutrient pollution is a leading cause of water quality impairments and degraded aquatic ecosystem condition. Reliable and reproducible indicators of ecosystem condition are needed to help manage nutrient pollution. The diatom component of periphyton has been used as a water quality indicator due to identifiable cell morphology and existence of relationships between nutrient concentration and diatom community composition. However, morphological identification of diatoms requires highly specialized personnel, is very time consuming, and can produce variable results, suggesting the need for alternative methods that are less expensive and more reproducible. DNA sequencing of the bacterial 16S rRNA gene is well documented and provides genus-level resolution of the community structure. The goal of this study was to evaluate the effects of nutrient loading and temperature on periphyton-associated bacterial communities using standard periphytometer techniques and next generation sequencing technologies. Continuous flow mesocosms were established in an eight tank system consisting of two temperature conditions (10°C and 20°C) and four nutrient conditions (1x to 6x ambient concentrations). Experimental conditions were replicated in July/August 2013 and September 2013. Replicate DNA samples were extracted and the 16S rRNA gene was sequenced using universal Bacterial primers. Initial analyses revealed strong differences in community structure based on temperature (p < 0.01, R = 0.997) and sampling month (p < 0.01, R = 0.993) while no significant differences were detected between nutrient treatments. These results suggest that the method can detect changes in periphyton associated bacterial communities based on temperature but a more refined approach, as might be based on functional genes instead of structural genes, may be needed to differentiate nutrient effects.

Design of experiment for optimization of plasma-polymerized octafluorocyclobutane coating on very high aspect ratio silicon molds.

PubMed

Yeo, L P; Yan, Y H; Lam, Y C; Chan-Park, Mary B

2006-11-21

As-fabricated deep reactive ion etched (DRIE) silicon mold with very high aspect ratio (>10) feature patterns is unsuitable for poly(dimethylsiloxane) (PDMS) replication because of the strong interaction between the Si surface and the replica and the corrugated mold sidewalls. The silicon mold can be conveniently passivated via plasma polymerization of octafluorocyclobutane (C4F8), which is also employed in the DRIE process itself, to enable the mold to be used repeatedly. To optimize the passivation conditions, we have undertaken a Box-Behnken experimental design on the basis of three passivation process parameters (plasma power, C4F8 flow rate, and deposition time). The measured responses were fluorinated film thickness, demolding status/success, demolding force, and fluorine/carbon ratio on the fifth replica surface. The optimal passivation process conditions were predicted to be an input power of 195 W, a C4F8 flow rate of 57 sccm, and a deposition time of 364 s; these were verified experimentally to have high accuracy. Demolding success requires medium-deposited film thickness (66-91 nm), and the thickness of the deposited films correlated strongly with deposition time. At moderate to high ranges, increased plasma power or gas flow rate promoted polymerization over reactive etching of the film. It was also found that small quantities of the fluorinated surface were transferred from the Si mold to the PDMS at each replication, entailing progressive wear of the fluorinated layer.

Increasing tobacco quitline calls from pregnant african american women: the "one tiny reason to quit" social marketing campaign.

PubMed

Kennedy, May G; Genderson, Maureen Wilson; Sepulveda, Allison L; Garland, Sheryl L; Wilson, Diane Baer; Stith-Singleton, Rose; Dubuque, Susan

2013-05-01

Pregnant African American women are at disproportionately high risk of premature birth and infant mortality, outcomes associated with cigarette smoking. Telephone-based, individual smoking cessation counseling has been shown to result in successful quit attempts in the general population and among pregnant women, but "quitlines" are underutilized. A social marketing campaign called One Tiny Reason to Quit (OTRTQ) promoted calling a quitline (1-800-QUIT-NOW) to pregnant, African American women in Richmond, Virginia, in 2009 and was replicated there 2 years later. The campaign disseminated messages via radio, interior bus ads, posters, newspaper ads, and billboards. Trained volunteers also delivered messages face-to-face and distributed branded give-away reminder items. The number of calls made from pregnant women in the Richmond area during summer 2009 was contrasted with (a) the number of calls during the seasons immediately before and after the campaign, and (b) the number of calls the previous summer. The replication used the same evaluation design. There were statistically significant spikes in calls from pregnant women during both campaign waves for both types of contrasts. A higher proportion of the calls from pregnant women were from African Americans during the campaign. A multimodal quitline promotion like OTRTQ should be considered for geographic areas with sizable African American populations and high rates of infant mortality.

A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom[C][W][OPEN

PubMed Central

Tulin, Frej; Cross, Frederick R.

2014-01-01

Research in yeast and animals has resulted in a well-supported consensus model for eukaryotic cell cycle control. The fit of this model to early diverging eukaryotes, such as the plant kingdom, remains unclear. Using the green alga Chlamydomonas reinhardtii, we developed an efficient pipeline, incorporating robotics, semiautomated image analysis, and deep sequencing, to molecularly identify >50 genes, mostly conserved in higher plants, specifically required for cell division but not cell growth. Mutated genes include the cyclin-dependent kinases CDKA (resembling yeast and animal Cdk1) and the plant-specific CDKB. The Chlamydomonas cell cycle consists of a long G1 during which cells can grow >10-fold, followed by multiple rapid cycles of DNA replication and segregation. CDKA and CDKB execute nonoverlapping functions: CDKA promotes transition between G1 and entry into the division cycle, while CDKB is essential specifically for spindle formation and nuclear division, but not for DNA replication, once CDKA-dependent initiation has occurred. The anaphase-promoting complex is required for similar steps in the Chlamydomonas cell cycle as in Opisthokonts; however, the spindle assembly checkpoint, which targets the APC in Opisthokonts, appears severely attenuated in Chlamydomonas, based on analysis of mutants affecting microtubule function. This approach allows unbiased integration of the consensus cell cycle control model with innovations specific to the plant lineage. PMID:25336509

Increasing Tobacco Quitline Calls from Pregnant African American Women: The “One Tiny Reason to Quit” Social Marketing Campaign

PubMed Central

Genderson, Maureen Wilson; Sepulveda, Allison L.; Garland, Sheryl L.; Wilson, Diane Baer; Stith-Singleton, Rose; Dubuque, Susan

2013-01-01

Abstract Introduction Pregnant African American women are at disproportionately high risk of premature birth and infant mortality, outcomes associated with cigarette smoking. Telephone-based, individual smoking cessation counseling has been shown to result in successful quit attempts in the general population and among pregnant women, but “quitlines” are underutilized. A social marketing campaign called One Tiny Reason to Quit (OTRTQ) promoted calling a quitline (1-800-QUIT-NOW) to pregnant, African American women in Richmond, Virginia, in 2009 and was replicated there 2 years later. Methods The campaign disseminated messages via radio, interior bus ads, posters, newspaper ads, and billboards. Trained volunteers also delivered messages face-to-face and distributed branded give-away reminder items. The number of calls made from pregnant women in the Richmond area during summer 2009 was contrasted with (a) the number of calls during the seasons immediately before and after the campaign, and (b) the number of calls the previous summer. The replication used the same evaluation design. Results There were statistically significant spikes in calls from pregnant women during both campaign waves for both types of contrasts. A higher proportion of the calls from pregnant women were from African Americans during the campaign. Conclusion A multimodal quitline promotion like OTRTQ should be considered for geographic areas with sizable African American populations and high rates of infant mortality. PMID:23621745

Hygienic effects and gas production of plastic bio-digesters under tropical conditions.

PubMed

Yen-Phi, Vo Thi; Clemens, Joachim; Rechenburg, Andrea; Vinneras, Björn; Lenssen, Christina; Kistemann, Thomas

2009-12-01

Plastic plug-flow bio-digesters have been promoted as a good option for improved treatment of manure and wastewater in developing countries although minimal information has been published on their hygienic status. This bench-scale study replicates bio-digester conditions to evaluate the reduction of pathogen and indicator microorganisms at three different hydraulic retention times (HRT) in the anaerobic treatment of pig manures at 30 degrees C for 50 days. Results showed that physicochemical values differed between HRTs. Gas production efficiency was better for longer HRTS. The accumulated sludge at the reactor's base increased with longer HRT. Phages and bacteria examined were reduced, but none was completely eliminated. Log10 reduction of bacteria ranged from 0.54 to 2.47. Phages ranged from 1.60 to 3.42. The reduction of organisms at HRT = 30 days was about one log10 unit higher than HRT = 15 days and about two log10 units higher than HRT = 3 days. The results indicate that the reduction of tested organisms increases with HRT. However the hygienic quality of the liquid effluent does not meet required quality values for surface and irrigation water. Longer HRTs are recommended to increase gas yield and achieve higher pathogen reduction. More barriers should be applied while handling bio-digester outputs to minimise risks to environmental and human health.

Gene-targeted mice lacking the Trex1 (DNase III) 3'-->5' DNA exonuclease develop inflammatory myocarditis.

PubMed

Morita, Masashi; Stamp, Gordon; Robins, Peter; Dulic, Anna; Rosewell, Ian; Hrivnak, Geza; Daly, Graham; Lindahl, Tomas; Barnes, Deborah E

2004-08-01

TREX1, originally designated DNase III, was isolated as a major nuclear DNA-specific 3'-->5' exonuclease that is widely distributed in both proliferating and nonproliferating mammalian tissues. The cognate cDNA shows homology to the editing subunit of the Escherichia coli replicative DNA polymerase III holoenzyme and encodes an exonuclease which was able to serve a DNA-editing function in vitro, promoting rejoining of a 3' mismatched residue in a reconstituted DNA base excision repair system. Here we report the generation of gene-targeted Trex1(-/-) mice. The null mice are viable and do not show the increase in spontaneous mutation frequency or cancer incidence that would be predicted if Trex1 served an obligatory role of editing mismatched 3' termini generated during DNA repair or DNA replication in vivo. Unexpectedly, Trex1(-/-) mice exhibit a dramatically reduced survival and develop inflammatory myocarditis leading to progressive, often dilated, cardiomyopathy and circulatory failure.

Antiretroviral therapy for adults infected with HIV: Guidelines for health care professionals from the Quebec HIV care committee

PubMed Central

Rouleau, Danielle; Fortin, Claude; Trottier, Benoît; Lalonde, Richard; Lapointe, Normand; Côté, Pierre; Routy, Jean-Pierre; Matte, Marie-France; Tsarevsky, Irina; Baril, Jean-Guy

2011-01-01

The appropriate use of antiretrovirals reduces morbidity and mortality caused by HIV infection. The present article provides health care professionals with a practical guide for the use of antiretrovirals. Therapy should be initiated based predominantly on clinical presentation and CD4 count, and should consist of three active drugs or at least two active drugs when this is not possible, as in cases of some treatment-experienced patients. This is the most effective way to achieve long-term suppression of viral replication. Selection of individual drugs in the regimen should consider the weight of the evidence supporting these choices, as well as their tolerability profiles and ease of use, the patients’ comorbidities and treatment history. Treatment interruption is not recommended, either in aviremic patients or in those who have experienced virological failure. Instead, the therapeutic regimen should be adjusted to minimize side effects, promote adherence and suppress viral replication. PMID:22654926

GADD45β, an anti-tumor gene, inhibits avian leukosis virus subgroup J replication in chickens.

PubMed

Zhang, Xinheng; Yan, Zhuanqiang; Li, Xinjian; Lin, Wencheng; Dai, Zhenkai; Yan, Yiming; Lu, Piaopiao; Chen, Weiguo; Zhang, Huanmin; Chen, Feng; Ma, Jingyun; Xie, Qingmei

2016-10-18

Avian leukosis virus subgroup J (ALV-J) is a retroviruses that induces neoplasia, hepatomegaly, immunosuppression and poor performance in chickens. The tumorigenic and pathogenic mechanisms of ALV-J remain a hot topic. To explore anti-tumor genes that promote resistance to ALV-J infection in chickens, we bred ALV-J resistant and susceptible chickens (F3 generation). RNA-sequencing (RNA-Seq) of liver tissue from the ALV-J resistant and susceptible chickens identified 216 differentially expressed genes; 88 of those genes were up-regulated in the ALV-J resistant chickens (compared to the susceptible ones). We screened for significantly up-regulated genes (P < 0.01) of interest in the ALV-J resistant chickens, based on their involvement in biological signaling pathways. Functional analyses showed that overexpression of GADD45β inhibited ALV-J replication. GADD45β could enhance defense against ALV-J infection and may be used as a molecular marker to identify ALV-J infections.

GADD45β, an anti-tumor gene, inhibits avian leukosis virus subgroup J replication in chickens

PubMed Central

Zhang, Xinheng; Yan, Zhuanqiang; Li, Xinjian; Lin, Wencheng; Dai, Zhenkai; Yan, Yiming; Lu, Piaopiao; Chen, Weiguo; Zhang, Huanmin; Chen, Feng; Ma, Jingyun; Xie, Qingmei

2016-01-01

Avian leukosis virus subgroup J (ALV-J) is a retroviruses that induces neoplasia, hepatomegaly, immunosuppression and poor performance in chickens. The tumorigenic and pathogenic mechanisms of ALV-J remain a hot topic. To explore anti-tumor genes that promote resistance to ALV-J infection in chickens, we bred ALV-J resistant and susceptible chickens (F3 generation). RNA-sequencing (RNA-Seq) of liver tissue from the ALV-J resistant and susceptible chickens identified 216 differentially expressed genes; 88 of those genes were up-regulated in the ALV-J resistant chickens (compared to the susceptible ones). We screened for significantly up-regulated genes (P < 0.01) of interest in the ALV-J resistant chickens, based on their involvement in biological signaling pathways. Functional analyses showed that overexpression of GADD45β inhibited ALV-J replication. GADD45β could enhance defense against ALV-J infection and may be used as a molecular marker to identify ALV-J infections. PMID:27655697

The APOBEC3 Family of Retroelement Restriction Factors

PubMed Central

Refsland, Eric W.; Harris, Reuben S.

2014-01-01

The ability to regulate and even target mutagenesis is an extremely valuable cellular asset. Enzyme-catalyzed DNA cytosine deamination is a molecular strategy employed by vertebrates to promote antibody diversity and defend against foreign nucleic acids. Ten years ago, a family of cellular enzymes was first described with several proving capable of deaminating DNA and inhibiting HIV-1 replication. Ensuing studies on the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) restriction factors have uncovered a broad-spectrum innate defense network that suppresses the replication of numerous endogenous and exogenous DNA-based parasites. Although many viruses possess equally elaborate counter-defense mechanisms, the APOBEC3 enzymes offer a tantalizing possibility of leveraging innate immunity to fend off viral infection. Here we focus on mechanisms of retroelement restriction by the APOBEC3 family of restriction enzymes and we consider the therapeutic benefits, as well as the possible pathological consequences, of arming cells with active DNA deaminases. PMID:23686230

Life in the Clouds of Venus? An Experimental Synthetic Biology Approach

NASA Technical Reports Server (NTRS)

Rothschild, L. J.; Paulino-Lima, I. G.; Amatya, D.; Bajar, B.; Geilich, B.; Hu, J.; Jackson, C. J.

2015-01-01

The surface of Venus constitutes the most hellish and biologically inhospitable planetary surface in our solar system, boasting a pH of 0, blistering winds that can melt lead, and pressures of 60 atm. However, during the earlier years of the solar system, without the runaway greenhouse effect that has plagued the planet, Venus potentially housed oceans and perhaps even life. There is a possibility that microbes could have retreated into hospitable niches in the atmosphere, as suggested by Carl Sagan as early as 1967 [1]. For example, 50 km above the raging hell of the Venusian surface, exists a relatively temperate environment that might serve as reservoir for life. This astrobiology project seeks to explore life at the extremes and to theorize whether microbial communities could not only survive but also reproduce in the Venusian atmosphere. Specifically, we ask: are aerosols viable microbial environments? But before we can test for life in the clouds, we have to develop a proper reporter to visualize cell growth in situ. For this purpose, we aimed to develop cell-growth dependent reporters to serve as remote biosensors for cell growth. We developed two using the polA promoter, a DNA-replication dependent promoter, and nrd operon promoter, a cell-cycle dependent promoter. Using these cell-growth reporters, the next step is to aerosolize microbes expressing these reporters in a suspension chamber adapted from a Millikan Drop Apparatus to assay reproduction in an aerosolized environment. Better yet is to test the reproduction of microbes in a microgravity regime such as on ISS.Approach: We engineered two cell-cycle dependent genetic reporters. One was the polA promoter which codes for DNA Polymerase I, a gene active in DNA replication [2]. The other was the nrdP. The activation of ribonucleotide reductase reduces ribonucleotides into deoxyribonucleotides and is involved in the bacterial cell cycle [3]. This promoter began activation during the initiation of DNA replication and is cell-cycle dependent [4]. These promoters were fused to a GFP reporter, transformed into E. coli. The constructs were deposited in the iGEM registry as K847210: Escherichia coli DNA-replication dependent polA promoter K847211: Escherichia coli cell-division dependent nrd promoter Results: Our constructs displayed fluorescence when transformed into NEB-5alpha competent cells. While nrdP-E0840 displayed sufficient fluorescence as verified by fluorescent microscopy, the original polAP-E0840 construct (which uses mut3b GFP) exhibited low expression; while fluorescence was visible under the microscope, the signal was too weak for the camera to recognize. The polA promoter was therefore digest-ed with EcoRI and SpeI then ligated into plasmid pNCS containing a RBS, Clover, and a terminator. Clover is a highly engineered green fluorescent protein that exhibits extreme brightness [5] Fluoresence time course data demonsrated that the genes were induced in a cell cycle dependant manner [6]. Our assays via microscopy and the bulk assay shows that our promoters are functional as cell cycle reporters.Conclusions: The application of such tools are widespread and not limited to astrobiology; nrdP could be used to determine doubling times empirically and could possibly extrapolate DNA content from intensity of signals expressed by polAP. However, we are pri-marily interested in its use in astrobiology.

Life as a cosmic imperative?

PubMed

de Duve, Christian

2011-02-13

The origin of life on Earth may be divided into two stages separated by the first appearance of replicable molecules, most probably of RNA. The first stage depended exclusively on chemistry. The second stage likewise involved chemistry, but with the additional participation of selection, a necessary concomitant of inevitable replication accidents. Consideration of these two processes suggests that the origin of life may have been close to obligatory under the physical-chemical conditions that prevailed at the site of its birth. Thus, an extrasolar planet in which those conditions were replicated appears as a probable site for the appearance of extra-terrestrial life.

Reference test methods for total water in lint cotton by Karl Fischer Titration and low temperature distillation

USDA-ARS?s Scientific Manuscript database

In a study of comparability of total water contents (%) of conditioned cottons by Karl Fischer Titration (KFT) and Low Temperature Distillation (LTD) reference methods, we demonstrated a match of averaged results based on a large number of replications and weighing the test specimens at the same tim...

Dynamics of aerial and terrestrial populations of Phytophthora ramorum in a California watershed under different climatic conditions

Treesearch

Catherine A. Eyre; Melina Kozanitas; Matteo Garbelotto

2013-01-01

We present a study of the epidemiology of sudden oak death (SOD) in California within a watershed based on temporally and spatially replicated surveys of symptoms, viability of the pathogen from symptomatic leaves, and genetic analyses using polymorphic SSR markers.Phytophthora ramorum is sensitive to climate; its...

Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria

PubMed Central

Taton, Arnaud; Unglaub, Federico; Wright, Nicole E.; Zeng, Wei Yue; Paz-Yepes, Javier; Brahamsha, Bianca; Palenik, Brian; Peterson, Todd C.; Haerizadeh, Farzad; Golden, Susan S.; Golden, James W.

2014-01-01

Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains. PMID:25074377

S-phase arrest after vincristine treatment may promote hepatitis B virus replication

PubMed Central

Xu, Lei; Tu, Zeng; Xu, Ge; Hu, Jie-Li; Cai, Xue-Fei; Zhan, Xing-Xing; Wang, Yu-Wei; Huang, Yuan; Chen, Juan; Huang, Ai-Long

2015-01-01

AIM: To observe the effect of vincristine on hepatitis B virus (HBV) replication in vitro and to study its possible mechanisms. METHODS: Vincristine was added to the cultures of two cell lines stably expressing HBV. Then, the levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core antigen (HBcAg) in the supernatants or cytoplasm were examined using by enzyme-linked immunosorbent assay and Western blot. The HBV pregenome RNA (pgRNA) was detected using reverse transcription-PCR and real-time fluorescent quantitative PCR (RT-qPCR), and viral DNA was detected using Southern blot and RT-qPCR. Cell proliferation after drug treatment was detected using the BrdU incorporation test and the trypan blue exclusion assay. Cell cycle and cell apoptosis were examined using flow cytometry and Western blot. RESULTS: Vincristine up-regulated HBV replication directly in vitro in a dose-dependent manner, and 24-h exposure to 0.1 μmol/L vincristine induced more than 4-fold and 3-fold increases in intracellular HBV DNA and the secretion of viral DNA, respectively. The expression of HBV pgRNA, intracellular HBsAg and HBcAg, and the secretion of HBeAg were also increased significantly after drug treatment. Most importantly, vincristine promoted the cell excretion of HBV nucleocapsids instead of HBV Dane particles, and the nucleocapsids are closely related to the HBV pathogenesis. Furthermore, vincristine inhibited the proliferation of cells stably expressing HBV. The higher the concentration of the drug, the more significant the inhibition of the cell proliferation and the stronger the HBV replication ability in cells. Flow cytometry indicated that cell cycle arrest at S-phase was responsible for the cell proliferation inhibition. CONCLUSION: Vincristine has a strong stimulatory effect on HBV replication and induces cell cycle arrest, and cell proliferation inhibition may be conducive to viral replication. PMID:25663769

MMSET is dynamically regulated during cell-cycle progression and promotes normal DNA replication.

PubMed

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.

Virus-specific antibodies allow viral replication in the marginal zone, thereby promoting CD8+ T-cell priming and viral control

PubMed Central

Duhan, Vikas; Khairnar, Vishal; Friedrich, Sarah-Kim; Zhou, Fan; Gassa, Asmae; Honke, Nadine; Shaabani, Namir; Gailus, Nicole; Botezatu, Lacramioara; Khandanpour, Cyrus; Dittmer, Ulf; Häussinger, Dieter; Recher, Mike; Hardt, Cornelia; Lang, Philipp A.; Lang, Karl S.

2016-01-01

Clinically used human vaccination aims to induce specific antibodies that can guarantee long-term protection against a pathogen. The reasons that other immune components often fail to induce protective immunity are still debated. Recently we found that enforced viral replication in secondary lymphoid organs is essential for immune activation. In this study we used the lymphocytic choriomeningitis virus (LCMV) to determine whether enforced virus replication occurs in the presence of virus-specific antibodies or virus-specific CD8+ T cells. We found that after systemic recall infection with LCMV-WE the presence of virus-specific antibodies allowed intracellular replication of virus in the marginal zone of spleen. In contrast, specific antibodies limited viral replication in liver, lung, and kidney. Upon recall infection with the persistent virus strain LCMV-Docile, viral replication in spleen was essential for the priming of CD8+ T cells and for viral control. In contrast to specific antibodies, memory CD8+ T cells inhibited viral replication in marginal zone but failed to protect mice from persistent viral infection. We conclude that virus-specific antibodies limit viral infection in peripheral organs but still allow replication of LCMV in the marginal zone, a mechanism that allows immune boosting during recall infection and thereby guarantees control of persistent virus. PMID:26805453

Polyploid titan cells produce haploid and aneuploid progeny to promote stress adaptation.

PubMed

Gerstein, Aleeza C; Fu, Man Shun; Mukaremera, Liliane; Li, Zhongming; Ormerod, Kate L; Fraser, James A; Berman, Judith; Nielsen, Kirsten

2015-10-13

Cryptococcus neoformans is a major life-threatening fungal pathogen. In response to the stress of the host environment, C. neoformans produces large polyploid titan cells. Titan cell production enhances the virulence of C. neoformans, yet whether the polyploid aspect of titan cells is specifically influential remains unknown. We show that titan cells were more likely to survive and produce offspring under multiple stress conditions than typical cells and that even their normally sized daughters maintained an advantage over typical cells in continued exposure to stress. Although polyploid titan cells generated haploid daughter cell progeny upon in vitro replication under nutrient-replete conditions, titan cells treated with the antifungal drug fluconazole produced fluconazole-resistant diploid and aneuploid daughter cells. Interestingly, a single titan mother cell was capable of generating multiple types of aneuploid daughter cells. The increased survival and genomic diversity of titan cell progeny promote rapid adaptation to new or high-stress conditions. The ability to adapt to stress is a key element for survival of pathogenic microbes in the host and thus plays an important role in pathogenesis. Here we investigated the predominantly haploid human fungal pathogen Cryptococcus neoformans, which is capable of ploidy and cell size increases during infection through production of titan cells. The enlarged polyploid titan cells are then able to rapidly undergo ploidy reduction to generate progeny with reduced ploidy and/or aneuploidy. Under stressful conditions, titan cell progeny have a growth and survival advantage over typical cell progeny. Understanding how titan cells enhance the rate of cryptococcal adaptation under stress conditions may assist in the development of novel drugs aimed at blocking ploidy transitions. Copyright © 2015 Gerstein et al.

Replication and robustness in developmental research.

PubMed

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

2014-11-01

Replications and robustness checks are key elements of the scientific method and a staple in many disciplines. However, leading journals in developmental psychology rarely include explicit replications of prior research conducted by different investigators, and few require authors to establish in their articles or online appendices that their key results are robust across estimation methods, data sets, and demographic subgroups. This article makes the case for prioritizing both explicit replications and, especially, within-study robustness checks in developmental psychology. It provides evidence on variation in effect sizes in developmental studies and documents strikingly different replication and robustness-checking practices in a sample of journals in developmental psychology and a sister behavioral science-applied economics. Our goal is not to show that any one behavioral science has a monopoly on best practices, but rather to show how journals from a related discipline address vital concerns of replication and generalizability shared by all social and behavioral sciences. We provide recommendations for promoting graduate training in replication and robustness-checking methods and for editorial policies that encourage these practices. Although some of our recommendations may shift the form and substance of developmental research articles, we argue that they would generate considerable scientific benefits for the field. (PsycINFO Database Record (c) 2014 APA, all rights reserved).

Persistence of an Oncogenic Papillomavirus Genome Requires cis Elements from the Viral Transcriptional Enhancer

PubMed Central

Van Doorslaer, Koenraad; Chen, Dan; Chapman, Sandra; Khan, Jameela

2017-01-01

ABSTRACT Human papillomavirus (HPV) genomes are replicated and maintained as extrachromosomal plasmids during persistent infection. The viral E2 proteins are thought to promote stable maintenance replication by tethering the viral DNA to host chromatin. However, this has been very difficult to prove genetically, as the E2 protein is involved in transcriptional regulation and initiation of replication, as well as its assumed role in genome maintenance. This makes mutational analysis of viral trans factors and cis elements in the background of the viral genome problematic and difficult to interpret. To circumvent this problem, we have developed a complementation assay in which the complete wild-type HPV18 genome is transfected into primary human keratinocytes along with subgenomic or mutated replicons that contain the minimal replication origin. The wild-type genome provides the E1 and E2 proteins in trans, allowing us to determine additional cis elements that are required for long-term replication and partitioning of the replicon. We found that, in addition to the core replication origin (and the three E2 binding sites located therein), additional sequences from the transcriptional enhancer portion of the URR (upstream regulatory region) are required in cis for long-term genome replication. PMID:29162712

Mouse Norovirus infection promotes autophagy induction to facilitate replication but prevents final autophagosome maturation

DOE Office of Scientific and Technical Information (OSTI.GOV)

O’Donnell, Tanya B.; Hyde, Jennifer L.; Mintern, Justine D.

Autophagy is a cellular process used to eliminate intracellular pathogens. Many viruses however are able to manipulate this cellular process for their own advantage. Here we demonstrate that Mouse Norovirus (MNV) infection induces autophagy but does not appear to utilise the autophagosomal membrane for establishment and formation of the viral replication complex. We have observed that MNV infection results in lipidation and recruitment of LC3 to the autophagosome membrane but prevents subsequent fusion of the autophagosomes with lysosomes, as SQSTM1 (an autophagy receptor) accumulates and Lysosome-Associated Membrane Protein1 is sequestered to the MNV replication complex (RC) rather than to autophagosomes.more » We have additionally observed that chemical modulation of autophagy differentially affects MNV replication. From this study we can conclude that MNV infection induces autophagy, however suppresses the final maturation step of this response, indicating that autophagy induction contributes to MNV replication independently of RC biogenesis. - Highlights: • MNV induces autophagy in infected murine macrophages. • MNV does not utilise autophagosomal membranes for replication. • The MNV-induced autophagosomes do not fuse with lysosomes. • MNV sequesters SQSTM1 to prevent autophagy degradation and turnover. • Chemical modulation of autophagy enhances MNV replication.« less

Identification of Novel Kaposi's Sarcoma-Associated Herpesvirus Orf50 Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential.

PubMed

Wakeman, Brian S; Izumiya, Yoshihiro; Speck, Samuel H

2017-01-01

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma and multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50 RTA is a known transactivator of multiple viral genes, allowing it to control the switch between latency and virus replication. We report here the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial), 3T12 (mouse fibroblast), and RAW 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 to 10 residues at the amino terminus of the protein. We show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation. Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma and multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV-infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication and is conserved among all known gammaherpesviruses. We show here for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of several isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression, leading to the efficient production of mature virions. Copyright © 2016 American Society for Microbiology.

Noise-induced bistability in the quasi-neutral coexistence of viral RNAs under different replication modes.

PubMed

Sardanyés, Josep; Arderiu, Andreu; Elena, Santiago F; Alarcón, Tomás

2018-05-01

Evolutionary and dynamical investigations into real viral populations indicate that RNA replication can range between the two extremes represented by so-called 'stamping machine replication' (SMR) and 'geometric replication' (GR). The impact of asymmetries in replication for single-stranded (+) sense RNA viruses has been mainly studied with deterministic models. However, viral replication should be better described by including stochasticity, as the cell infection process is typically initiated with a very small number of RNA macromolecules, and thus largely influenced by intrinsic noise. Under appropriate conditions, deterministic theoretical descriptions of viral RNA replication predict a quasi-neutral coexistence scenario, with a line of fixed points involving different strands' equilibrium ratios depending on the initial conditions. Recent research into the quasi-neutral coexistence in two competing populations reveals that stochastic fluctuations fundamentally alter the mean-field scenario, and one of the two species outcompetes the other. In this article, we study this phenomenon for viral RNA replication modes by means of stochastic simulations and a diffusion approximation. Our results reveal that noise has a strong impact on the amplification of viral RNAs, also causing the emergence of noise-induced bistability. We provide analytical criteria for the dominance of (+) sense strands depending on the initial populations on the line of equilibria, which are in agreement with direct stochastic simulation results. The biological implications of this noise-driven mechanism are discussed within the framework of the evolutionary dynamics of RNA viruses with different modes of replication. © 2018 The Author(s).

Plasmodium falciparum CRK4 directs continuous rounds of DNA replication during schizogony.

PubMed

Ganter, Markus; Goldberg, Jonathan M; Dvorin, Jeffrey D; Paulo, Joao A; King, Jonas G; Tripathi, Abhai K; Paul, Aditya S; Yang, Jing; Coppens, Isabelle; Jiang, Rays H Y; Elsworth, Brendan; Baker, David A; Dinglasan, Rhoel R; Gygi, Steven P; Duraisingh, Manoj T

2017-02-17

Plasmodium parasites, the causative agents of malaria, have evolved a unique cell division cycle in the clinically relevant asexual blood stage of infection 1 . DNA replication commences approximately halfway through the intracellular development following invasion and parasite growth. The schizont stage is associated with multiple rounds of DNA replication and nuclear division without cytokinesis, resulting in a multinucleated cell. Nuclei divide asynchronously through schizogony, with only the final round of DNA replication and segregation being synchronous and coordinated with daughter cell assembly 2,3 . However, the control mechanisms for this divergent mode of replication are unknown. Here, we show that the Plasmodium-specific kinase PfCRK4 is a key cell-cycle regulator that orchestrates multiple rounds of DNA replication throughout schizogony in Plasmodium falciparum. PfCRK4 depletion led to a complete block in nuclear division and profoundly inhibited DNA replication. Quantitative phosphoproteomic profiling identified a set of PfCRK4-regulated phosphoproteins with greatest functional similarity to CDK2 substrates, particularly proteins involved in the origin of replication firing. PfCRK4 was required for initial and subsequent rounds of DNA replication during schizogony and, in addition, was essential for development in the mosquito vector. Our results identified an essential S-phase promoting factor of the unconventional P. falciparum cell cycle. PfCRK4 is required for both a prolonged period of the intraerythrocytic stage of Plasmodium infection, as well as for transmission, revealing a broad window for PfCRK4-targeted chemotherapeutics.

Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice

PubMed Central

Hogan, Chad H.; Oldenburg, Darby G.; Kara, Mehmet

2018-01-01

Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host. PMID:29390024

Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation.

PubMed

Lu, Canhua; Nakayasu, Ernesto S; Zhang, Li-Qun; Luo, Zhao-Qing

2016-01-26

The morphology of bacterial cells is important for virulence, evasion of the host immune system, and coping with environmental stresses. The widely distributed Fic proteins (filamentation induced by cAMP) are annotated as proteins involved in cell division because of the presence of the HPFx[D/E]GN[G/K]R motif. We showed that the presence of Fic-1 from Pseudomonas fluorescens significantly reduced the yield of plasmid DNA when expressed in Escherichia coli or P. fluorescens. Fic-1 interacted with GyrB, a subunit of DNA gyrase, which is essential for bacterial DNA replication. Fic-1 catalyzed the AMPylation of GyrB at Tyr(109), a residue critical for binding ATP, and exhibited auto-AMPylation activity. Mutation of the Fic-1 auto-AMPylated site greatly reduced AMPylation activity toward itself and toward GyrB. Fic-1-dependent AMPylation of GyrB triggered the SOS response, indicative of DNA replication stress or DNA damage. Fic-1 also promoted the formation of elongated cells when the SOS response was blocked. We identified an α-inhibitor protein that we named anti-Fic-1 (AntF), encoded by a gene immediately upstream of Fic-1. AntF interacted with Fic-1, inhibited the AMPylation activity of Fic-1 for GyrB in vitro, and blocked Fic-1-mediated inhibition of DNA replication in bacteria, suggesting that Fic-1 and AntF comprise a toxin-antitoxin module. Our work establishes Fic-1 as an AMPylating enzyme that targets GyrB to inhibit DNA replication and may target other proteins to regulate bacterial morphology. Copyright © 2016, American Association for the Advancement of Science.

Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice.

PubMed

Van Skike, Nick D; Minkah, Nana K; Hogan, Chad H; Wu, Gary; Benziger, Peter T; Oldenburg, Darby G; Kara, Mehmet; Kim-Holzapfel, Deborah M; White, Douglas W; Tibbetts, Scott A; French, Jarrod B; Krug, Laurie T

2018-02-01

Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host.

Genome-wide mapping of nuclear mitochondrial DNA sequences links DNA replication origins to chromosomal double-strand break formation in Schizosaccharomyces pombe

PubMed Central

Lenglez, Sandrine; Hermand, Damien; Decottignies, Anabelle

2010-01-01

Chromosomal double-strand breaks (DSBs) threaten genome integrity and repair of these lesions is often mutagenic. How and where DSBs are formed is a major question conveniently addressed in simple model organisms like yeast. NUMTs, nuclear DNA sequences of mitochondrial origin, are present in most eukaryotic genomes and probably result from the capture of mitochondrial DNA (mtDNA) fragments into chromosomal breaks. NUMT formation is ongoing and was reported to cause de novo human genetic diseases. Study of NUMTs is likely to contribute to the understanding of naturally occurring chromosomal breaks. We show that Schizosaccharomyces pombe NUMTs are exclusively located in noncoding regions with no preference for gene promoters and, when located into promoters, do not affect gene transcription level. Strikingly, most noncoding regions comprising NUMTs are also associated with a DNA replication origin (ORI). Chromatin immunoprecipitation experiments revealed that chromosomal NUMTs are probably not acting as ORI on their own but that mtDNA insertions occurred directly next to ORIs, suggesting that these loci may be prone to DSB formation. Accordingly, induction of excessive DNA replication origin firing, a phenomenon often associated with human tumor formation, resulted in frequent nucleotide deletion events within ORI3001 subtelomeric chromosomal locus, illustrating a novel aspect of DNA replication-driven genomic instability. How mtDNA is fragmented is another important issue that we addressed by sequencing experimentally induced NUMTs. This highlighted regions of S. pombe mtDNA prone to breaking. Together with an analysis of human NUMTs, we propose that these fragile sites in mtDNA may correspond to replication pause sites. PMID:20688779

Mitochondrial fission proteins Fis1 and Mdv1, but not Dnm1, play a role in maintenance of heteroplasmy in budding yeast.

PubMed

Bradshaw, Elliot; Yoshida, Minoru; Ling, Feng

2012-04-24

In budding yeast, the mitochondrial DNA (mtDNA) replication pathway involving the homologous DNA pairing protein Mhr1 promotes mitochondrial allele segregation. Mitochondrial fusion facilitates the recombination-mediated replication pathway; however, the role of fission remains largely unknown. By monitoring mitochondrial allele segregation during zygotic division, we found that the absence of fission proteins Fis1 or Mdv1, but not Dnm1, resulted in increased initial homoplasmy levels and decreased mtDNA copy number. However, decreases in mtDNA copy number alone were not sufficient for rapid establishment of homoplasmy, suggesting that inhibiting the activities of certain fission proteins promotes homoplasmy by reducing the number of mtDNA segregation units. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Recombination Promoted by DNA Viruses: Phage λ to Herpes Simplex Virus

PubMed Central

Weller, Sandra K.; Sawitzke, James A.

2015-01-01

The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies. PMID:25002096

MicroRNA-33 promotes the replicative senescence of mouse embryonic fibroblasts by suppressing CDK6

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Shun; Huang, Haijiao; Li, Nanhong

2016-05-13

MicroRNAs are a large class of tiny noncoding RNAs, which have emerged as critical regulators of gene expression, and thus are involved in multiple cellular processes, including cellular senescence. MicroRNA-33 has previously been established to exert crucial effect on cell proliferation, lipid metabolism and cholesterol metabolism. Nonetheless, the association between microRNA-33 and cellular senescence and its underlying molecular mechanism are far to be elucidated. The present study has attempted to probe into the effect of microRNA-33 on MEFs senescence. Our data unveiled that microRNA-33 was dramatically down-regulated in senescent MEFs compared to the young MEFs, and ectopic expression of microRNA-33more » promoted MEFs senescence, while knock-down of microRNA-33 exhibited a protective effect against senescence phenotype. Moreover, we verified CDK6 as a direct target of microRNA-33 in mouse. Silencing of CDK6 induced the premature senescence phenotype of MEFs similarly as microRNA-33, while enforced expression of CDK6 significantly reverse the senescence-induction effect of microRNA-33. Taken together, our results suggested that microRNA-33 enhanced the replicative senescence of MEFs potentially by suppressing CDK6 expression. -- Highlights: •MicroRNA-33 was dramatically down-regulated in senescent MEF cells. •Altered expression of microRNA-33 exerted a critical role in MEFs senescence. •MicroRNA-33 promoted the replicative senescence of MEFs via targeting of CDK6.« less

Sulphur responsiveness of the Chlamydomonas reinhardtii LHCBM9 promoter.

PubMed

Sawyer, Anne L; Hankamer, Ben D; Ross, Ian L

2015-05-01

A 44-base-pair region in the Chlamydomonas reinhardtii LHCBM9 promoter is essential for sulphur responsiveness. The photosynthetic light-harvesting complex (LHC) proteins play essential roles both in light capture, the first step of photosynthesis, and in photoprotective mechanisms. In contrast to the other LHC proteins and the majority of photosynthesis proteins, the Chlamydomonas reinhardtii photosystem II-associated LHC protein, LHCBM9, was recently reported to be up-regulated under sulphur deprivation conditions, which also induce hydrogen production. Here, we examined the sulphur responsiveness of the LHCBM9 gene at the transcriptional level, through promoter deletion analysis. The LHCBM9 promoter was found to be responsive to sulphur deprivation, with a 44-base-pair region between nucleotide positions -136 and -180 relative to the translation start site identified as essential for this response. Anaerobiosis was found to enhance promoter activity under sulphur deprivation conditions, however, alone was unable to induce promoter activity. The study of LHCBM9 is of biological and biotechnological importance, as its expression is linked to photobiological hydrogen production, theoretically the most efficient process for biofuel production, while the simplicity of using an S-deprivation trigger enables the development of a novel C. reinhardtii-inducible promoter system based on LHCBM9.

The Yeast Forkhead Transcription Factors Fkh1 and Fkh2 Regulate Lifespan and Stress Response Together with the Anaphase-Promoting Complex

PubMed Central

Postnikoff, Spike D. L.; Malo, Mackenzie E.; Wong, Berchman; Harkness, Troy A. A.

2012-01-01

Forkhead box O (FOXO) transcription factors have a conserved function in regulating metazoan lifespan. A key function in this process involves the regulation of the cell cycle and stress responses including free radical scavenging. We employed yeast chronological and replicative lifespan assays, as well as oxidative stress assays, to explore the potential evolutionary conservation of function between the FOXOs and the yeast forkhead box transcription factors FKH1 and FKH2. We report that the deletion of both FKH genes impedes normal lifespan and stress resistance, particularly in stationary phase cells, which are non-responsive to caloric restriction. Conversely, increased expression of the FKHs leads to extended lifespan and improved stress response. Here we show the Anaphase-Promoting Complex (APC) genetically interacts with the Fkh pathway, likely working in a linear pathway under normal conditions, as fkh1Δ fkh2Δ post-mitotic survival is epistatic to that observed in apc5CA mutants. However, under stress conditions, post-mitotic survival is dramatically impaired in apc5CA fkh1Δ fkh2Δ, while increased expression of either FKH rescues APC mutant growth defects. This study establishes the FKHs role as evolutionarily conserved regulators of lifespan in yeast and identifies the APC as a novel component of this mechanism under certain conditions, likely through combined regulation of stress response, genomic stability, and cell cycle regulation. PMID:22438832

Assessing the Use of School Public Address Systems to Deliver Nutrition Messages to Children: Shape Up Somerville--Audio Adventures

ERIC Educational Resources Information Center

Folta, Sara C.; Goldberg, Jeanne P.; Economos, Christina; Bell, Rick; Landers, Stewart; Hyatt, Raymond

2006-01-01

Given the current childhood obesity epidemic, it is especially important to find effective ways to promote healthful foods to children. School public address (PA) systems represent an inexpensive and a replicable way of reaching children with health messages. To test the effectiveness of this channel, messages were created to promote 2 dried bean…

Two host microRNAs influence WSSV replication via STAT gene regulation.

PubMed

Huang, Ying; Wang, Wen; Ren, Qian

2016-03-31

MicroRNAs (miRNAs) have important roles in post-transcriptional regulation of gene expression. During viral infection, viruses utilize hosts to enhance their replication by altering cellular miRNAs. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays crucial roles in the antiviral responses. In this study, two miRNAs (miR-9041 and miR-9850) from Macrobrachium rosenbergii were found to promote white spot syndrome virus (WSSV) replication. The up-regulation of miR-9041 or miR-9850 suppresses STAT expression in the gills of M. rosenbergii, which subsequently down-regulates the expression of its downstream dynamin (Dnm) genes: Dnm1, Dnm2, and Dnm3. Knockdown of miR-9041 and miR-9850 restricts WSSV replication by up-regulating STAT and Dnm gene expression. The silencing of STAT, Dnm1, Dnm2, or Dnm3 led to an increase of the number of WSSV copies in shrimp. The injection of recombinant Dnm1, Dnm2, or Dnm3 proteins could inhibit WSSV replication in vivo. Overall, our research indicates the roles of host miRNAs in the enhancement of WSSV replication by regulating the host JAK/STAT pathway.

CDC-48/p97 coordinates CDT-1 degradation with GINS chromatin dissociation to ensure faithful DNA replication.

PubMed

Franz, André; Orth, Michael; Pirson, Paul A; Sonneville, Remi; Blow, J Julian; Gartner, Anton; Stemmann, Olaf; Hoppe, Thorsten

2011-10-07

Faithful transmission of genomic information requires tight spatiotemporal regulation of DNA replication factors. In the licensing step of DNA replication, CDT-1 is loaded onto chromatin to subsequently promote the recruitment of additional replication factors, including CDC-45 and GINS. During the elongation step, the CDC-45/GINS complex moves with the replication fork; however, it is largely unknown how its chromatin association is regulated. Here, we show that the chaperone-like ATPase CDC-48/p97 coordinates degradation of CDT-1 with release of the CDC-45/GINS complex. C. elegans embryos lacking CDC-48 or its cofactors UFD-1/NPL-4 accumulate CDT-1 on mitotic chromatin, indicating a critical role of CDC-48 in CDT-1 turnover. Strikingly, CDC-48(UFD-1/NPL-4)-deficient embryos show persistent chromatin association of CDC-45/GINS, which is a consequence of CDT-1 stabilization. Moreover, our data confirmed a similar regulation in Xenopus egg extracts, emphasizing a conserved coordination of licensing and elongation events during eukaryotic DNA replication by CDC-48/p97. Copyright © 2011 Elsevier Inc. All rights reserved.

Methylation of DNA Ligase 1 by G9a/GLP Recruits UHRF1 to Replicating DNA and Regulates DNA Methylation.

PubMed

Ferry, Laure; Fournier, Alexandra; Tsusaka, Takeshi; Adelmant, Guillaume; Shimazu, Tadahiro; Matano, Shohei; Kirsh, Olivier; Amouroux, Rachel; Dohmae, Naoshi; Suzuki, Takehiro; Filion, Guillaume J; Deng, Wen; de Dieuleveult, Maud; Fritsch, Lauriane; Kudithipudi, Srikanth; Jeltsch, Albert; Leonhardt, Heinrich; Hajkova, Petra; Marto, Jarrod A; Arita, Kyohei; Shinkai, Yoichi; Defossez, Pierre-Antoine

2017-08-17

DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance. Copyright © 2017 Elsevier Inc. All rights reserved.

Temperature and nutrient effects on periphyton associated ...

EPA Pesticide Factsheets

Nutrient pollution is a leading cause of water quality impairments and degraded aquatic ecosystem condition. Reliable and reproducible indicators of ecosystem condition are needed to help manage nutrient pollution. The diatom component of periphyton has been used as a water quality indicator due to identifiable cell morphology and existence of relationships between nutrient concentration and diatom community composition. However, morphological identification of diatoms requires highly specialized personnel, is very time consuming, and can produce variable results, suggesting the need for alternative methods that are less expensive and more reproducible. DNA sequencing of the bacterial 16S rRNA gene is well documented and provides genus-level resolution of the community structure. The goal of this study was to evaluate the effects of nutrient loading and temperature on periphyton-associated bacterial communities using standard periphytometer techniques and next generation sequencing technologies. Continuous flow mesocosms were established in an eight tank system consisting of two temperature conditions (10°C and 20°C) and four nutrient conditions (1x to 6x ambient concentrations). Experimental conditions were replicated in July/August 2013 and September 2013. Replicate DNA samples were extracted and the 16S rRNA gene was sequenced using universal Bacterial primers. Initial analyses revealed strong differences in community structure based on temperature (p <

Gene Rearrangement Attenuates Expression and Lethality of a Nonsegmented Negative Strand RNA Virus

NASA Astrophysics Data System (ADS)

Williams Wertz, Gail; Perepelitsa, Victoria P.; Ball, L. Andrew

1998-03-01

The nonsegmented negative strand RNA viruses comprise hundreds of human, animal, insect, and plant pathogens. Gene expression of these viruses is controlled by the highly conserved order of genes relative to the single transcriptional promoter. We utilized this regulatory mechanism to alter gene expression levels of vesicular stomatitis virus by rearranging the gene order. This report documents that gene expression levels and the viral phenotype can be manipulated in a predictable manner. Translocation of the promoter-proximal nucleocapsid protein gene N, whose product is required stoichiometrically for genome replication, to successive positions down the genome reduced N mRNA and protein expression in a stepwise manner. The reduction in N gene expression resulted in a stepwise decrease in genomic RNA replication. Translocation of the N gene also attenuated the viruses to increasing extents for replication in cultured cells and for lethality in mice, without compromising their ability to elicit protective immunity. Because monopartite negative strand RNA viruses have not been reported to undergo homologous recombination, gene rearrangement should be irreversible and may provide a rational strategy for developing stably attenuated live vaccines against this type of virus.

Sequential folding of UmuC by the Hsp70 and Hsp60 chaperone complexes of Escherichia coli.

PubMed

Petit, M A; Bedale, W; Osipiuk, J; Lu, C; Rajagopalan, M; McInerney, P; Goodman, M F; Echols, H

1994-09-23

Replication-blocking lesions generate a signal in Escherichia coli that leads to the induction of the multigene SOS response. Among the SOS-induced genes are umuD and umuC, whose products are necessary for the increased mutation rate in induced bacteria. The mutations are likely to result from replication across the DNA lesion, and such a bypass event has been reconstituted in vitro (Rajagopalan, M., L, C., Woodgate, R., O'Donnel, M., Goodman, M. F., Echols, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10777-10781). In this work, we show that the chaperone proteins promote the proper folding of UmuC protein in vitro. We treated purified and inactive UmuC with Hsp70 and Hsp60. After Hsp70 treatment, the DNA binding activity of UmuC was recovered, but the ability to promote replication across DNA lesions was not. However, lesion bypass activity was recovered upon further treatment with Hsp60. The biological significance of such a folding pathway for UmuC protein is strengthened by in vivo evidence for a role of DnaK in UV-induced mutagenesis.

Miscoding and mutagenic properties of 8-oxoguanine and abasic sites: Ubiquitous lesions in damaged DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grollman, A.P.; Takeshita, Masaru

1995-12-31

More than twenty oxidatively-damaged bases, including 8-oxoguanine, have been found to occur in genomic DNA. Some of these lesions block DNA replication and are potentially lethal; others generate mutations which can initiate carcinogenesis and promote cellular aging. In this report, the authors focus attention on the mutagenicity and repair of 8-oxoguanine. Kasai and Nishimura`s discovery that hydroxyl radicals react with guanine residues in DNA to form 8-oxoguanine and the development of sensitive methods for the detection and quantitation of this modified base led to the observation that approximately 1 in 10{sup 5} guanine residues in mammalian DNA are oxidized atmore » the C-8 position. DNA containing 8-oxoguanine and synthetic analogs of the abasic site have been used to investigate the miscoding and mutagenic potential of these ubiquitous lesions. Studies in the laboratory were facilitated by the development of solid state synthetic methods by which these lesions could be introduced at defined positions in DNA. In this paper, the authors review studies in which 8-oxoguanine and abasic sites have been used in model systems to explore various early events in the replication of selectively damaged DNA.« less

Mechanism of error-free DNA synthesis across N1-methyl-deoxyadenosine by human DNA polymerase-ι

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jain, Rinku; Choudhury, Jayati Roy; Buku, Angeliki

N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the synmore » conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a “foothold” and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity.« less

Credibility, Replicability, and Reproducibility in Simulation for Biomedicine and Clinical Applications in Neuroscience

PubMed Central

Mulugeta, Lealem; Drach, Andrew; Erdemir, Ahmet; Hunt, C. A.; Horner, Marc; Ku, Joy P.; Myers Jr., Jerry G.; Vadigepalli, Rajanikanth; Lytton, William W.

2018-01-01

Modeling and simulation in computational neuroscience is currently a research enterprise to better understand neural systems. It is not yet directly applicable to the problems of patients with brain disease. To be used for clinical applications, there must not only be considerable progress in the field but also a concerted effort to use best practices in order to demonstrate model credibility to regulatory bodies, to clinics and hospitals, to doctors, and to patients. In doing this for neuroscience, we can learn lessons from long-standing practices in other areas of simulation (aircraft, computer chips), from software engineering, and from other biomedical disciplines. In this manuscript, we introduce some basic concepts that will be important in the development of credible clinical neuroscience models: reproducibility and replicability; verification and validation; model configuration; and procedures and processes for credible mechanistic multiscale modeling. We also discuss how garnering strong community involvement can promote model credibility. Finally, in addition to direct usage with patients, we note the potential for simulation usage in the area of Simulation-Based Medical Education, an area which to date has been primarily reliant on physical models (mannequins) and scenario-based simulations rather than on numerical simulations. PMID:29713272

Credibility, Replicability, and Reproducibility in Simulation for Biomedicine and Clinical Applications in Neuroscience.

PubMed

Mulugeta, Lealem; Drach, Andrew; Erdemir, Ahmet; Hunt, C A; Horner, Marc; Ku, Joy P; Myers, Jerry G; Vadigepalli, Rajanikanth; Lytton, William W

2018-01-01

Modeling and simulation in computational neuroscience is currently a research enterprise to better understand neural systems. It is not yet directly applicable to the problems of patients with brain disease. To be used for clinical applications, there must not only be considerable progress in the field but also a concerted effort to use best practices in order to demonstrate model credibility to regulatory bodies, to clinics and hospitals, to doctors, and to patients. In doing this for neuroscience, we can learn lessons from long-standing practices in other areas of simulation (aircraft, computer chips), from software engineering, and from other biomedical disciplines. In this manuscript, we introduce some basic concepts that will be important in the development of credible clinical neuroscience models: reproducibility and replicability; verification and validation; model configuration; and procedures and processes for credible mechanistic multiscale modeling. We also discuss how garnering strong community involvement can promote model credibility. Finally, in addition to direct usage with patients, we note the potential for simulation usage in the area of Simulation-Based Medical Education, an area which to date has been primarily reliant on physical models (mannequins) and scenario-based simulations rather than on numerical simulations.

Structure and Dynamics of Replication-Mutation Systems

NASA Astrophysics Data System (ADS)

Schuster, Peter

1987-03-01

The kinetic equations of polynucleotide replication can be brought into fairly simple form provided certain environmental conditions are fulfilled. Two flow reactors, the continuously stirred tank reactor (CSTR) and a special dialysis reactor are particularly suitable for the analysis of replication kinetics. An experimental setup to study the chemical reaction network of RNA synthesis was derived from the bacteriophage Qβ. It consists of a virus specific RNA polymerase, Qβ replicase, the activated ribonucleosides GTP, ATP, CTP and UTP as well as a template suitable for replication. The ordinary differential equations for replication and mutation under the conditions of the flow reactors were analysed by the qualitative methods of bifurcation theory as well as by numerical integration. The various kinetic equations are classified according to their dynamical properties: we distinguish "quasilinear systems" which have uniquely stable point attractors and "nonlinear systems" with inherent nonlinearities which lead to multiple steady states, Hopf bifuractions, Feigenbaum-like sequences and chaotic dynamics for certain parameter ranges. Some examples which are relevant in molecular evolution and population genetics are discussed in detail.

Measuring DNA Replication in Hypoxic Conditions.

PubMed

Foskolou, Iosifina P; Biasoli, Deborah; Olcina, Monica M; Hammond, Ester M

2016-01-01

It is imperative that dividing cells maintain replication fork integrity in order to prevent DNA damage and cell death. The investigation of DNA replication is of high importance as alterations in this process can lead to genomic instability, a known causative factor of tumor development. A simple, sensitive, and informative technique which enables the study of DNA replication, is the DNA fiber assay, an adaptation of which is described in this chapter. The DNA fiber method is a powerful tool, which allows the quantitative and qualitative analysis of DNA replication at the single molecule level. The sequential pulse labeling of live cells with two thymidine analogues and the subsequent detection with specific antibodies and fluorescence imaging allows direct examination of sites of DNA synthesis. In this chapter, we describe how this assay can be performed in conditions of low oxygen levels (hypoxia)-a physiologically relevant stress that occurs in most solid tumors. Moreover, we suggest ways on how to overcome the technical problems that arise while using the hypoxic chambers.

Exploring the roles of DNA methylation in the metal-reducing bacterium Shewanella oneidensis MR-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bendall, Matthew L.; Luong, Khai; Wetmore, Kelly M.

2013-08-30

We performed whole genome analyses of DNA methylation in Shewanella 17 oneidensis MR-1 to examine its possible role in regulating gene expression and 18 other cellular processes. Single-Molecule Real Time (SMRT) sequencing 19 revealed extensive methylation of adenine (N6mA) throughout the 20 genome. These methylated bases were located in five sequence motifs, 21 including three novel targets for Type I restriction/modification enzymes. The 22 sequence motifs targeted by putative methyltranferases were determined via 23 SMRT sequencing of gene knockout mutants. In addition, we found S. 24 oneidensis MR-1 cultures grown under various culture conditions displayed 25 different DNA methylation patterns.more » However, the small number of differentially 26 methylated sites could not be directly linked to the much larger number of 27 differentially expressed genes in these conditions, suggesting DNA methylation is 28 not a major regulator of gene expression in S. oneidensis MR-1. The enrichment 29 of methylated GATC motifs in the origin of replication indicate DNA methylation 30 may regulate genome replication in a manner similar to that seen in Escherichia 31 coli. Furthermore, comparative analyses suggest that many 32 Gammaproteobacteria, including all members of the Shewanellaceae family, may 33 also utilize DNA methylation to regulate genome replication.« less

Bacillus spp. from rainforest soil promote plant growth under limited nitrogen conditions.

PubMed

Huang, X-F; Zhou, D; Guo, J; Manter, D K; Reardon, K F; Vivanco, J M

2015-03-01

The aim of this study was to evaluate effects of PGPR (plant growth-promoting rhizobacteria) isolated from rainforest soil on different plants under limited nitrogen conditions. Bacterial isolates from a Peruvian rainforest soil were screened for plant growth-promoting effects on Arabidopsis (Col-0). Four selected isolates including one Bacillus subtilis, two B. atrophaeus and one B. pumilus significantly promoted growth of Zea mays L. and Solanum lycopersicum under greenhouse conditions. Moreover, the PGPRs significantly promoted growth of S. lycopersicum in both low and nitrogen-amended soil conditions. These PGPR strains were further studied to obtain insights into possible mechanisms of plant growth promotion. Volatile chemicals from those isolates promoted Arabidopsis growth, and the expression of genes related to IAA production was induced in the Arabidopsis plants treated with PGPRs. Further, selected PGPR strains triggered induced systemic resistance (ISR) against Pseudomonas syringae pv tomato DC3000 in Arabidopsis. PGPR strains isolated from the rainforest soil promoted the plant growth of Arabidopsis, corn and tomato. New PGPR that have wider adaptability to different crops, soils and environmental conditions are needed to decrease our reliance on agricultural amendments derived from fossil-based fuels. The PGPRs isolated from a nonagricultural site constitute new plant growth-promoting strains that could be developed for agricultural uses. © 2014 The Society for Applied Microbiology.

Optimization of an RNA-Seq Differential Gene Expression Analysis Depending on Biological Replicate Number and Library Size

PubMed Central

Lamarre, Sophie; Frasse, Pierre; Zouine, Mohamed; Labourdette, Delphine; Sainderichin, Elise; Hu, Guojian; Le Berre-Anton, Véronique; Bouzayen, Mondher; Maza, Elie

2018-01-01

RNA-Seq is a widely used technology that allows an efficient genome-wide quantification of gene expressions for, for example, differential expression (DE) analysis. After a brief review of the main issues, methods and tools related to the DE analysis of RNA-Seq data, this article focuses on the impact of both the replicate number and library size in such analyses. While the main drawback of previous relevant studies is the lack of generality, we conducted both an analysis of a two-condition experiment (with eight biological replicates per condition) to compare the results with previous benchmark studies, and a meta-analysis of 17 experiments with up to 18 biological conditions, eight biological replicates and 100 million (M) reads per sample. As a global trend, we concluded that the replicate number has a larger impact than the library size on the power of the DE analysis, except for low-expressed genes, for which both parameters seem to have the same impact. Our study also provides new insights for practitioners aiming to enhance their experimental designs. For instance, by analyzing both the sensitivity and specificity of the DE analysis, we showed that the optimal threshold to control the false discovery rate (FDR) is approximately 2−r, where r is the replicate number. Furthermore, we showed that the false positive rate (FPR) is rather well controlled by all three studied R packages: DESeq, DESeq2, and edgeR. We also analyzed the impact of both the replicate number and library size on gene ontology (GO) enrichment analysis. Interestingly, we concluded that increases in the replicate number and library size tend to enhance the sensitivity and specificity, respectively, of the GO analysis. Finally, we recommend to RNA-Seq practitioners the production of a pilot data set to strictly analyze the power of their experimental design, or the use of a public data set, which should be similar to the data set they will obtain. For individuals working on tomato research, on the basis of the meta-analysis, we recommend at least four biological replicates per condition and 20 M reads per sample to be almost sure of obtaining about 1000 DE genes if they exist. PMID:29491871

Proteasome-dependent degradation of replisome components regulates faithful DNA replication.

PubMed

Roseaulin, Laura C; Noguchi, Chiaki; Noguchi, Eishi

2013-08-15

The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF(Pof3) (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.

Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome

PubMed Central

De Nicola, Beatrice; Lech, Christopher J.; Heddi, Brahim; Regmi, Sagar; Frasson, Ilaria; Perrone, Rosalba; Richter, Sara N.; Phan, Anh Tuân

2016-01-01

The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics. PMID:27298260

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

PubMed

Harley, Margaret E; Murina, Olga; Leitch, Andrea; Higgs, Martin R; Bicknell, Louise S; Yigit, Gökhan; Blackford, Andrew N; Zlatanou, Anastasia; Mackenzie, Karen J; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A M; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B; Nürnberg, Peter; Jackson, Stephen P; Hurles, Matthew E; Wollnik, Bernd; Stewart, Grant S; Jackson, Andrew P

2016-01-01

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

Autophagic machinery activated by dengue virus enhances virus replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Y.-R.; Lei, H.-Y.; Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan

2008-05-10

Autophagy is a cellular response against stresses which include the infection of viruses and bacteria. We unravel that Dengue virus-2 (DV2) can trigger autophagic process in various infected cell lines demonstrated by GFP-LC3 dot formation and increased LC3-II formation. Autophagosome formation was also observed under the transmission electron microscope. DV2-induced autophagy further enhances the titers of extracellular and intracellular viruses indicating that autophagy can promote viral replication in the infected cells. Moreover, our data show that ATG5 protein is required to execute DV2-induced autophagy. All together, we are the first to demonstrate that DV can activate autophagic machinery that ismore » favorable for viral replication.« less

Self-replication: Nanostructure evolution

NASA Astrophysics Data System (ADS)

Simmel, Friedrich C.

2017-10-01

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

Use of the bovine leukaemia virus LTR U3 promoter for expressing antisense antiviral RNAs and competitive inhibition of viral infection in cell culture.

PubMed

Shayakhmetov, D; Kovalenko, D; Yurov, G; Borisenko, A; Tikchonenko, T

1997-08-01

Use of viral inducible promoters which can be activated by virus-specific transactivator proteins to drive expression of antisense (as)RNA genes appears to be an attractive approach to inhibit virus infections in vivo. To this end, we have constructed an asRNA gene expressed from the bovine leukaemia virus (BLV) U3 promoter that is complementary to the R-U5 region of the BLV genome. This is the region that is most susceptible to inhibition by asRNA. With plasmid pLU, which expresses the asRNA gene under the control of the BLV U3 promoter, 75% inhibition of virus replication was attained in CC81 cells (the molar ratio of pLU DNA over BLV proviral DNA in the transfection mixture was 5:1). Plasmid pLT, which contains only the BLV U3 promoter without any asRNA-coding region, also efficiently (up to 60%) inhibited virus replication when cotransfected with BLV proviral DNA at a ratio of 20:1. It was suggested that competition between functional and 'empty' viral promoters for the viral transactivator protein p38tax could account for this inhibition. An immunoblotting assay showed that in the presence of nuclear extracts from CC81 cells exogenous BLV p38tax specifically associates with its responsive sequence located in the BLV U3 promoter. Moreover, the additional expression of p38tax in CC81 cells abolishes the inhibitory effect of the empty viral promoter. These observations suggest a new mechanism of BLV inhibition caused, most probably, by sequestering of the viral transactivator protein.

Salmonella exploits the host endolysosomal tethering factor HOPS complex to promote its intravacuolar replication

PubMed Central

Sindhwani, Aastha; Kaur, Harmeet; Tuli, Amit

2017-01-01

Salmonella enterica serovar typhimurium extensively remodels the host late endocytic compartments to establish its vacuolar niche within the host cells conducive for its replication, also known as the Salmonella-containing vacuole (SCV). By maintaining a prolonged interaction with late endosomes and lysosomes of the host cells in the form of interconnected network of tubules (Salmonella-induced filaments or SIFs), Salmonella gains access to both membrane and fluid-phase cargo from these compartments. This is essential for maintaining SCV membrane integrity and for bacterial intravacuolar nutrition. Here, we have identified the multisubunit lysosomal tethering factor—HOPS (HOmotypic fusion and Protein Sorting) complex as a crucial host factor facilitating delivery of late endosomal and lysosomal content to SCVs, providing membrane for SIF formation, and nutrients for intravacuolar bacterial replication. Accordingly, depletion of HOPS subunits significantly reduced the bacterial load in non-phagocytic and phagocytic cells as well as in a mouse model of Salmonella infection. We found that Salmonella effector SifA in complex with its binding partner; SKIP, interacts with HOPS subunit Vps39 and mediates recruitment of this tethering factor to SCV compartments. The lysosomal small GTPase Arl8b that binds to, and promotes membrane localization of Vps41 (and other HOPS subunits) was also required for HOPS recruitment to SCVs and SIFs. Our findings suggest that Salmonella recruits the host late endosomal and lysosomal membrane fusion machinery to its vacuolar niche for access to host membrane and nutrients, ensuring its intracellular survival and replication. PMID:29084291

A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins

PubMed Central

de Wilde, Adriaan H.; Wannee, Kazimier F.; Scholte, Florine E. M.; Goeman, Jelle J.; ten Dijke, Peter; Snijder, Eric J.

2015-01-01

ABSTRACT To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the β2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a >2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy. IMPORTANCE Replication of all viruses, including SARS-CoV, depends on and is influenced by cellular pathways. Although substantial progress has been made in dissecting the coronavirus replicative cycle, our understanding of the host factors that stimulate (proviral factors) or restrict (antiviral factors) infection remains far from complete. To study the role of host proteins in SARS-CoV infection, we set out to systematically identify kinase-regulated processes that influence virus replication. Protein kinases are key regulators in signal transduction, controlling a wide variety of cellular processes, and many of them are targets of approved drugs and other compounds. Our screen identified a variety of hits and will form the basis for more detailed follow-up studies that should contribute to a better understanding of SARS-CoV replication and coronavirus-host interactions in general. The identified factors could be interesting targets for the development of host-directed antiviral therapy to treat infections with SARS-CoV or other pathogenic coronaviruses. PMID:26041291

Single Cell Analysis of Human RAD18-Dependent DNA Post-Replication Repair by Alkaline Bromodeoxyuridine Comet Assay

PubMed Central

Mórocz, Mónika; Gali, Himabindu; Raskó, István; Downes, C. Stephen; Haracska, Lajos

2013-01-01

Damage to DNA can block replication progression resulting in gaps in the newly synthesized DNA. Cells utilize a number of post-replication repair (PRR) mechanisms such as the RAD18 controlled translesion synthesis or template switching to overcome the discontinuities formed opposite the DNA lesions and to complete DNA replication. Gaining more insights into the role of PRR genes promotes better understanding of DNA damage tolerance and of how their malfunction can lead to increased genome instability and cancer. However, a simple and efficient method to characterise gene specific PRR deficiencies at a single cell level has not been developed. Here we describe the so named BrdU comet PRR assay to test the contribution of human RAD18 to PRR at a single cell level, by which we kinetically characterized the consequences of the deletion of human RAD18 on the replication of UV-damaged DNA. Moreover, we demonstrate the capability of our method to evaluate PRR at a single cell level in unsynchronized cell population. PMID:23936422

Autophagy postpones apoptotic cell death in PRRSV infection through Bad-Beclin1 interaction.

PubMed

Zhou, Ao; Li, Shuaifeng; Khan, Faheem Ahmed; Zhang, Shujun

2016-01-01

Autophagy and apoptosis play significant roles in PRRSV infection and replication. However, the interaction between these 2 processes in PRRSV replication is still far from been completely understood. In our studies, the exposure of MARC-145 cells to PRRSV confirmed the activation of autophagy and subsequent induction of apoptosis. The inhibition of autophagy by 3-methyladenine (3-MA) caused a significant increase in PRRSV-induced apoptosis, showing a potential connection between both mechanisms. Moreover, we observed an increase in Bad expression (a pro-apoptotic protein) and Beclin1 (an autophagy regulator) in virus-infected cells up to 36h. Co-immunoprecipitation assays showed the formation of Bad and Beclin1 complex in PRRSV infected cells. Accordingly, Bad co-localized with Beclin1 in MARC-145 infected cells. Knockdown of Beclin1 significantly decreased PRRSV replication and PRRSV-induced autophagy, while Bad silencing resulted in increased autophagy and enhanced viral replication. Furthermore, PRRSV infection phosphorylated Bad (Ser112) to promote cellular survival. These results demonstrate that autophagy can favor PRRSV replication by postponing apoptosis through the formation of a Bad-Beclin1 complex.

H4 replication-dependent diacetylation and Hat1 promote S-phase chromatin assembly in vivo

PubMed Central

Ejlassi-Lassallette, Aïda; Mocquard, Eloïse; Arnaud, Marie-Claire; Thiriet, Christophe

2011-01-01

While specific posttranslational modification patterns within the H3 and H4 tail domains are associated with the S-phase, their actual functions in replication-dependent chromatin assembly have not yet been defined. Here we used incorporation of trace amounts of recombinant proteins into naturally synchronous macroplasmodia of Physarum polycephalum to examine the function of H3 and H4 tail domains in replication-coupled chromatin assembly. We found that the H3/H4 complex lacking the H4 tail domain was not efficiently recovered in nuclei, whereas depletion of the H3 tail domain did not impede nuclear import but chromatin assembly failed. Furthermore, our results revealed that the proper pattern of acetylation on the H4 tail domain is required for nuclear import and chromatin assembly. This is most likely due to binding of Hat1, as coimmunoprecipitation experiments showed Hat1 associated with predeposition histones in the cytoplasm and with replicating chromatin. These results suggest that the type B histone acetyltransferase assists in shuttling the H3/H4 complex from cytoplasm to the replication forks. PMID:21118997

Cyclophilin A Interacts with Viral VP4 and Inhibits the Replication of Infectious Bursal Disease Virus.

PubMed

Wang, Nian; Zhang, Lizhou; Chen, Yuming; Lu, Zhen; Gao, Li; Wang, Yongqiang; Gao, Yulong; Gao, Honglei; Cui, Hongyu; Li, Kai; Liu, Changjun; Zhang, Yanping; Qi, Xiaole; Wang, Xiaomei

2015-01-01

Nonstructural protein VP4, a serine protease of infectious bursal disease virus (IBDV) that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 to form the viral proteins VP2, VP4, and VP3, is essential to the replication of IBDV. However, the interacting partners of VP4 in host cells and the effects of the interaction on the IBDV lifecycle remain incompletely elucidated. In this study, using the yeast two-hybrid system, the putative VP4-interacting partner cyclophilin A (CypA) was obtained from a chicken embryo fibroblast (CEF) expression library. CypA was further confirmed to interact with VP4 of IBDV using co-immunoprecipitation (CO-IP), GST pull-down, and confocal microscopy assays. Moreover, we found that the overexpression of CypA suppressed IBDV replication, whereas the knock-down of CypA by small interfering RNAs promoted the replication of IBDV. Taken together, our findings indicate that the host cell protein CypA interacts with viral VP4 and inhibits the replication of IBDV.

Autophagy postpones apoptotic cell death in PRRSV infection through Bad-Beclin1 interaction

PubMed Central

Zhou, Ao; Li, Shuaifeng; Khan, Faheem Ahmed; Zhang, Shujun

2016-01-01

Autophagy and apoptosis play significant roles in PRRSV infection and replication. However, the interaction between these 2 processes in PRRSV replication is still far from been completely understood. In our studies, the exposure of MARC-145 cells to PRRSV confirmed the activation of autophagy and subsequent induction of apoptosis. The inhibition of autophagy by 3-methyladenine (3-MA) caused a significant increase in PRRSV-induced apoptosis, showing a potential connection between both mechanisms. Moreover, we observed an increase in Bad expression (a pro-apoptotic protein) and Beclin1 (an autophagy regulator) in virus-infected cells up to 36h. Co-immunoprecipitation assays showed the formation of Bad and Beclin1 complex in PRRSV infected cells. Accordingly, Bad co-localized with Beclin1 in MARC-145 infected cells. Knockdown of Beclin1 significantly decreased PRRSV replication and PRRSV-induced autophagy, while Bad silencing resulted in increased autophagy and enhanced viral replication. Furthermore, PRRSV infection phosphorylated Bad (Ser112) to promote cellular survival. These results demonstrate that autophagy can favor PRRSV replication by postponing apoptosis through the formation of a Bad-Beclin1 complex. PMID:26670824

Perturbation of ribosome biogenesis drives cells into senescence through 5S RNP-mediated p53 activation.

PubMed

Nishimura, Kazuho; Kumazawa, Takuya; Kuroda, Takao; Katagiri, Naohiro; Tsuchiya, Mai; Goto, Natsuka; Furumai, Ryohei; Murayama, Akiko; Yanagisawa, Junn; Kimura, Keiji

2015-03-03

The 5S ribonucleoprotein particle (RNP) complex, consisting of RPL11, RPL5, and 5S rRNA, is implicated in p53 regulation under ribotoxic stress. Here, we show that the 5S RNP contributes to p53 activation and promotes cellular senescence in response to oncogenic or replicative stress. Oncogenic stress accelerates rRNA transcription and replicative stress delays rRNA processing, resulting in RPL11 and RPL5 accumulation in the ribosome-free fraction, where they bind MDM2. Experimental upregulation of rRNA transcription or downregulation of rRNA processing, mimicking the nucleolus under oncogenic or replicative stress, respectively, also induces RPL11-mediated p53 activation and cellular senescence. We demonstrate that exogenous expression of certain rRNA-processing factors rescues the processing defect, attenuates p53 accumulation, and increases replicative lifespan. To summarize, the nucleolar-5S RNP-p53 pathway functions as a senescence inducer in response to oncogenic and replicative stresses. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

SEE locomotor behavior test discriminates C57BL/6J and DBA/2J mouse inbred strains across laboratories and protocol conditions.

PubMed

Kafkafi, Neri; Lipkind, Dina; Benjamini, Yoav; Mayo, Cheryl L; Elmer, Gregory I; Golani, Ilan

2003-06-01

Conventional tests of behavioral phenotyping frequently have difficulties differentiating certain genotypes and replicating these differences across laboratories and protocol conditions. This study explores the hypothesis that automated tests can be designed to quantify ethologically relevant behavior patterns that more readily characterize heritable and replicable phenotypes. It used SEE (Strategy for the Exploration of Exploration) to phenotype the locomotor behavior of the C57BL/6 and DBA/2 mouse inbred strains across 3 laboratories. The 2 genotypes differed in 15 different measures of behavior, none of which had a significant genotype-laboratory interaction. Within the same laboratory, most of these differences were replicated in additional experiments despite the test photoperiod phase being changed and saline being injected. Results suggest that well-designed tests may considerably enhance replicability across laboratories.

Agents of Reconciliation: Agency-Affirmation Promotes Constructive Tendencies Following Transgressions in Low-Commitment Relationships.

PubMed

SimanTov-Nachlieli, Ilanit; Shnabel, Nurit; Mori-Hoffman, Anael

2017-02-01

Conflicting parties experience threats to both their agency and morality, but the experience of agency-threat exerts more influence on their behavior, leading to relationship-destructive tendencies. Whereas high-commitment relationships facilitate constructive tendencies despite the conflict, we theorized that in low-commitment relationships, affirming the adversary's agency is a prerequisite for facilitating more constructive tendencies. Focusing on sibling conflicts, Study 1 found that when commitment was low (rather than high), agency-affirmation increased participants' constructive tendencies toward their brother/sister compared with a control/no-affirmation condition. A corresponding morality-affirmation did not affect participants' tendencies. Study 2 replicated these results in workplace conflicts and further found that the positive effect of agency-affirmation in low-commitment relationships was mediated by participants' wish to restore their morality. Study 3 induced a conflict between lab participants and manipulated their commitment. Again, in the low- (rather than high-) commitment condition, agency-affirmation increased participants' wish to restore their morality, leading to constructive behavior.

HIV-1 Vif promotes the formation of high molecular mass APOBEC3G complexes

PubMed Central

Goila-Gaur, Ritu; Khan, Mohammad A.; Miyagi, Eri; Kao, Sandra; Opi, Sandrine; Takeuchi, Hiroaki; Strebel, Klaus

2008-01-01

HIV-1 Vif inhibits the antiviral activity of APOBEC3G (APO3G) by inducing proteasomal degradation. Here, we studied the effects of Vif on APO3G in vitro. In this system, Vif did not cause APO3G degradation. Instead, Vif induced changes in APO3G that affected immunoprecipitation of the native protein. This effect required wt Vif and was reversed by heat-denaturation of APO3G. Sucrose gradient analysis demonstrated that wt Vif induced the gradual transition of APO3G translated in vitro or expressed in HeLa cells from a low molecular mass conformation to puromycin-sensitive high molecular mass (HMM) complexes. In the absence of Vif or the presence of biologically inactive Vif APO3G failed to form HMM complexes. Our results expose a novel function of Vif that promotes the assembly of APO3G into presumably packaging-incompetent HMM complexes and may explain how Vif can overcome the APO3G-imposed block to HIV replication under conditions of no or inefficient APO3G degradation. PMID:18023836

Local application of periodontal ligament stromal cells promotes soft tissue regeneration.

PubMed

Baik, H S; Park, J; Lee, K J; Chung, C

2014-09-01

To test the potential stimulatory effect of local application of periodontal ligament (PDL) stromal cells on soft tissue regeneration. Fluorescently labeled PDL cells outgrown from extracted human premolars or phosphate-buffered saline were locally injected to the cutaneous wounds created on mice. Soft tissue regeneration was evaluated for 14 days using photographs and histomorphometry. PDL cell engraftment was tracked with confocal microscopy. To detect the paracrine effect of the PDL cells on soft tissue regeneration, PDL cell-conditioned medium (CM) was evaluated for the concentration of secretory factors, transforming growth factor-beta 1 (TGFβ1). The effect of PDL CM on the proliferation and migration of dermal fibroblast and keratinocyte was tested using MTT assay and migration assay. The application of PDL cells significantly promoted soft tissue regeneration compared with the application of PBS. Self-replicating PDL cells were engrafted into the hair follicles of the host tissue. Dermal fibroblast proliferation and keratinocyte migration were significantly enhanced by the treatment with PDL CM. Physiologically significant amount of TGFβ1 was secreted from PDL cells into the CM. Local injection of PDL cells promoted soft tissue regeneration in part by the enhancement of fibroblast proliferation and keratinocyte migration through a paracrine mechanism. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.