Random mutagenesis by error-prone pol plasmid replication in Escherichia coli.

PubMed

Alexander, David L; Lilly, Joshua; Hernandez, Jaime; Romsdahl, Jillian; Troll, Christopher J; Camps, Manel

2014-01-01

Directed evolution is an approach that mimics natural evolution in the laboratory with the goal of modifying existing enzymatic activities or of generating new ones. The identification of mutants with desired properties involves the generation of genetic diversity coupled with a functional selection or screen. Genetic diversity can be generated using PCR or using in vivo methods such as chemical mutagenesis or error-prone replication of the desired sequence in a mutator strain. In vivo mutagenesis methods facilitate iterative selection because they do not require cloning, but generally produce a low mutation density with mutations not restricted to specific genes or areas within a gene. For this reason, this approach is typically used to generate new biochemical properties when large numbers of mutants can be screened or selected. Here we describe protocols for an advanced in vivo mutagenesis method that is based on error-prone replication of a ColE1 plasmid bearing the gene of interest. Compared to other in vivo mutagenesis methods, this plasmid-targeted approach allows increased mutation loads and facilitates iterative selection approaches. We also describe the mutation spectrum for this mutagenesis methodology in detail, and, using cycle 3 GFP as a target for mutagenesis, we illustrate the phenotypic diversity that can be generated using our method. In sum, error-prone Pol I replication is a mutagenesis method that is ideally suited for the evolution of new biochemical activities when a functional selection is available.

Selection Bias, Vote Counting, and Money-Priming Effects: A Comment on Rohrer, Pashler, and Harris (2015) and Vohs (2015)

PubMed Central

2016-01-01

When a series of studies fails to replicate a well-documented effect, researchers might be tempted to use a “vote counting” approach to decide whether the effect is reliable—that is, simply comparing the number of successful and unsuccessful replications. Vohs’s (2015) response to the absence of money priming effects reported by Rohrer, Pashler, and Harris (2015) provides an example of this approach. Unfortunately, vote counting is a poor strategy to assess the reliability of psychological findings because it neglects the impact of selection bias and questionable research practices. In the present comment, we show that a range of meta-analytic tools indicate irregularities in the money priming literature discussed by Rohrer et al. and Vohs, which all point to the conclusion that these effects are distorted by selection bias, reporting biases, or p-hacking. This could help to explain why money-priming effects have proven unreliable in a number of direct replication attempts in which biases have been minimized through preregistration or transparent reporting. Our major conclusion is that the simple proportion of significant findings is a poor guide to the reliability of research and that preregistered replications are an essential means to assess the reliability of money-priming effects. PMID:27077759

Selection bias, vote counting, and money-priming effects: A comment on Rohrer, Pashler, and Harris (2015) and Vohs (2015).

PubMed

Vadillo, Miguel A; Hardwicke, Tom E; Shanks, David R

2016-05-01

When a series of studies fails to replicate a well-documented effect, researchers might be tempted to use a "vote counting" approach to decide whether the effect is reliable-that is, simply comparing the number of successful and unsuccessful replications. Vohs's (2015) response to the absence of money priming effects reported by Rohrer, Pashler, and Harris (2015) provides an example of this approach. Unfortunately, vote counting is a poor strategy to assess the reliability of psychological findings because it neglects the impact of selection bias and questionable research practices. In the present comment, we show that a range of meta-analytic tools indicate irregularities in the money priming literature discussed by Rohrer et al. and Vohs, which all point to the conclusion that these effects are distorted by selection bias, reporting biases, or p-hacking. This could help to explain why money-priming effects have proven unreliable in a number of direct replication attempts in which biases have been minimized through preregistration or transparent reporting. Our major conclusion is that the simple proportion of significant findings is a poor guide to the reliability of research and that preregistered replications are an essential means to assess the reliability of money-priming effects. (c) 2016 APA, all rights reserved).

Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication.

PubMed

Brödel, Andreas K; Jaramillo, Alfonso; Isalan, Mark

2017-09-01

Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. These include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. A typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (PM) library expressing variants of the gene of interest (GOI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. The system has been developed for the selection of new orthogonal transcription factors (TFs) but is capable of evolving any gene-or gene circuit function-that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage λ cI TF against two synthetic bidirectional promoters. The evolved TF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. This protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ∼7 weeks.

Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome

PubMed Central

Erliandri, Indri; Fu, Haiqing; Nakano, Megumi; Kim, Jung-Hyun; Miga, Karen H.; Liskovykh, Mikhail; Earnshaw, William C.; Masumoto, Hiroshi; Kouprina, Natalay; Aladjem, Mirit I.; Larionov, Vladimir

2014-01-01

In human chromosomes, centromeric regions comprise megabase-size arrays of 171 bp alpha-satellite DNA monomers. The large distances spanned by these arrays preclude their replication from external sites and imply that the repetitive monomers contain replication origins. However, replication within these arrays has not previously been profiled and the role of alpha-satellite DNA in initiation of DNA replication has not yet been demonstrated. Here, replication of alpha-satellite DNA in endogenous human centromeric regions and in de novo formed Human Artificial Chromosome (HAC) was analyzed. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized into centrochromatin occurred earlier than those organized into heterochromatin. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions. Depletion of CENP-B, a kinetochore protein that binds directly to a 17 bp CENP-B box motif common to alpha-satellite DNA, resulted in enrichment of alpha-satellite sequences for proteins of the ORC complex, suggesting that CENP-B may have a role in regulating the replication of centromeric regions. Mapping of replication initiation sites in the HAC revealed that replication preferentially initiated in transcriptionally active regions. PMID:25228468

Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

NASA Astrophysics Data System (ADS)

Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

1990-12-01

Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

Neuronal boost to evolutionary dynamics

PubMed Central

de Vladar, Harold P.; Szathmáry, Eörs

2015-01-01

Standard evolutionary dynamics is limited by the constraints of the genetic system. A central message of evolutionary neurodynamics is that evolutionary dynamics in the brain can happen in a neuronal niche in real time, despite the fact that neurons do not reproduce. We show that Hebbian learning and structural synaptic plasticity broaden the capacity for informational replication and guided variability provided a neuronally plausible mechanism of replication is in place. The synergy between learning and selection is more efficient than the equivalent search by mutation selection. We also consider asymmetric landscapes and show that the learning weights become correlated with the fitness gradient. That is, the neuronal complexes learn the local properties of the fitness landscape, resulting in the generation of variability directed towards the direction of fitness increase, as if mutations in a genetic pool were drawn such that they would increase reproductive success. Evolution might thus be more efficient within evolved brains than among organisms out in the wild. PMID:26640653

The alien replicon: Artificial genetic constructs to direct the synthesis of transmissible self-replicating RNAs: In vivo synthesised heterologous (alien) RNA constructs are capable of initiating self-replication following transmission to the host organism.

PubMed

Kochetov, Alex V

2014-12-01

Artificial genetic constructs that direct the synthesis of self-replicating RNA molecules are used widely to induce gene silencing, for bioproduction, and for vaccination. Interestingly, one variant of the self-replicon has not been discussed in the literature: namely, transgenic organisms that synthesise alien replicons. For example, plant cells may be easily genetically modified to produce bacteriophages or insect viruses. Alien replicon-producing organisms (ARPOs) may serve as a unique tool for biocontrol or to selectively influence the characteristics of a target organism. The ARPO approach would have to meet strict biosafety criteria, and its practical applications are problematic. However, a discussion on ARPO applicability would be valuable to outline the full set of options available in the bioengineering toolbox. In this paper, RNA replicons for bioengineering are reviewed briefly, and the ARPO approach is discussed. © 2014 WILEY Periodicals, Inc.

Five rules for the evolution of cooperation.

PubMed

Nowak, Martin A

2006-12-08

Cooperation is needed for evolution to construct new levels of organization. Genomes, cells, multicellular organisms, social insects, and human society are all based on cooperation. Cooperation means that selfish replicators forgo some of their reproductive potential to help one another. But natural selection implies competition and therefore opposes cooperation unless a specific mechanism is at work. Here I discuss five mechanisms for the evolution of cooperation: kin selection, direct reciprocity, indirect reciprocity, network reciprocity, and group selection. For each mechanism, a simple rule is derived that specifies whether natural selection can lead to cooperation.

Five Rules for the Evolution of Cooperation

NASA Astrophysics Data System (ADS)

Nowak, Martin A.

2006-12-01

Cooperation is needed for evolution to construct new levels of organization. Genomes, cells, multicellular organisms, social insects, and human society are all based on cooperation. Cooperation means that selfish replicators forgo some of their reproductive potential to help one another. But natural selection implies competition and therefore opposes cooperation unless a specific mechanism is at work. Here I discuss five mechanisms for the evolution of cooperation: kin selection, direct reciprocity, indirect reciprocity, network reciprocity, and group selection. For each mechanism, a simple rule is derived that specifies whether natural selection can lead to cooperation.

Human embryonic stem cells fail to activate CHK1 and commit to apoptosis in response to DNA replication stress.

PubMed

Desmarais, Joëlle A; Hoffmann, Michele J; Bingham, Gregg; Gagou, Mary E; Meuth, Mark; Andrews, Peter W

2012-07-01

Pluripotent cells of the early embryo, to which embryonic stem cells (ESCs) correspond, give rise to all the somatic cells of the developing fetus. Any defects that occur in their genome or epigenome would have devastating consequences. Genetic and epigenetic change in human ESCs appear to be an inevitable consequence of long-term culture, driven by selection of variant cells that have a higher propensity for self-renewal rather than either differentiation or death. Mechanisms underlying the potentially separate events of mutation and subsequent selection of variants are poorly understood. Here, we show that human ESCs and their malignant counterpart, embryonal carcinoma (EC) cells, both fail to activate critical S-phase checkpoints when exposed to DNA replication inhibitors and commit to apoptosis instead. Human ESCs and EC cells also fail to form replication protein A, γH2AX, or RAD51 foci or load topoisomerase (DNA) II binding protein 1 onto chromatin in response to replication inhibitors. Furthermore, direct measurements of single-stranded DNA (ssDNA) show that these cells fail to generate the ssDNA regions in response to replication stress that are necessary for the activation of checkpoints and the initiation of homologous recombination repair to protect replication fork integrity and restart DNA replication. Taken together, our data suggest that pluripotent cells control genome integrity by the elimination of damaged cells through apoptosis rather than DNA repair, and therefore, mutations or epigenetic modifications resulting in an imbalance in cell death control could lead to genetic instability. Copyright © 2012 AlphaMed Press.

Local neutral networks help maintain inaccurately replicating ribozymes.

PubMed

Szilágyi, András; Kun, Ádám; Szathmáry, Eörs

2014-01-01

The error threshold of replication limits the selectively maintainable genome size against recurrent deleterious mutations for most fitness landscapes. In the context of RNA replication a distinction between the genotypic and the phenotypic error threshold has been made; where the latter concerns the maintenance of secondary structure rather than sequence. RNA secondary structure is treated as a proxy for function. The phenotypic error threshold allows higher per digit mutation rates than its genotypic counterpart, and is known to increase with the frequency of neutral mutations in sequence space. Here we show that the degree of neutrality, i.e. the frequency of nearest-neighbour (one-step) neutral mutants is a remarkably accurate proxy for the overall frequency of such mutants in an experimentally verifiable formula for the phenotypic error threshold; this we achieve by the full numerical solution for the concentration of all sequences in mutation-selection balance up to length 16. We reinforce our previous result that currently known ribozymes could be selectively maintained by the accuracy known from the best available polymerase ribozymes. Furthermore, we show that in silico stabilizing selection can increase the mutational robustness of ribozymes due to the fact that they were produced by artificial directional selection in the first place. Our finding offers a better understanding of the error threshold and provides further insight into the plausibility of an ancient RNA world.

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts.

PubMed

Bernstein, Diana L; Le Lay, John E; Ruano, Elena G; Kaestner, Klaus H

2015-05-01

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator-like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics.

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts

PubMed Central

Bernstein, Diana L.; Le Lay, John E.; Ruano, Elena G.; Kaestner, Klaus H.

2015-01-01

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator–like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics. PMID:25866970

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.

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

STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function

PubMed Central

Bhattacharjee, Anukana; Stewart, Jason; Chaiken, Mary; Price, Carolyn M.

2016-01-01

Mammalian CST (CTC1-STN1-TEN1) participates in multiple aspects of telomere replication and genome-wide recovery from replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Conservation between CTC1 and RPA1 is less apparent. Currently the mechanism underlying CST action is largely unknown. Here we address CST mechanism by using a DNA-binding mutant, (STN1 OB-fold mutant, STN1-OBM) to examine the relationship between DNA binding and CST function. In vivo, STN1-OBM affects resolution of endogenous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. These selective effects indicate mechanistic differences in CST action during resolution of different replication problems. In vitro binding studies show that STN1 directly engages both short and long ssDNA oligonucleotides, however STN1-OBM preferentially destabilizes binding to short substrates. The finding that STN1-OBM affects binding to only certain substrates starts to explain the in vivo separation of function observed in STN1-OBM expressing cells. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve different replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The in vitro studies also provide insight into CST binding mechanism. Like RPA, CST likely contacts DNA via multiple OB folds. However, the importance of STN1 for binding short substrates indicates differences in the architecture of CST and RPA DNA-protein complexes. Based on our results, we propose a dynamic DNA binding model that provides a general mechanism for CST action at diverse forms of replication stress. PMID:27690379

The cosmological model of eternal inflation and the transition from chance to biological evolution in the history of life

PubMed Central

Koonin, Eugene V

2007-01-01

Background Recent developments in cosmology radically change the conception of the universe as well as the very notions of "probable" and "possible". The model of eternal inflation implies that all macroscopic histories permitted by laws of physics are repeated an infinite number of times in the infinite multiverse. In contrast to the traditional cosmological models of a single, finite universe, this worldview provides for the origin of an infinite number of complex systems by chance, even as the probability of complexity emerging in any given region of the multiverse is extremely low. This change in perspective has profound implications for the history of any phenomenon, and life on earth cannot be an exception. Hypothesis Origin of life is a chicken and egg problem: for biological evolution that is governed, primarily, by natural selection, to take off, efficient systems for replication and translation are required, but even barebones cores of these systems appear to be products of extensive selection. The currently favored (partial) solution is an RNA world without proteins in which replication is catalyzed by ribozymes and which serves as the cradle for the translation system. However, the RNA world faces its own hard problems as ribozyme-catalyzed RNA replication remains a hypothesis and the selective pressures behind the origin of translation remain mysterious. Eternal inflation offers a viable alternative that is untenable in a finite universe, i.e., that a coupled system of translation and replication emerged by chance, and became the breakthrough stage from which biological evolution, centered around Darwinian selection, took off. A corollary of this hypothesis is that an RNA world, as a diverse population of replicating RNA molecules, might have never existed. In this model, the stage for Darwinian selection is set by anthropic selection of complex systems that rarely but inevitably emerge by chance in the infinite universe (multiverse). Conclusion The plausibility of different models for the origin of life on earth directly depends on the adopted cosmological scenario. In an infinite universe (multiverse), emergence of highly complex systems by chance is inevitable. Therefore, under this cosmology, an entity as complex as a coupled translation-replication system should be considered a viable breakthrough stage for the onset of biological evolution. Reviewers This article was reviewed by Eric Bapteste, David Krakauer, Sergei Maslov, and Itai Yanai. PMID:17540027

The cosmological model of eternal inflation and the transition from chance to biological evolution in the history of life.

PubMed

Koonin, Eugene V

2007-05-31

Recent developments in cosmology radically change the conception of the universe as well as the very notions of "probable" and "possible". The model of eternal inflation implies that all macroscopic histories permitted by laws of physics are repeated an infinite number of times in the infinite multiverse. In contrast to the traditional cosmological models of a single, finite universe, this worldview provides for the origin of an infinite number of complex systems by chance, even as the probability of complexity emerging in any given region of the multiverse is extremely low. This change in perspective has profound implications for the history of any phenomenon, and life on earth cannot be an exception. Origin of life is a chicken and egg problem: for biological evolution that is governed, primarily, by natural selection, to take off, efficient systems for replication and translation are required, but even barebones cores of these systems appear to be products of extensive selection. The currently favored (partial) solution is an RNA world without proteins in which replication is catalyzed by ribozymes and which serves as the cradle for the translation system. However, the RNA world faces its own hard problems as ribozyme-catalyzed RNA replication remains a hypothesis and the selective pressures behind the origin of translation remain mysterious. Eternal inflation offers a viable alternative that is untenable in a finite universe, i.e., that a coupled system of translation and replication emerged by chance, and became the breakthrough stage from which biological evolution, centered around Darwinian selection, took off. A corollary of this hypothesis is that an RNA world, as a diverse population of replicating RNA molecules, might have never existed. In this model, the stage for Darwinian selection is set by anthropic selection of complex systems that rarely but inevitably emerge by chance in the infinite universe (multiverse). The plausibility of different models for the origin of life on earth directly depends on the adopted cosmological scenario. In an infinite universe (multiverse), emergence of highly complex systems by chance is inevitable. Therefore, under this cosmology, an entity as complex as a coupled translation-replication system should be considered a viable breakthrough stage for the onset of biological evolution. This article was reviewed by Eric Bapteste, David Krakauer, Sergei Maslov, and Itai Yanai.

Five rules for the evolution of cooperation

PubMed Central

Nowak, Martin A.

2011-01-01

Cooperation is needed for evolution to construct new levels of organization. The emergence of genomes, cells, multi-cellular organisms, social insects and human society are all based on cooperation. Cooperation means that selfish replicators forgo some of their reproductive potential to help one another. But natural selection implies competition and therefore opposes cooperation unless a specific mechanism is at work. Here I discuss five mechanisms for the evolution of cooperation: kin selection, direct reciprocity, indirect reciprocity, network reciprocity and group selection. For each mechanism, a simple rule is derived which specifies whether natural selection can lead to cooperation. PMID:17158317

The Influence of Primary and Secondary DNA Structure in Deletion and Duplication between Direct Repeats in Escherichia Coli

PubMed Central

Trinh, T. Q.; Sinden, R. R.

1993-01-01

We describe a system to measure the frequency of both deletions and duplications between direct repeats. Short 17- and 18-bp palindromic and nonpalindromic DNA sequences were cloned into the EcoRI site within the chloramphenicol acetyltransferase gene of plasmids pBR325 and pJT7. This creates an insert between direct repeated EcoRI sites and results in a chloramphenicol-sensitive phenotype. Selection for chloramphenicol resistance was utilized to select chloramphenicol resistant revertants that included those with precise deletion of the insert from plasmid pBR325 and duplication of the insert in plasmid pJT7. The frequency of deletion or duplication varied more than 500-fold depending on the sequence of the short sequence inserted into the EcoRI site. For the nonpalindromic inserts, multiple internal direct repeats and the length of the direct repeats appear to influence the frequency of deletion. Certain palindromic DNA sequences with the potential to form DNA hairpin structures that might stabilize the misalignment of direct repeats had a high frequency of deletion. Other DNA sequences with the potential to form structures that might destabilize misalignment of direct repeats had a very low frequency of deletion. Duplication mutations occurred at the highest frequency when the DNA between the direct repeats contained no direct or inverted repeats. The presence of inverted repeats dramatically reduced the frequency of duplications. The results support the slippage-misalignment model, suggesting that misalignment occurring during DNA replication leads to deletion and duplication mutations. The results also support the idea that the formation of DNA secondary structures during DNA replication can facilitate and direct specific mutagenic events. PMID:8325478

Single-Molecule FISH Reveals Non-selective Packaging of Rift Valley Fever Virus Genome Segments

PubMed Central

Wichgers Schreur, Paul J.; Kortekaas, Jeroen

2016-01-01

The bunyavirus genome comprises a small (S), medium (M), and large (L) RNA segment of negative polarity. Although genome segmentation confers evolutionary advantages by enabling genome reassortment events with related viruses, genome segmentation also complicates genome replication and packaging. Accumulating evidence suggests that genomes of viruses with eight or more genome segments are incorporated into virions by highly selective processes. Remarkably, little is known about the genome packaging process of the tri-segmented bunyaviruses. Here, we evaluated, by single-molecule RNA fluorescence in situ hybridization (FISH), the intracellular spatio-temporal distribution and replication kinetics of the Rift Valley fever virus (RVFV) genome and determined the segment composition of mature virions. The results reveal that the RVFV genome segments start to replicate near the site of infection before spreading and replicating throughout the cytoplasm followed by translocation to the virion assembly site at the Golgi network. Despite the average intracellular S, M and L genome segments approached a 1:1:1 ratio, major differences in genome segment ratios were observed among cells. We also observed a significant amount of cells lacking evidence of M-segment replication. Analysis of two-segmented replicons and four-segmented viruses subsequently confirmed the previous notion that Golgi recruitment is mediated by the Gn glycoprotein. The absence of colocalization of the different segments in the cytoplasm and the successful rescue of a tri-segmented variant with a codon shuffled M-segment suggested that inter-segment interactions are unlikely to drive the copackaging of the different segments into a single virion. The latter was confirmed by direct visualization of RNPs inside mature virions which showed that the majority of virions lack one or more genome segments. Altogether, this study suggests that RVFV genome packaging is a non-selective process. PMID:27548280

Mutation rate evolution in replicator dynamics.

PubMed

Allen, Benjamin; Rosenbloom, Daniel I Scholes

2012-11-01

The mutation rate of an organism is itself evolvable. In stable environments, if faithful replication is costless, theory predicts that mutation rates will evolve to zero. However, positive mutation rates can evolve in novel or fluctuating environments, as analytical and empirical studies have shown. Previous work on this question has focused on environments that fluctuate independently of the evolving population. Here we consider fluctuations that arise from frequency-dependent selection in the evolving population itself. We investigate how the dynamics of competing traits can induce selective pressure on the rates of mutation between these traits. To address this question, we introduce a theoretical framework combining replicator dynamics and adaptive dynamics. We suppose that changes in mutation rates are rare, compared to changes in the traits under direct selection, so that the expected evolutionary trajectories of mutation rates can be obtained from analysis of pairwise competition between strains of different rates. Depending on the nature of frequency-dependent trait dynamics, we demonstrate three possible outcomes of this competition. First, if trait frequencies are at a mutation-selection equilibrium, lower mutation rates can displace higher ones. Second, if trait dynamics converge to a heteroclinic cycle-arising, for example, from "rock-paper-scissors" interactions-mutator strains succeed against non-mutators. Third, in cases where selection alone maintains all traits at positive frequencies, zero and nonzero mutation rates can coexist indefinitely. Our second result suggests that relatively high mutation rates may be observed for traits subject to cyclical frequency-dependent dynamics.

Structural basis of the 3′-end recognition of a leading strand in stalled replication forks by PriA

PubMed Central

Sasaki, Kaori; Ose, Toyoyuki; Okamoto, Naoaki; Maenaka, Katsumi; Tanaka, Taku; Masai, Hisao; Saito, Mihoko; Shirai, Tsuyoshi; Kohda, Daisuke

2007-01-01

In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3′ end of a nascent leading DNA strand in the stalled replication forks. The crystal structures in complexes with oligonucleotides and the combination of fluorescence correlation spectroscopy and mutagenesis reveal that the N-terminal domain of PriA possesses a binding pocket for the 3′-terminal nucleotide residue of DNA. The interaction with the deoxyribose 3′-OH is essential for the 3′-terminal recognition. In contrast, the direct interaction with 3′-end nucleobase is unexpected, considering the same affinity for oligonucleotides carrying the four bases at the 3′ end. Thus, the N-terminal domain of PriA recognizes the 3′-end base in a base-non-selective manner, in addition to the deoxyribose and 5′-side phosphodiester group, of the 3′-terminal nucleotide to acquire both sufficient affinity and non-selectivity to find all of the stalled replication forks generated during DNA duplication. This unique feature is prerequisite for the proper positioning of the helicase domain of PriA on the unreplicated double-stranded DNA. PMID:17464287

Directional selection in temporally replicated studies is remarkably consistent.

PubMed

Morrissey, Michael B; Hadfield, Jarrod D

2012-02-01

Temporal variation in selection is a fundamental determinant of evolutionary outcomes. A recent paper presented a synthetic analysis of temporal variation in selection in natural populations. The authors concluded that there is substantial variation in the strength and direction of selection over time, but acknowledged that sampling error would result in estimates of selection that were more variable than the true values. We reanalyze their dataset using techniques that account for the necessary effect of sampling error to inflate apparent levels of variation and show that directional selection is remarkably constant over time, both in magnitude and direction. Thus we cannot claim that the available data support the existence of substantial temporal heterogeneity in selection. Nonetheless, we conject that temporal variation in selection could be important, but that there are good reasons why it may not appear in the available data. These new analyses highlight the importance of applying techniques that estimate parameters of the distribution of selection, rather than parameters of the distribution of estimated selection (which will reflect both sampling error and "real" variation in selection); indeed, despite availability of methods for the former, focus on the latter has been common in synthetic reviews of the aspects of selection in nature, and can lead to serious misinterpretations. © 2011 The Author(s). Evolution© 2011 The Society for the Study of Evolution.

Dengue virus replicates and accumulates in Aedes aegypti salivary glands

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

Raquin, Vincent, E-mail: vincent.raquin@univ-lyon1

Dengue virus (DENV) is an RNA virus transmitted among humans by mosquito vectors, mainly Aedes aegypti. DENV transmission requires viral dissemination from the mosquito midgut to the salivary glands. During this process the virus undergoes several population bottlenecks, which are stochastic reductions in population size that restrict intra-host viral genetic diversity and limit the efficiency of natural selection. Despite the implications for virus transmission and evolution, DENV replication in salivary glands has not been directly demonstrated. Here, we used a strand-specific quantitative RT-PCR assay to demonstrate that negative-strand DENV RNA is produced in Ae. aegypti salivary glands, providing conclusive evidencemore » that viral replication occurs in this tissue. Furthermore, we showed that the concentration of DENV genomic RNA in salivary glands increases significantly over time, indicating that active replication likely replenishes DENV genetic diversity prior to transmission. These findings improve our understanding of the biological determinants of DENV fitness and evolution. - Highlights: •Strand-specific RT-qPCR allows accurate quantification of DENV (-) RNA in mosquito tissues. •Detection of DENV (-) RNA in salivary glands provides evidence of viral replication in this tissue. •Viral replication in salivary glands likely replenishes DENV genetic diversity prior to transmission.« less

Replication stress-induced chromosome breakage is correlated with replication fork progression and is preceded by single-stranded DNA formation.

PubMed

Feng, Wenyi; Di Rienzi, Sara C; Raghuraman, M K; Brewer, Bonita J

2011-10-01

Chromosome breakage as a result of replication stress has been hypothesized to be the direct consequence of defective replication fork progression, or "collapsed" replication forks. However, direct and genome-wide evidence that collapsed replication forks give rise to chromosome breakage is still lacking. Previously we showed that a yeast replication checkpoint mutant mec1-1, after transient exposure to replication impediment imposed by hydroxyurea (HU), failed to complete DNA replication, accumulated single-stranded DNA (ssDNA) at the replication forks, and fragmented its chromosomes. In this study, by following replication fork progression genome-wide via ssDNA detection and by direct mapping of chromosome breakage after HU exposure, we have tested the hypothesis that the chromosome breakage in mec1 cells occurs at collapsed replication forks. We demonstrate that sites of chromosome breakage indeed correlate with replication fork locations. Moreover, ssDNA can be detected prior to chromosome breakage, suggesting that ssDNA accumulation is the common precursor to double strand breaks at collapsed replication forks.

The origin of polynucleotide-directed protein synthesis

NASA Technical Reports Server (NTRS)

Orgel, Leslie E.

1989-01-01

If protein synthesis evolved in an RNA world it was probably preceded by simpler processes by means of which interaction with amino acids conferred selective advantage on replicating RNA molecules. It is suggested that at first the simple attachment of amino acids to the 2'(3') termini of RNA templates favored initiation of replication at the end of the template rather than at internal positions. The second stage in the evolution of protein synthesis would probably have been the association of pairs of charged RNA adaptors in such a way as to favor noncoded formation of peptides. Only after this process had become efficient could coded synthesis have begun.

Host-directed combinatorial RNAi improves inhibition of diverse strains of influenza A virus in human respiratory epithelial cells.

PubMed

Estrin, Michael A; Hussein, Islam T M; Puryear, Wendy B; Kuan, Anne C; Artim, Stephen C; Runstadler, Jonathan A

2018-01-01

Influenza A virus infections are important causes of morbidity and mortality worldwide, and currently available prevention and treatment methods are suboptimal. In recent years, genome-wide investigations have revealed numerous host factors that are required for influenza to successfully complete its life cycle. However, only a select, small number of influenza strains were evaluated using this platform, and there was considerable variation in the genes identified across different investigations. In an effort to develop a universally efficacious therapeutic strategy with limited potential for the emergence of resistance, this study was performed to investigate the effect of combinatorial RNA interference (RNAi) on inhibiting the replication of diverse influenza A virus subtypes and strains. Candidate genes were selected for targeting based on the results of multiple previous independent genome-wide studies. The effect of single and combinatorial RNAi on the replication of 12 diverse influenza A viruses, including three strains isolated from birds and one strain isolated from seals, was then evaluated in primary normal human bronchial epithelial cells. After excluding overly toxic siRNA, two siRNA combinations were identified that reduced mean viral replication by greater than 79 percent in all mammalian strains, and greater than 68 percent in all avian strains. Host-directed combinatorial RNAi effectively prevents growth of a broad range of influenza virus strains in vitro, and is a potential therapeutic candidate for further development and future in vivo studies.

Precise replication of antireflective nanostructures from biotemplates

NASA Astrophysics Data System (ADS)

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

2007-03-01

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

Exponential growth and selection in self-replicating materials from DNA origami rafts

NASA Astrophysics Data System (ADS)

He, Xiaojin; Sha, Ruojie; Zhuo, Rebecca; Mi, Yongli; Chaikin, Paul M.; Seeman, Nadrian C.

2017-10-01

Self-replication and evolution under selective pressure are inherent phenomena in life, and but few artificial systems exhibit these phenomena. We have designed a system of DNA origami rafts that exponentially replicates a seed pattern, doubling the copies in each diurnal-like cycle of temperature and ultraviolet illumination, producing more than 7 million copies in 24 cycles. We demonstrate environmental selection in growing populations by incorporating pH-sensitive binding in two subpopulations. In one species, pH-sensitive triplex DNA bonds enable parent-daughter templating, while in the second species, triplex binding inhibits the formation of duplex DNA templating. At pH 5.3, the replication rate of species I is ~1.3-1.4 times faster than that of species II. At pH 7.8, the replication rates are reversed. When mixed together in the same vial, the progeny of species I replicate preferentially at pH 7.8 similarly at pH 5.3, the progeny of species II take over the system. This addressable selectivity should be adaptable to the selection and evolution of multi-component self-replicating materials in the nanoscopic-to-microscopic size range.

An ultra-precision tool nanoindentation instrument for replication of single point diamond tool cutting edges

NASA Astrophysics Data System (ADS)

Cai, Yindi; Chen, Yuan-Liu; Xu, Malu; Shimizu, Yuki; Ito, So; Matsukuma, Hiraku; Gao, Wei

2018-05-01

Precision replication of the diamond tool cutting edge is required for non-destructive tool metrology. This paper presents an ultra-precision tool nanoindentation instrument designed and constructed for replication of the cutting edge of a single point diamond tool onto a selected soft metal workpiece by precisely indenting the tool cutting edge into the workpiece surface. The instrument has the ability to control the indentation depth with a nanometric resolution, enabling the replication of tool cutting edges with high precision. The motion of the diamond tool along the indentation direction is controlled by the piezoelectric actuator of a fast tool servo (FTS). An integrated capacitive sensor of the FTS is employed to detect the displacement of the diamond tool. The soft metal workpiece is attached to an aluminum cantilever whose deflection is monitored by another capacitive sensor, referred to as an outside capacitive sensor. The indentation force and depth can be accurately evaluated from the diamond tool displacement, the cantilever deflection and the cantilever spring constant. Experiments were carried out by replicating the cutting edge of a single point diamond tool with a nose radius of 2.0 mm on a copper workpiece surface. The profile of the replicated tool cutting edge was measured using an atomic force microscope (AFM). The effectiveness of the instrument in precision replication of diamond tool cutting edges is well-verified by the experimental results.

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

PubMed Central

Etheridge, Thomas J.; Boulineau, Rémi L.; Herbert, Alex; Watson, Adam T.; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest; Osborne, Mark A.; Klenerman, David; Lee, Steven F.; Carr, Antony M.

2014-01-01

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds. PMID:25106872

Cryo-EM of dynamic protein complexes in eukaryotic DNA replication.

PubMed

Sun, Jingchuan; Yuan, Zuanning; Bai, Lin; Li, Huilin

2017-01-01

DNA replication in Eukaryotes is a highly dynamic process that involves several dozens of proteins. Some of these proteins form stable complexes that are amenable to high-resolution structure determination by cryo-EM, thanks to the recent advent of the direct electron detector and powerful image analysis algorithm. But many of these proteins associate only transiently and flexibly, precluding traditional biochemical purification. We found that direct mixing of the component proteins followed by 2D and 3D image sorting can capture some very weakly interacting complexes. Even at 2D average level and at low resolution, EM images of these flexible complexes can provide important biological insights. It is often necessary to positively identify the feature-of-interest in a low resolution EM structure. We found that systematically fusing or inserting maltose binding protein (MBP) to selected proteins is highly effective in these situations. In this chapter, we describe the EM studies of several protein complexes involved in the eukaryotic DNA replication over the past decade or so. We suggest that some of the approaches used in these studies may be applicable to structural analysis of other biological systems. © 2016 The Protein Society.

Tigers on trails: occupancy modeling for cluster sampling.

PubMed

Hines, J E; Nichols, J D; Royle, J A; MacKenzie, D I; Gopalaswamy, A M; Kumar, N Samba; Karanth, K U

2010-07-01

Occupancy modeling focuses on inference about the distribution of organisms over space, using temporal or spatial replication to allow inference about the detection process. Inference based on spatial replication strictly requires that replicates be selected randomly and with replacement, but the importance of these design requirements is not well understood. This paper focuses on an increasingly popular sampling design based on spatial replicates that are not selected randomly and that are expected to exhibit Markovian dependence. We develop two new occupancy models for data collected under this sort of design, one based on an underlying Markov model for spatial dependence and the other based on a trap response model with Markovian detections. We then simulated data under the model for Markovian spatial dependence and fit the data to standard occupancy models and to the two new models. Bias of occupancy estimates was substantial for the standard models, smaller for the new trap response model, and negligible for the new spatial process model. We also fit these models to data from a large-scale tiger occupancy survey recently conducted in Karnataka State, southwestern India. In addition to providing evidence of a positive relationship between tiger occupancy and habitat, model selection statistics and estimates strongly supported the use of the model with Markovian spatial dependence. This new model provides another tool for the decomposition of the detection process, which is sometimes needed for proper estimation and which may also permit interesting biological inferences. In addition to designs employing spatial replication, we note the likely existence of temporal Markovian dependence in many designs using temporal replication. The models developed here will be useful either directly, or with minor extensions, for these designs as well. We believe that these new models represent important additions to the suite of modeling tools now available for occupancy estimation in conservation monitoring. More generally, this work represents a contribution to the topic of cluster sampling for situations in which there is a need for specific modeling (e.g., reflecting dependence) for the distribution of the variable(s) of interest among subunits.

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

Evolution of the additive genetic variance–covariance matrix under continuous directional selection on a complex behavioural phenotype

PubMed Central

Careau, Vincent; Wolak, Matthew E.; Carter, Patrick A.; Garland, Theodore

2015-01-01

Given the pace at which human-induced environmental changes occur, a pressing challenge is to determine the speed with which selection can drive evolutionary change. A key determinant of adaptive response to multivariate phenotypic selection is the additive genetic variance–covariance matrix (G). Yet knowledge of G in a population experiencing new or altered selection is not sufficient to predict selection response because G itself evolves in ways that are poorly understood. We experimentally evaluated changes in G when closely related behavioural traits experience continuous directional selection. We applied the genetic covariance tensor approach to a large dataset (n = 17 328 individuals) from a replicated, 31-generation artificial selection experiment that bred mice for voluntary wheel running on days 5 and 6 of a 6-day test. Selection on this subset of G induced proportional changes across the matrix for all 6 days of running behaviour within the first four generations. The changes in G induced by selection resulted in a fourfold slower-than-predicted rate of response to selection. Thus, selection exacerbated constraints within G and limited future adaptive response, a phenomenon that could have profound consequences for populations facing rapid environmental change. PMID:26582016

HIV-1 protease inhibitor mutations affect the development of HIV-1 resistance to the maturation inhibitor bevirimat.

PubMed

Fun, Axel; van Maarseveen, Noortje M; Pokorná, Jana; Maas, Renée Em; Schipper, Pauline J; Konvalinka, Jan; Nijhuis, Monique

2011-08-24

Maturation inhibitors are an experimental class of antiretrovirals that inhibit Human Immunodeficiency Virus (HIV) particle maturation, the structural rearrangement required to form infectious virus particles. This rearrangement is triggered by the ordered cleavage of the precursor Gag polyproteins into their functional counterparts by the viral enzyme protease. In contrast to protease inhibitors, maturation inhibitors impede particle maturation by targeting the substrate of protease (Gag) instead of the protease enzyme itself. Direct cross-resistance between protease and maturation inhibitors may seem unlikely, but the co-evolution of protease and its substrate, Gag, during protease inhibitor therapy, could potentially affect future maturation inhibitor therapy. Previous studies showed that there might also be an effect of protease inhibitor resistance mutations on the development of maturation inhibitor resistance, but the exact mechanism remains unclear. We used wild-type and protease inhibitor resistant viruses to determine the impact of protease inhibitor resistance mutations on the development of maturation inhibitor resistance. Our resistance selection studies demonstrated that the resistance profiles for the maturation inhibitor bevirimat are more diverse for viruses with a mutated protease compared to viruses with a wild-type protease. Viral replication did not appear to be a major factor during emergence of bevirimat resistance. In all in vitro selections, one of four mutations was selected: Gag V362I, A364V, S368N or V370A. The impact of these mutations on maturation inhibitor resistance and viral replication was analyzed in different protease backgrounds. The data suggest that the protease background affects development of HIV-1 resistance to bevirimat and the replication profiles of bevirimat-selected HIV-1. The protease-dependent bevirimat resistance and replication levels can be explained by differences in CA/p2 cleavage processing by the different proteases. These findings highlight the complicated interactions between the viral protease and its substrate. By providing a better understanding of these interactions, we aim to help guide the development of second generation maturation inhibitors.

Changes in transcriptional orientation are associated with increases in evolutionary rates of enterobacterial genes.

PubMed

Lin, Chieh-Hua; Lian, Chun-Yi; Hsiung, Chao Agnes; Chen, Feng-Chi

2011-10-05

Changes in transcriptional orientation ("CTOs") occur frequently in prokaryotic genomes. Such changes usually result from genomic inversions, which may cause a conflict between the directions of replication and transcription and an increase in mutation rate. However, CTOs do not always lead to the replication-transcription confrontation. Furthermore, CTOs may cause deleterious disruptions of operon structure and/or gene regulations. The currently existing CTOs may indicate relaxation of selection pressure. Therefore, it is of interest to investigate whether CTOs have an independent effect on the evolutionary rates of the affected genes, and whether these genes are subject to any type of selection pressure in prokaryotes. Three closely related enterbacteria, Escherichia coli, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium, were selected for comparisons of synonymous (dS) and nonsynonymous (dN) substitution rate between the genes that have experienced changes in transcriptional orientation (changed-orientation genes, "COGs") and those that do not (same-orientation genes, "SOGs"). The dN/dS ratio was also derived to evaluate the selection pressure on the analyzed genes. Confounding factors in the estimation of evolutionary rates, such as gene essentiality, gene expression level, replication-transcription confrontation, and decreased dS at gene terminals were controlled in the COG-SOG comparisons. We demonstrate that COGs have significantly higher dN and dS than SOGs when a series of confounding factors are controlled. However, the dN/dS ratios are similar between the two gene groups, suggesting that the increase in dS can sufficiently explain the increase in dN in COGs. Therefore, the increases in evolutionary rates in COGs may be mainly mutation-driven. Here we show that CTOs can increase the evolutionary rates of the affected genes. This effect is independent of the replication-transcription confrontation, which is suggested to be the major cause of inversion-associated evolutionary rate increases. The real cause of such evolutionary rate increases remains unclear but is worth further explorations.

Compartmentalized self-replication: a novel method for the directed evolution of polymerases and other enzymes.

PubMed

Ghadessy, Farid J; Holliger, Philipp

2007-01-01

Compartmentalized self-replication (CSR) is a novel method for the directed evolution of enzymes and, in particular, polymerases. In its simplest form, CSR consists of a simple feedback loop involving a polymerase that replicates only its own encoding gene (self-replication). Self-replication occurs in discrete, spatially separate, noncommunicating compartments formed by a heat-stable water-in-oil emulsion. Compartmentalization ensures the linkage of phenotype and genotype (i.e., it ensures that each polymerase replicates only its own encoding gene to the exclusion of those in the other compartments). As a result, adaptive gains by the polymerase directly (and proportionally) translate into genetic amplification of the encoding polymerase gene. CSR has proven to be a useful strategy for the directed evolution of polymerases directly from diverse repertoires of polymerase genes. In this chapter, we describe some of the CSR protocols used successfully to evolve variants of T. aquaticus Pol I (Taq) polymerase with novel and useful properties, such as increased thermostability or resistance to the potent inhibitor, heparin, from a repertoire of randomly mutated Taq polymerase genes.

Analysis of 60 reported glioma risk SNPs replicates published GWAS findings but fails to replicate associations from published candidate-gene studies.

PubMed

Walsh, Kyle M; Anderson, Erik; Hansen, Helen M; Decker, Paul A; Kosel, Matt L; Kollmeyer, Thomas; Rice, Terri; Zheng, Shichun; Xiao, Yuanyuan; Chang, Jeffrey S; McCoy, Lucie S; Bracci, Paige M; Wiemels, Joe L; Pico, Alexander R; Smirnov, Ivan; Lachance, Daniel H; Sicotte, Hugues; Eckel-Passow, Jeanette E; Wiencke, John K; Jenkins, Robert B; Wrensch, Margaret R

2013-02-01

Genomewide association studies (GWAS) and candidate-gene studies have implicated single-nucleotide polymorphisms (SNPs) in at least 45 different genes as putative glioma risk factors. Attempts to validate these associations have yielded variable results and few genetic risk factors have been consistently replicated. We conducted a case-control study of Caucasian glioma cases and controls from the University of California San Francisco (810 cases, 512 controls) and the Mayo Clinic (852 cases, 789 controls) in an attempt to replicate previously reported genetic risk factors for glioma. Sixty SNPs selected from the literature (eight from GWAS and 52 from candidate-gene studies) were successfully genotyped on an Illumina custom genotyping panel. Eight SNPs in/near seven different genes (TERT, EGFR, CCDC26, CDKN2A, PHLDB1, RTEL1, TP53) were significantly associated with glioma risk in the combined dataset (P < 0.05), with all associations in the same direction as in previous reports. Several SNP associations showed considerable differences across histologic subtype. All eight successfully replicated associations were first identified by GWAS, although none of the putative risk SNPs from candidate-gene studies was associated in the full case-control sample (all P values > 0.05). Although several confirmed associations are located near genes long known to be involved in gliomagenesis (e.g., EGFR, CDKN2A, TP53), these associations were first discovered by the GWAS approach and are in noncoding regions. These results highlight that the deficiencies of the candidate-gene approach lay in selecting both appropriate genes and relevant SNPs within these genes. © 2012 WILEY PERIODICALS, INC.

Isolation and Characterization of Highly Replicable Hepatitis C Virus Genotype 1a Strain HCV-RMT

PubMed Central

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

2013-01-01

Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV) clone called H77. The replication ability of some of these clones has been confirmed in vitro and in vivo, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient’s serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between in vitro and in vivo replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication in vitro and in vivo. PMID:24358200

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

PubMed

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

2013-01-01

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

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

NASA Astrophysics Data System (ADS)

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

1994-07-01

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

Direct NMR Evidence that Transient Tautomeric and Anionic States in dG·dT Form Watson-Crick-like Base Pairs.

PubMed

Szymanski, Eric S; Kimsey, Isaac J; Al-Hashimi, Hashim M

2017-03-29

The replicative and translational machinery utilizes the unique geometry of canonical G·C and A·T/U Watson-Crick base pairs to discriminate against DNA and RNA mismatches in order to ensure high fidelity replication, transcription, and translation. There is growing evidence that spontaneous errors occur when mismatches adopt a Watson-Crick-like geometry through tautomerization and/or ionization of the bases. Studies employing NMR relaxation dispersion recently showed that wobble dG·dT and rG·rU mismatches in DNA and RNA duplexes transiently form tautomeric and anionic species with probabilities (≈0.01-0.40%) that are in concordance with replicative and translational errors. Although computational studies indicate that these exceptionally short-lived and low-abundance species form Watson-Crick-like base pairs, their conformation could not be directly deduced from the experimental data, and alternative pairing geometries could not be ruled out. Here, we report direct NMR evidence that the transient tautomeric and anionic species form hydrogen-bonded Watson-Crick-like base pairs. A guanine-to-inosine substitution, which selectively knocks out a Watson-Crick-type (G)N2H 2 ···O2(T) hydrogen bond, significantly destabilized the transient tautomeric and anionic species, as assessed by lack of any detectable chemical exchange by imino nitrogen rotating frame spin relaxation (R 1ρ ) experiments. An 15 N R 1ρ NMR experiment targeting the amino nitrogen of guanine (dG-N2) provides direct evidence for Watson-Crick (G)N2H 2 ···O2(T) hydrogen bonding in the transient tautomeric state. The strategy presented in this work can be generally applied to examine hydrogen-bonding patterns in nucleic acid transient states including in other tautomeric and anionic species that are postulated to play roles in replication and translational errors.

Down-regulate of Djrfc2 causes tissues hypertrophy during planarian regeneration.

PubMed

Guo, Qi; Zhao, Guixia; Ni, Jiajia; Guo, Yanan; Zhang, Yizhe; Tian, Qingnan; Zhang, Shoutao

2017-11-25

Planarians are an ideal model organism for regeneration research due to their amazing ability to regenerate. DNA replication is crucial for genome stability. Replication factor C (RFC), which is a replication factor C-like complex and plays an important role during DNA replication in eukaryotes, has been reported as a wound response factor during planarian regeneration. However, how RFC controls regeneration in planarians by regulating DNA replication remains to be explained. Here, we used a two-dimensional electrophoresis (2-DE) proteomic approach to identify differentially expressed proteins in intact and regenerated planarians. Approximately 132 protein spots showed differences between intact and regenerative tissues. We selected 21 significantly expressed protein spots and processed them using TOF MS analysis. Finally, we cloned three of these candidate genes (Djhsp70, Djrfc2, Djfaim), focusing on the function of Djrfc2 during regeneration. We found that the distribution of Djrfc2 tends toward the wound site. RNA interference (RNAi) of Djrfc2 increases the number of dividing cells and the expression level of planarian neoblast marker genes, which may result in hyper-proliferation. Our studies use an available approach to directly study the regeneration dynamic at the protein level and provide further evidence to support a function of Djrfc2 in planarian regeneration. Copyright © 2017. Published by Elsevier Inc.

Lambda Red Mediated Gap Repair Utilizes a Novel Replicative Intermediate in Escherichia coli

PubMed Central

Reddy, Thimma R.; Fevat, Léna M. S.; Munson, Sarah E.; Stewart, A. Francis; Cowley, Shaun M.

2015-01-01

The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction. Consequently, our findings have important implications for the understanding of E. coli replication and Red recombination. PMID:25803509

Enantiomeric Cross-Inhibition in the Synthesis of Oligonucleotides on a Nonchiral Template

NASA Technical Reports Server (NTRS)

Schmidt, Jurgen G.; Nielsen, Peter E.; Orgel, Leslie E.

1997-01-01

Prebiotic syntheses of chiral monomers always yield racemic mixtures. Living systems, however, utilize L-amino acids and D-nucleotides in their biopolymers. The generation of optical asymmetry by selection and amplification in an autocatalytic process is, therefore, an important element in many theories of the origin of life. Replication of polynucleotides in template-directed syntheses is an obvious candidate for such an amplification step in a pre-'RNA world'. A serious objection to this suggestion is the observation that the efficiency of template-directed syntheses of RNA is limited by enantiomeric cross-inhibition. Peptide Nucleic Acids (PNAs), amide-linked, nonchiral analogues of RNA, have been 'copied' into RNA and constitute an alternative to chiral polynucleotides as an informational replicating system. Here, we use PNA as model for a hypothetical, nonchiral precursor of RNA in experiments re-examining enantiomeric cross-inhibition. We find that enantiomeric cross-inhibition is as serious in the polymerization of nucleotides on a PNA template as it is on a conventional RNA or DNA template.

A New Replicator: A theoretical framework for analysing replication

PubMed Central

2010-01-01

Background Replicators are the crucial entities in evolution. The notion of a replicator, however, is far less exact than the weight of its importance. Without identifying and classifying multiplying entities exactly, their dynamics cannot be determined appropriately. Therefore, it is importance to decide the nature and characteristics of any multiplying entity, in a detailed and formal way. Results Replication is basically an autocatalytic process which enables us to rest on the notions of formal chemistry. This statement has major implications. Simple autocatalytic cycle intermediates are considered as non-informational replicators. A consequence of which is that any autocatalytically multiplying entity is a replicator, be it simple or overly complex (even nests). A stricter definition refers to entities which can inherit acquired changes (informational replicators). Simple autocatalytic molecules (and nests) are excluded from this group. However, in turn, any entity possessing copiable information is to be named a replicator, even multicellular organisms. In order to deal with the situation, an abstract, formal framework is presented, which allows the proper identification of various types of replicators. This sheds light on the old problem of the units and levels of selection and evolution. A hierarchical classification for the partition of the replicator-continuum is provided where specific replicators are nested within more general ones. The classification should be able to be successfully applied to known replicators and also to future candidates. Conclusion This paper redefines the concept of the replicator from a bottom-up theoretical approach. The formal definition and the abstract models presented can distinguish between among all possible replicator types, based on their quantity of variable and heritable information. This allows for the exact identification of various replicator types and their underlying dynamics. The most important claim is that replication, in general, is basically autocatalysis, with a specific defined environment and selective force. A replicator is not valid unless its working environment, and the selective force to which it is subject, is specified. PMID:20219099

Benchmarking surface selective vacuum ultraviolet and thermal postprocessing of thermoplastics for ultrasmooth 3-D-printed micro-optics

NASA Astrophysics Data System (ADS)

Kirchner, Robert; Chidambaram, Nachiappan; Schift, Helmut

2018-04-01

State-of-the-art, polymeric, refractive micro-optics simultaneously require an ultrasmooth three-dimensional (3-D) surface and a precise geometry for excellent optical performance with minimal stray light. In earlier work, we have established a surface finishing process for thermoplastic polymer master structures that is only effective on the surface and does not affect the designed optical geometry, thus enabling polishing without touching. Therewith, the high curvature corners of a 50-μm-tall optical diffuser device were maintained while the surface roughness was reduced to about 10-nm root mean square. For this, 3-D master structures were first fabricated by direct write laser-lithography with two-photon polymerization. The master structures were replicated into poly(methyl methacrylate) through a poly(dimethyl siloxane) intermediate replication stamp. Finally, all structures were surface-polished by selective high-energy photon exposure and thermal postprocessing. In this work, we focus on the comparison of the surface smoothening using either postprocessing or dedicated direct writing strategies. For this comparison, strategies for modifying the exposed voxel size and the writing discretization being the primary source of roughness were tested by sweeping the laser exposure dose for two different resist materials and objectives. In conclusion, the postprocessing smoothening resulted in a lower roughness compared to a direct writing strategy-even when 50-nm vertical discretization steps were used-and still enabled 10 times shorter writing times.

Designing herpes viruses as oncolytics

PubMed Central

Peters, Cole; Rabkin, Samuel D

2015-01-01

Oncolytic herpes simplex virus (oHSV) was one of the first genetically-engineered oncolytic viruses. Because HSV is a natural human pathogen that can cause serious disease, it is incumbent that it can be genetically-engineered or significantly attenuated for safety. Here, we present a detailed explanation of the functions of HSV-1 genes frequently mutated to endow oncolytic activity. These genes are nonessential for growth in tissue culture cells but are important for growth in postmitotic cells, interfering with intrinsic antiviral and innate immune responses or causing pathology, functions dispensable for replication in cancer cells. Understanding the function of these genes leads to informed creation of new oHSVs with better therapeutic efficacy. Virus infection and replication can also be directed to cancer cells through tumor-selective receptor binding and transcriptional- or post-transcriptional miRNA-targeting, respectively. In addition to the direct effects of oHSV on infected cancer cells and tumors, oHSV can be “armed” with transgenes that are: reporters, to track virus replication and spread; cytotoxic, to kill uninfected tumor cells; immune modulatory, to stimulate antitumor immunity; or tumor microenvironment altering, to enhance virus spread or to inhibit tumor growth. In addition to HSV-1, other alphaherpesviruses are also discussed for their oncolytic activity. PMID:26462293

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

PubMed

Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

2017-05-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

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

Canonical and Non-Canonical Autophagy in HIV-1 Replication Cycle

PubMed Central

Leymarie, Olivier; Lepont, Leslie; Berlioz-Torrent, Clarisse

2017-01-01

Autophagy is a lysosomal-dependent degradative process essential for maintaining cellular homeostasis, and is a key player in innate and adaptive immune responses to intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1). In HIV-1 target cells, autophagy mechanisms can (i) selectively direct viral proteins and viruses for degradation; (ii) participate in the processing and presentation of viral-derived antigens through major histocompatibility complexes; and (iii) contribute to interferon production in response to HIV-1 infection. As a consequence, HIV-1 has evolved different strategies to finely regulate the autophagy pathway to favor its replication and dissemination. HIV-1 notably encodes accessory genes encoding Tat, Nef and Vpu proteins, which are able to perturb and hijack canonical and non-canonical autophagy mechanisms. This review outlines the current knowledge on the complex interplay between autophagy and HIV-1 replication cycle, providing an overview of the autophagy-mediated molecular processes deployed both by infected cells to combat the virus and by HIV-1 to evade antiviral response. PMID:28946621

Directional selection has shaped the oral jaws of Lake Malawi cichlid fishes.

PubMed

Albertson, R Craig; Streelman, J Todd; Kocher, Thomas D

2003-04-29

East African cichlid fishes represent one of the most striking examples of rapid and convergent evolutionary radiation among vertebrates. Models of ecological speciation would suggest that functional divergence in feeding morphology has contributed to the origin and maintenance of cichlid species diversity. However, definitive evidence for the action of natural selection has been missing. Here we use quantitative genetics to identify regions of the cichlid genome responsible for functionally important shape differences in the oral jaw apparatus. The consistent direction of effects for individual quantitative trait loci suggest that cichlid jaws and teeth evolved in response to strong, divergent selection. Moreover, several chromosomal regions contain a disproportionate number of quantitative trait loci, indicating a prominent role for pleiotropy or genetic linkage in the divergence of this character complex. Of particular interest are genomic intervals with concerted effects on both the length and height of the lower jaw. Coordinated changes in this area of the oral jaw apparatus are predicted to have direct consequences for the speed and strength of jaw movement. Taken together, our results imply that the rapid and replicative nature of cichlid trophic evolution is the result of directional selection on chromosomal packages that encode functionally linked aspects of the craniofacial skeleton.

Inhibition of duck hepatitis B virus replication by mimic peptides in vitro

PubMed Central

JIA, HONGYU; LIU, CHANGHONG; YANG, YING; ZHU, HAIHONG; CHEN, FENG; LIU, JIHONG; ZHOU, LINFU

2015-01-01

The aim of the present study was to investigate the inhibitory effect of specific mimic peptides targeting duck hepatitis B virus polymerase (DHBVP) on duck hepatitis B virus (DHBV) replication in primary duck hepatocytes. Phage display technology (PDT) was used to screen for mimic peptides specifically targeting DHBVP and the associated coding sequences were determined using DNA sequencing. The selected mimic peptides were then used to treat primary duck hepatocytes infected with DHBV in vitro. Infected hepatocytes expressing the mimic peptides intracellularly were also prepared. The cells were divided into mimic peptide groups (EXP groups), an entecavir-treated group (positive control) and a negative control group. The medium was changed every 48 h. Following a 10-day incubation, the cell supernatants were collected. DHBV-DNA in the cellular nucleus, cytoplasm and culture supernatant was analyzed by quantitative polymerase chain reaction (qPCR). Eight mimic peptides were selected following three PDT screening rounds for investigation in the DHBV-infected primary duck hepatocytes. The qPCR results showed that following direct treatment with mimic peptide 2 or 7, intracellular expression of mimic peptide 2 or 7, or treatment with entecavir, the DHBV-DNA levels in the culture supernatant and cytoplasm of duck hepatocytes were significantly lower than those in the negative control (P<0.05). The cytoplasmic DHBV-DNA content of the cells treated with mimic peptide 7 was lower than that in the other groups (P<0.05). In addition, the DHBV-DNA content of the nuclear fractions following the intracellular expression of mimic peptide 7 was significantly lower than that in the other groups (P<0.05). Mimic peptides specifically targeting DHBVP, administered directly or expressed intracellularly, can significantly inhibit DHBV replication in vitro. PMID:26640539

Inhibition of duck hepatitis B virus replication by mimic peptides in vitro.

PubMed

Jia, Hongyu; Liu, Changhong; Yang, Ying; Zhu, Haihong; Chen, Feng; Liu, Jihong; Zhou, Linfu

2015-11-01

The aim of the present study was to investigate the inhibitory effect of specific mimic peptides targeting duck hepatitis B virus polymerase (DHBVP) on duck hepatitis B virus (DHBV) replication in primary duck hepatocytes. Phage display technology (PDT) was used to screen for mimic peptides specifically targeting DHBVP and the associated coding sequences were determined using DNA sequencing. The selected mimic peptides were then used to treat primary duck hepatocytes infected with DHBV in vitro. Infected hepatocytes expressing the mimic peptides intracellularly were also prepared. The cells were divided into mimic peptide groups (EXP groups), an entecavir-treated group (positive control) and a negative control group. The medium was changed every 48 h. Following a 10-day incubation, the cell supernatants were collected. DHBV-DNA in the cellular nucleus, cytoplasm and culture supernatant was analyzed by quantitative polymerase chain reaction (qPCR). Eight mimic peptides were selected following three PDT screening rounds for investigation in the DHBV-infected primary duck hepatocytes. The qPCR results showed that following direct treatment with mimic peptide 2 or 7, intracellular expression of mimic peptide 2 or 7, or treatment with entecavir, the DHBV-DNA levels in the culture supernatant and cytoplasm of duck hepatocytes were significantly lower than those in the negative control (P<0.05). The cytoplasmic DHBV-DNA content of the cells treated with mimic peptide 7 was lower than that in the other groups (P<0.05). In addition, the DHBV-DNA content of the nuclear fractions following the intracellular expression of mimic peptide 7 was significantly lower than that in the other groups (P<0.05). Mimic peptides specifically targeting DHBVP, administered directly or expressed intracellularly, can significantly inhibit DHBV replication in vitro .

Evolution of the additive genetic variance-covariance matrix under continuous directional selection on a complex behavioural phenotype.

PubMed

Careau, Vincent; Wolak, Matthew E; Carter, Patrick A; Garland, Theodore

2015-11-22

Given the pace at which human-induced environmental changes occur, a pressing challenge is to determine the speed with which selection can drive evolutionary change. A key determinant of adaptive response to multivariate phenotypic selection is the additive genetic variance-covariance matrix ( G: ). Yet knowledge of G: in a population experiencing new or altered selection is not sufficient to predict selection response because G: itself evolves in ways that are poorly understood. We experimentally evaluated changes in G: when closely related behavioural traits experience continuous directional selection. We applied the genetic covariance tensor approach to a large dataset (n = 17 328 individuals) from a replicated, 31-generation artificial selection experiment that bred mice for voluntary wheel running on days 5 and 6 of a 6-day test. Selection on this subset of G: induced proportional changes across the matrix for all 6 days of running behaviour within the first four generations. The changes in G: induced by selection resulted in a fourfold slower-than-predicted rate of response to selection. Thus, selection exacerbated constraints within G: and limited future adaptive response, a phenomenon that could have profound consequences for populations facing rapid environmental change. © 2015 The Author(s).

The Emergence of Life as a First-Order Phase Transition

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

Mathis, Cole; Bhattacharya, Tanmoy; Walker, Sara Imari

It is well known that life on Earth alters its environment over evolutionary and geological timescales. An important open question is whether this is a result of evolutionary optimization or a universal feature of life. In the latter case, the origin of life would be coincident with a shift in environmental conditions. Here in this paper we present a model for the emergence of life in which replicators are explicitly coupled to their environment through the recycling of a finite supply of resources. The model exhibits a dynamic, first-order phase transition from nonlife to life, where the life phase ismore » distinguished by selection on replicators. We show that environmental coupling plays an important role in the dynamics of the transition. The transition corresponds to a redistribution of matter in replicators and their environment, driven by selection on replicators, exhibiting an explosive growth in diversity as replicators are selected. The transition is accurately tracked by the mutual information shared between replicators and their environment. In the absence of successfully repartitioning system resources, the transition fails to complete, leading to the possibility of many frustrated trials before life first emerges. Often, the replicators that initiate the transition are not those that are ultimately selected. The results are consistent with the view that life's propensity to shape its environment is indeed a universal feature of replicators, characteristic of the transition from nonlife to life. We discuss the implications of these results for understanding life's emergence and evolutionary transitions more broadly.« less

The Emergence of Life as a First-Order Phase Transition

DOE PAGES

Mathis, Cole; Bhattacharya, Tanmoy; Walker, Sara Imari

2017-03-01

It is well known that life on Earth alters its environment over evolutionary and geological timescales. An important open question is whether this is a result of evolutionary optimization or a universal feature of life. In the latter case, the origin of life would be coincident with a shift in environmental conditions. Here in this paper we present a model for the emergence of life in which replicators are explicitly coupled to their environment through the recycling of a finite supply of resources. The model exhibits a dynamic, first-order phase transition from nonlife to life, where the life phase ismore » distinguished by selection on replicators. We show that environmental coupling plays an important role in the dynamics of the transition. The transition corresponds to a redistribution of matter in replicators and their environment, driven by selection on replicators, exhibiting an explosive growth in diversity as replicators are selected. The transition is accurately tracked by the mutual information shared between replicators and their environment. In the absence of successfully repartitioning system resources, the transition fails to complete, leading to the possibility of many frustrated trials before life first emerges. Often, the replicators that initiate the transition are not those that are ultimately selected. The results are consistent with the view that life's propensity to shape its environment is indeed a universal feature of replicators, characteristic of the transition from nonlife to life. We discuss the implications of these results for understanding life's emergence and evolutionary transitions more broadly.« less

The Emergence of Life as a First-Order Phase Transition

NASA Astrophysics Data System (ADS)

Mathis, Cole; Bhattacharya, Tanmoy; Imari Walker, Sara

2017-03-01

It is well known that life on Earth alters its environment over evolutionary and geological timescales. An important open question is whether this is a result of evolutionary optimization or a universal feature of life. In the latter case, the origin of life would be coincident with a shift in environmental conditions. Here we present a model for the emergence of life in which replicators are explicitly coupled to their environment through the recycling of a finite supply of resources. The model exhibits a dynamic, first-order phase transition from nonlife to life, where the life phase is distinguished by selection on replicators. We show that environmental coupling plays an important role in the dynamics of the transition. The transition corresponds to a redistribution of matter in replicators and their environment, driven by selection on replicators, exhibiting an explosive growth in diversity as replicators are selected. The transition is accurately tracked by the mutual information shared between replicators and their environment. In the absence of successfully repartitioning system resources, the transition fails to complete, leading to the possibility of many frustrated trials before life first emerges. Often, the replicators that initiate the transition are not those that are ultimately selected. The results are consistent with the view that life's propensity to shape its environment is indeed a universal feature of replicators, characteristic of the transition from nonlife to life. We discuss the implications of these results for understanding life's emergence and evolutionary transitions more broadly.

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

Directed evolution of polymerase function by compartmentalized self-replication.

PubMed

Ghadessy, F J; Ong, J L; Holliger, P

2001-04-10

We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Taq DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptides cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases, as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication.

Problem solving during artificial selection of self-replicating loops

NASA Astrophysics Data System (ADS)

Chou, Hui-Hsien; Reggia, James A.

1998-05-01

Past cellular automata models of self-replication have generally done only one thing: replicate themselves. However, it has recently been demonstrated that such self-replicating structures can be programmed to also carry out a task during the replication process. Past models of this sort have been limited in that the “program” involved is copied unchanged from parent to child, so that each generation of replicants is executing exactly the same program on exactly the same data. Here we take a different approach in which each replicant receives a distinct partial solution that is modified during replication. Under artificial selection, replicants with promising solutions proliferate while those with failed solutions are lost. We show that this approach can be applied successfully to solve an NP-complete problem, the satisfiability problem. Bounds are given on the cellular space size and time needed to solve a given problem, and simulations demonstrate that this approach works effectively. These and other recent results raise the possibility of evolving self-replicating structures that have a simulated metabolism or that carry out useful tasks.

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex

PubMed Central

Ganaie, Safder S.; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve

2017-01-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases. PMID:28459842

Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.

PubMed

Kaul, Artur; Stauffer, Sarah; Berger, Carola; Pertel, Thomas; Schmitt, Jennifer; Kallis, Stephanie; Zayas, Margarita; Lopez, Margarita Zayas; Lohmann, Volker; Luban, Jeremy; Bartenschlager, Ralf

2009-08-01

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication.

Isolation and molecular cloning of a fast-growing strain of human hepatitis A virus from its double-stranded replicative form.

PubMed Central

Venuti, A; Di Russo, C; del Grosso, N; Patti, A M; Ruggeri, F; De Stasio, P R; Martiniello, M G; Pagnotti, P; Degener, A M; Midulla, M

1985-01-01

A fast-growing strain of human hepatitis A virus was selected and characterized. The virus has the unusual property of developing a strong cytopathic effect in tissue culture in 7 to 10 days. Sequences of the viral genome were cloned into recombinant plasmids with the double-stranded replicative form as a template for the reverse transcription of cDNA. Restriction analysis and direct sequencing indicate that this strain is different from that described by Ticehurst et al. (Proc. Natl. Acad. Sci. USA 80:5885-5889, 1983) in the region that presumptively codes for the major capsid protein VP1, but both isolates have conserved large areas of homology in the untranslated 5'-terminal sequences of the genome. Images PMID:2997478

Ease of articulation: A replication.

PubMed

Shuster, Linda I; Cottrill, Claire

2015-01-01

Researchers, as well as the lay public and the popular press, have become increasingly concerned about the lack of reproducibility of research findings. Despite this concern, research has shown that replications of previously published work comprise a very small proportion of published studies. Moreover, there are fewer published direct replications of research studies by independent investigators, and this type of replication is much less likely to confirm the results of the original research than are replications by the original investigator or conceptual replications. A search of the communication disorders research literature reveals that direct replications by independent investigators are virtually non-existent. The purpose of this project was to describe the major issues related to research reproducibility and report the results of a direct replication of a study by Locke (1972) regarding ease of articulation. Two methods for rating ease of articulation were employed. We were able to reproduce the results of the original study for the first method, obtaining a moderate positive correlation between our rankings of phoneme difficulty and Locke's rankings. We obtained a very high positive correlation between our phoneme rankings and rankings obtained in the original study for the second method. Moreover, we found a higher correlation between difficulty rankings and order of speech sound acquisition for American English than was found in the original study. Direct replication is not necessary for all studies in communication disorders, but should be considered for high impact studies, treatment studies, and those that provide data to support models and theories. The reader will be able to: (1) describe the major concerns related to the replicability of research findings; (2) describe the status of research replications in communication disorders; (3) describe how ease of articulation may relate to the order of speech sound acquisition in children; (4) list some types/areas of research that might be candidates for replication in the field of communication disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

PubMed

Kuriya, Kenji; Higashiyama, Eriko; Avşar-Ban, Eriko; Tamaru, Yutaka; Ogata, Shin; Takebayashi, Shin-ichiro; Ogata, Masato; Okumura, Katsuzumi

2015-12-01

Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

Affect-related related behaviors in mice selectively bred for high and low voluntary alcohol consumption

PubMed Central

Can, Adem; Grahame, Nicholas J.; Gould, Todd D.

2016-01-01

There is considerable evidence for the existence of comorbidity between alcohol-use disorders and depression in humans. One strategy to elucidate hereditary factors affecting the comorbidity of these disorders is to use genetic animal models, such as mouse lines selectively bred for voluntary ethanol consumption. We hypothesized that mice from lines that were bred for high-alcohol preference would manifest increased depression-like phenotypes compared to low-alcohol preferring mice. Mice that were bi-directionally selected and bred on the basis of their High-(HAP) or Low-Alcohol Preference (LAP) were tested in the open-field (OFT), dark-light box (DLB), forced swim (FST), and learned helplessness tests (LH). The study was conducted in two independently derived replicates. In the OFT, both HAP2 and HAP3 mice showed higher levels of general locomotion compared to LAP mice. However, only HAP2 mice spent more time in the center compared to LAP2 mice. In the DLB, there was a slightly higher anxiety-like phenotype in HAP mice. In both FST and LH, we observed higher depression-like behaviors in HAP mice compared to LAP mice, but this was limited to the Replicate 2 mice. Overall, we identified affect-related behavioral changes in mouse lines bred for high-alcohol preference. Notably, the Replicate 3 lines that showed fewer depression-like behaviors also manifest smaller differences in alcohol intake. These data suggest that there may be overlap between genes that predispose to excessive alcohol intake and those underlying affective-related behaviors in the mouse. PMID:21989731

Affect-related behaviors in mice selectively bred for high and low voluntary alcohol consumption.

PubMed

Can, Adem; Grahame, Nicholas J; Gould, Todd D

2012-03-01

There is considerable evidence for the existence of comorbidity between alcohol-use disorders and depression in humans. One strategy to elucidate hereditary factors affecting the comorbidity of these disorders is to use genetic animal models, such as mouse lines selectively bred for voluntary ethanol consumption. We hypothesized that mice from lines that were bred for high-alcohol preference would manifest increased depression-like phenotypes compared to low-alcohol preferring mice. Mice that were bi-directionally selected and bred on the basis of their High- (HAP) or Low-Alcohol Preference (LAP) were tested in the open-field (OFT), dark-light box (DLB), forced swim (FST), and learned helplessness tests (LH). The study was conducted in two independently derived replicates. In the OFT, both HAP2 and HAP3 mice showed higher levels of general locomotion compared to LAP mice. However, only HAP2 mice spent more time in the center compared to LAP2 mice. In the DLB, there was a slightly higher anxiety-like phenotype in HAP mice. In both FST and LH, we observed higher depression-like behaviors in HAP mice compared to LAP mice, but this was limited to the Replicate 2 mice. Overall, we identified affect-related behavioral changes in mouse lines bred for high-alcohol preference. Notably, the Replicate 3 lines that showed fewer depression-like behaviors also manifest smaller differences in alcohol intake. These data suggest that there may be overlap between genes that predispose to excessive alcohol intake and those underlying affect-related behaviors in the mouse.

Eukaryotic Replicative Helicase Subunit Interaction with DNA and Its Role in DNA Replication

PubMed Central

Martinez, Matthew P.; Wacker, Amanda L.; Bruck, Irina; Kaplan, Daniel L.

2017-01-01

The replicative helicase unwinds parental double-stranded DNA at a replication fork to provide single-stranded DNA templates for the replicative polymerases. In eukaryotes, the replicative helicase is composed of the Cdc45 protein, the heterohexameric ring-shaped Mcm2-7 complex, and the tetrameric GINS complex (CMG). The CMG proteins bind directly to DNA, as demonstrated by experiments with purified proteins. The mechanism and function of these DNA-protein interactions are presently being investigated, and a number of important discoveries relating to how the helicase proteins interact with DNA have been reported recently. While some of the protein-DNA interactions directly relate to the unwinding function of the enzyme complex, other protein-DNA interactions may be important for minichromosome maintenance (MCM) loading, origin melting or replication stress. This review describes our current understanding of how the eukaryotic replicative helicase subunits interact with DNA structures in vitro, and proposed models for the in vivo functions of replicative helicase-DNA interactions are also described. PMID:28383499

Simple systems that exhibit self-directed replication

NASA Technical Reports Server (NTRS)

Reggia, James A.; Armentrout, Steven L.; Chou, Hui-Hsien; Peng, Yun

1993-01-01

Biological experience and intuition suggest that self-replication is an inherently complex phenomenon, and early cellular automata models support that conception. More recently, simpler computational models of self-directed replication called sheathed loops have been developed. It is shown here that 'unsheathing' these structures and altering certain assumptions about the symmetry of their components leads to a family of nontrivial self-replicating structures some substantially smaller and simpler than those previously reported. The dependence of replication time and transition function complexity on initial structure size, cell state symmetry, and neighborhood are examined. These results support the view that self-replication is not an inherently complex phenomenon but rather an emergent property arising from local interactions in systems that can be much simpler than is generally believed.

Changes in transcriptional orientation are associated with increases in evolutionary rates of enterobacterial genes

PubMed Central

2011-01-01

Background Changes in transcriptional orientation (“CTOs”) occur frequently in prokaryotic genomes. Such changes usually result from genomic inversions, which may cause a conflict between the directions of replication and transcription and an increase in mutation rate. However, CTOs do not always lead to the replication-transcription confrontation. Furthermore, CTOs may cause deleterious disruptions of operon structure and/or gene regulations. The currently existing CTOs may indicate relaxation of selection pressure. Therefore, it is of interest to investigate whether CTOs have an independent effect on the evolutionary rates of the affected genes, and whether these genes are subject to any type of selection pressure in prokaryotes. Methods Three closely related enterbacteria, Escherichia coli, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium, were selected for comparisons of synonymous (dS) and nonsynonymous (dN) substitution rate between the genes that have experienced changes in transcriptional orientation (changed-orientation genes, “COGs”) and those that do not (same-orientation genes, “SOGs”). The dN/dS ratio was also derived to evaluate the selection pressure on the analyzed genes. Confounding factors in the estimation of evolutionary rates, such as gene essentiality, gene expression level, replication-transcription confrontation, and decreased dS at gene terminals were controlled in the COG-SOG comparisons. Results We demonstrate that COGs have significantly higher dN and dS than SOGs when a series of confounding factors are controlled. However, the dN/dS ratios are similar between the two gene groups, suggesting that the increase in dS can sufficiently explain the increase in dN in COGs. Therefore, the increases in evolutionary rates in COGs may be mainly mutation-driven. Conclusions Here we show that CTOs can increase the evolutionary rates of the affected genes. This effect is independent of the replication-transcription confrontation, which is suggested to be the major cause of inversion-associated evolutionary rate increases. The real cause of such evolutionary rate increases remains unclear but is worth further explorations. PMID:22152004

Selective activity of several cholic acid derivatives against human immunodeficiency virus replication in vitro.

PubMed

Baba, M; Schols, D; Nakashima, H; Pauwels, R; Parmentier, G; Meijer, D K; De Clercq, E

1989-01-01

Several cholic acid derivatives such as taurolithocholic acid, lithocholic acid 3-sulfate, taurolithocholic acid 3-sulfate, and glycolithocholic acid 3-sulfate were shown to inhibit selectively the replication of human immunodeficiency virus type 1 (HIV-1) in vitro. These compounds completely protected MT-4 cells against HIV-1-induced cytopathogenicity at a concentration of 100 micrograms/ml, whereas no toxicity for the host cells was observed at 200 micrograms/ml. They also inhibited HIV-1 antigen expression in HIV-1-infected CEM cells. The bile acids (cholic acid, deoxycholic acid, chenodeoxycholic acid, and lithocholic acid) did not show any inhibitory effect on HIV-1 replication at concentrations that were not toxic to the host (MT-4) cells. From a structure-function analysis of a number of cholic acid derivatives, the presence of either a sulfonate (as in the tauro conjugates) or a sulfate group as well as the "litho" configuration appeared to be necessary for the expression of anti-HIV-1 activity. The active cholic acid derivatives did not directly inactivate the virus particles at the concentrations that were not toxic to the host cells. Lithocholic acid 3-sulfate, taurolithocholic acid 3-sulfate, and glycolithocholic acid 3-sulfate, but not taurolithocholic acid, partially inhibited virus adsorption to MT-4 cells. These three compounds were also inhibitory to the reverse transcriptase activity associated with HIV-1.

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

NASA Technical Reports Server (NTRS)

Kanavarioti, Anastassia; Bernasconi, Claude F.

1997-01-01

This project is 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 were: (a) To elucidate the mechanistic aspects of template-directed (TD) chemistry, (b) to identify the conditions, environmental and other, that favor "organized chemistry" and stereo selective polymerization of nucleotides and (c) to search and, hopefully, find catalysts that will improve the efficiency of these reactions. Enhanced efficiency is expected to facilitate the road towards a self-replicating chemical system based on all four nucleic acid bases. During the first nine months of the granting period from January 1997 to October 1997, we have made substantial progress towards the first two objectives. During this period our activities were directed towards (1) synthesizing activated nucleotides to be used as substrates, (2) using these substrates in order to determine the effect of the leaving group (imidazole (Im), 2-methylimidazole (2-MeIm), and 2,4-dimethylimidazole (2,4-diMeIm)) in the product distribution, (3) developing techniques for analysis of mixtures by LC/MS, (4) creating a protocol in order to obtain kinetic parameters of the dimerization reaction and (5) analyzing kinetic data obtained with the poly(C)/2-MeImpG system. With the exception of item (5), the experimental work for the projects (1) - (4) is still in progress. A list of publications and manuscripts resulted from this research is enclosed.

Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance.

PubMed

Arezi, Bahram; McKinney, Nancy; Hansen, Connie; Cayouette, Michelle; Fox, Jeffrey; Chen, Keith; Lapira, Jennifer; Hamilton, Sarah; Hogrefe, Holly

2014-01-01

Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR) to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd ) for primed template and a moderate (2-fold) increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes.

Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition

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

Ciomperlik, Jessica J.; Basta, Holly A.; Palmenberg, Ann C., E-mail: acpalmen@wisc.edu

2016-01-15

Cardiovirus Leader proteins (L{sub X}) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus L{sub M} structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus L{sub E}, and also larger complexes with L{sub E}:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant L{sub S} and L{sub T} from Theiloviruses. When mutations were introduced tomore » alter the L{sub E} zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on L{sub E} must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by L{sub E}.« less

High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

PubMed

Bi, Yanwei; Sun, Le; Gao, Dandan; Ding, Chen; Li, Zhihua; Li, Yadong; Cun, Wei; Li, Qihan

2014-05-01

A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)) RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

High-Efficiency Targeted Editing of Large Viral Genomes by RNA-Guided Nucleases

PubMed Central

Gao, Dandan; Ding, Chen; Li, Zhihua; Li, Yadong; Cun, Wei; Li, Qihan

2014-01-01

A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)) RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses. PMID:24788700

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

Chemical Evolution and the Evolutionary Definition of Life.

PubMed

Higgs, Paul G

2017-06-01

Darwinian evolution requires a mechanism for generation of diversity in a population, and selective differences between individuals that influence reproduction. In biology, diversity is generated by mutations and selective differences arise because of the encoded functions of the sequences (e.g., ribozymes or proteins). Here, I draw attention to a process that I will call chemical evolution, in which the diversity is generated by random chemical synthesis instead of (or in addition to) mutation, and selection acts on physicochemical properties, such as hydrolysis, photolysis, solubility, or surface binding. Chemical evolution applies to short oligonucleotides that can be generated by random polymerization, as well as by template-directed replication, and which may be too short to encode a specific function. Chemical evolution is an important stage on the pathway to life, between the stage of "just chemistry" and the stage of full biological evolution. A mathematical model is presented here that illustrates the differences between these three stages. Chemical evolution leads to much larger differences in molecular concentrations than can be achieved by selection without replication. However, chemical evolution is not open-ended, unlike biological evolution. The ability to undergo Darwinian evolution is often considered to be a defining feature of life. Here, I argue that chemical evolution, although Darwinian, does not quite constitute life, and that a good place to put the conceptual boundary between non-life and life is between chemical and biological evolution.

Exploiting replication in distributed systems

NASA Technical Reports Server (NTRS)

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

1989-01-01

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

Selective positioning and integration of individual single-walled carbon nanotubes.

PubMed

Jiao, Liying; Xian, Xiaojun; Wu, Zhongyun; Zhang, Jin; Liu, Zhongfan

2009-01-01

We present a general selective positioning and integration technique for fabricating single-walled carbon nanotube (SWNT) circuits with preselected individual SWNTs as building blocks by utilizing poly(methyl methacrylate) (PMMA) thin film as a macroscopically handlable mediator. The transparency and marker-replicating capability of PMMA mediator allow the selective placement of chirality-specific nanotubes onto predesigned patterned surfaces with a resolution of ca. 1 mum. This technique is compatible with multiple operations and p-n conversion by chemical doping, which enables the construction of complex and logic circuits. As demonstrations of building SWNTs circuits, we fabricated a field effect inverter, a 2 x 2 all-SWNT crossbar field effect transistor (FET), and flexible FETs on plastic with this technique. This selective positioning approach can also be extended to construct purpose-directed architecture with various nanoscale building blocks.

Intersubtype Differences in the Effect of a Rare p24 Gag Mutation on HIV-1 Replicative Fitness

PubMed Central

Chopera, Denis R.; Cotton, Laura A.; Zawaira, Alexander; Mann, Jaclyn K.; Ngandu, Nobubelo K.; Ntale, Roman; Carlson, Jonathan M.; Mlisana, Koleka; Woodman, Zenda; de Assis Rosa, Debra; Martin, Eric; Miura, Toshiyuki; Pereyra, Florencia; Walker, Bruce D.; Gray, Clive M.; Martin, Darren P.; Ndung'u, Thumbi; Brockman, Mark A.; Karim, Salim Abdool

2012-01-01

Certain immune-driven mutations in HIV-1, such as those arising in p24Gag, decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24Gag M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (P = 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (P < 0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces in vitro viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24Gag codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited in vitro replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness. PMID:23015721

Registered Report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.

PubMed

Sharma, Vidhu; Young, Lisa; Cavadas, Miguel; Owen, Kate

2016-03-21

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "COT drives resistance to RAF inhibition through MAPK pathway reactivation" by Johannessen and colleagues, published in Nature in 2010 (Johannessen et al., 2010). The key experiments to be replicated are those reported in Figures 3B, 3D-E, 3I, and 4E-F. In Figures 3B, D-E, RPMI-7951 and OUMS023 cells were reported to exhibit robust ERK/MEK activity concomitant with reduced growth sensitivity in the presence of the BRAF inhibitor PLX4720. MAP3K8 (COT/TPL2) directly regulated MEK/ERK phosphorylation, as the treatment of RPMI-7951 cells with a MAP3K8 kinase inhibitor resulted in a dose-dependent suppression of MEK/ERK activity (Figure 3I). In contrast, MAP3K8-deficient A375 cells remained sensitive to BRAF inhibition, exhibiting reduced growth and MEK/ERK activity during inhibitor treatment. To determine if RAF and MEK inhibitors together can overcome single-agent resistance, MAP3K8-expressing A375 cells treated with PLX4720 along with MEK inhibitors significantly inhibited both cell viability and ERK activation compared to treatment with PLX4720 alone, as reported in Figures 4E-F. The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

Darwinian Evolution of Mutualistic RNA Replicators with Different Genes

NASA Astrophysics Data System (ADS)

Mizuuchi, R.; Ichihashi, N.

2017-07-01

We report a sustainable long-term replication and evolution of two distinct cooperative RNA replicators encoding different genes. One of the RNAs evolved to maintain or increase the cooperativity, despite selective advantage of selfish mutations.

Selective Attention Deficits in High Test Anxious Children: A Failure to Replicate.

ERIC Educational Resources Information Center

Ford, Carol E.; And Others

1985-01-01

Results of a study involving 5 high test-anxious poor readers failed to replicate an earlier study reporting selective attention deficits in the population. Further, there was a marginal tendency for older poor readers to be high test-anxious. (Author/CL)

Role of Replication and CpG Methylation in Fragile X Syndrome CGG Deletions in Primate Cells

PubMed Central

Nichol Edamura, Kerrie; Leonard, Michelle R.; Pearson, Christopher E.

2005-01-01

Instability of the fragile X CGG repeat involves both maternally derived expansions and deletions in the gametes of full-mutation males. It has also been suggested that the absence of aberrant CpG methylation may enhance repeat deletions through an unknown process. The effect of CGG tract length, DNA replication direction, location of replication initiation, and CpG methylation upon CGG stability were investigated using an SV40 primate replication system. Replication-dependant deletions with 53 CGG repeats were observed when replication was initiated proximal to the repeat, with CGG as the lagging-strand template. When we initiated replication further from the repeat, while maintaining CGG as the lagging-strand template or using CCG as the lagging-strand template, significant instability was not observed. CpG methylation of the unstable template stabilized the repeat, decreasing both the frequency and the magnitude of deletion events. Furthermore, CpG methylation slowed the efficiency of replication for all templates. Interestingly, replication forks displayed no evidence of a block at the CGG repeat tract, regardless of replication direction or CpG methylation status. Templates with 20 CGG repeats were stable under all circumstances. These results reveal that CGG deletions occur during replication and are sensitive to replication-fork dynamics, tract length, and CpG methylation. PMID:15625623

DNA replication machinery is required for development in Drosophila.

PubMed

Kohzaki, Hidetsugu; Asano, Maki; Murakami, Yota

2018-01-01

 In Drosophila , some factors involved in chromosome replication seem to be involved in gene amplification and endoreplication, which are actively utilized in particular tissue development, but direct evidence has not been shown. Therefore, we examined the effect of depletion of replication factors on these processes. First, we confirmed RNAi knockdown can be used for the depletion of replication factors by comparing the phenotypes of RNAi knockdown and deletion or point mutants of the components of DNA licensing factor, MCM2, MCM4 and Cdt1. Next, we found that tissue-specific RNAi knockdown of replication factors caused tissue-specific defects, probably due to defects in DNA replication. In particular, we found that depletion inhibited gene amplification of the chorion gene in follicle cells and endoreplication in salivary glands, showing that chromosomal DNA replication factors are required for these processes. Finally, using RNAi, we screened the genes for chromosomal DNA replication that affected tissue development. Interestingly, wing specific knockdown of Mcm10 induced wing formation defects. These results suggest that some components of chromosomal replication machinery are directly involved in tissue development.

Mutagenic Effects of Ribavirin on Hepatitis E Virus-Viral Extinction versus Selection of Fitness-Enhancing Mutations.

PubMed

Todt, Daniel; Walter, Stephanie; Brown, Richard J P; Steinmann, Eike

2016-10-13

Hepatitis E virus (HEV), an important agent of viral hepatitis worldwide, can cause severe courses of infection in pregnant women and immunosuppressed patients. To date, HEV infections can only be treated with ribavirin (RBV). Major drawbacks of this therapy are that RBV is not approved for administration to pregnant women and that the virus can acquire mutations, which render the intra-host population less sensitive or even resistant to RBV. One of the proposed modes of action of RBV is a direct mutagenic effect on viral genomes, inducing mismatches and subsequent nucleotide substitutions. These transition events can drive the already error-prone viral replication beyond an error threshold, causing viral population extinction. In contrast, the expanded heterogeneous viral population can facilitate selection of mutant viruses with enhanced replication fitness. Emergence of these mutant viruses can lead to therapeutic failure. Consequently, the onset of RBV treatment in chronically HEV-infected individuals can result in two divergent outcomes: viral extinction versus selection of fitness-enhanced viruses. Following an overview of RNA viruses treated with RBV in clinics and a summary of the different antiviral modes of action of this drug, we focus on the mutagenic effect of RBV on HEV intrahost populations, and how HEV is able to overcome lethal mutagenesis.

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.

Comparison of Marker-Based Genomic Estimated Breeding Values and Phenotypic Evaluation for Selection of Bacterial Spot Resistance in Tomato.

PubMed

Liabeuf, Debora; Sim, Sung-Chur; Francis, David M

2018-03-01

Bacterial spot affects tomato crops (Solanum lycopersicum) grown under humid conditions. Major genes and quantitative trait loci (QTL) for resistance have been described, and multiple loci from diverse sources need to be combined to improve disease control. We investigated genomic selection (GS) prediction models for resistance to Xanthomonas euvesicatoria and experimentally evaluated the accuracy of these models. The training population consisted of 109 families combining resistance from four sources and directionally selected from a population of 1,100 individuals. The families were evaluated on a plot basis in replicated inoculated trials and genotyped with single nucleotide polymorphisms (SNP). We compared the prediction ability of models developed with 14 to 387 SNP. Genomic estimated breeding values (GEBV) were derived using Bayesian least absolute shrinkage and selection operator regression (BL) and ridge regression (RR). Evaluations were based on leave-one-out cross validation and on empirical observations in replicated field trials using the next generation of inbred progeny and a hybrid population resulting from selections in the training population. Prediction ability was evaluated based on correlations between GEBV and phenotypes (r g ), percentage of coselection between genomic and phenotypic selection, and relative efficiency of selection (r g /r p ). Results were similar with BL and RR models. Models using only markers previously identified as significantly associated with resistance but weighted based on GEBV and mixed models with markers associated with resistance treated as fixed effects and markers distributed in the genome treated as random effects offered greater accuracy and a high percentage of coselection. The accuracy of these models to predict the performance of progeny and hybrids exceeded the accuracy of phenotypic selection.

Effect of Load History on Fatigue Life.

DTIC Science & Technology

1980-06-01

emission 166 6.4.4 Edge replication 176 6.4.5 Stiffness monitoring 177 6.4.6 Temperature monitoring 179 6.5 Selection of NDI Techniques for Tasks II and III...composites of T300/5208 and T300/934 in room temperature, laboratory at R = 0.0 139 66 Schematic of acoustic emission event 151 67 Schematic diagram of...acoustic emission system 152 68 Cross section in the 00 direction of a coupon loaded statically to 60% of the Average Ultimate Tensile Strength 158

Binding of Glutathione to Enterovirus Capsids Is Essential for Virion Morphogenesis

PubMed Central

Thibaut, Hendrik Jan; Thys, Bert; Canela, María-Dolores; Aguado, Leire; Wimmer, Eckard; Paul, Aniko; Pérez-Pérez, María-Jesús; van Kuppeveld, Frank J. M.; Neyts, Johan

2014-01-01

Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis. PMID:24722756

Low Dose Vaccination with Attenuated Francisella tularensis Strain SchuS4 Mutants Protects against Tularemia Independent of the Route of Vaccination

PubMed Central

Rockx-Brouwer, Dedeke; Chong, Audrey; Wehrly, Tara D.; Child, Robert; Crane, Deborah D.

2012-01-01

Tularemia, caused by the Gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia. PMID:22662210

DNA Replication Arrest and DNA Damage Responses Induced by Alkylating Minor Groove Binders

DTIC Science & Technology

2001-05-01

We are interested in the molecular mechanisms involved in DNA replication arrest by the S phase DNA damage checkpoints. Using in vitro simian virus...40 DNA replication assays, we have found three factors that directly contribute to DNA damage-induced DNA replication arrest: Replication Protein A...trans-acting inhibitors. RPA is the major eukaryotic single-stranded DNA binding protein required for DNA replication , repair and recombination. Upon DNA

Evolving a polymerase for hydrophobic base analogues.

PubMed

Loakes, David; Gallego, José; Pinheiro, Vitor B; Kool, Eric T; Holliger, Philipp

2009-10-21

Hydrophobic base analogues (HBAs) have shown great promise for the expansion of the chemical and coding potential of nucleic acids but are generally poor polymerase substrates. While extensive synthetic efforts have yielded examples of HBAs with favorable substrate properties, their discovery has remained challenging. Here we describe a complementary strategy for improving HBA substrate properties by directed evolution of a dedicated polymerase using compartmentalized self-replication (CSR) with the archetypal HBA 5-nitroindole (d5NI) and its derivative 5-nitroindole-3-carboxamide (d5NIC) as selection substrates. Starting from a repertoire of chimeric polymerases generated by molecular breeding of DNA polymerase genes from the genus Thermus, we isolated a polymerase (5D4) with a generically enhanced ability to utilize HBAs. The selected polymerase. 5D4 was able to form and extend d5NI and d5NIC (d5NI(C)) self-pairs as well as d5NI(C) heteropairs with all four bases with efficiencies approaching, or exceeding, those of the cognate Watson-Crick pairs, despite significant distortions caused by the intercalation of the d5NI(C) heterocycles into the opposing strand base stack, as shown by nuclear magnetic resonance spectroscopy (NMR). Unlike Taq polymerase, 5D4 was also able to extend HBA pairs such as Pyrene: varphi (abasic site), d5NI: varphi, and isocarbostyril (ICS): 7-azaindole (7AI), allowed bypass of a chemically diverse spectrum of HBAs, and enabled PCR amplification with primers comprising multiple d5NI(C)-substitutions, while maintaining high levels of catalytic activity and fidelity. The selected polymerase 5D4 promises to expand the range of nucleobase analogues amenable to replication and should find numerous applications, including the synthesis and replication of nucleic acid polymers with expanded chemical and functional diversity.

Herpes Simplex Virus Type 2 Suppressive Therapy with Acyclovir or Valacyclovir Does Not Select for Specific HIV-1 Resistance in HIV-1/HSV-2 Dually Infected Persons

PubMed Central

Lingappa, Jairam; Beck, Ingrid; Frenkel, Lisa M.; Pepper, Gregory; Celum, Connie; Wald, Anna; Fife, Kenneth H.; Were, Edwin; Mugo, Nelly; Sanchez, Jorge; Essex, Myron; Makhema, Joseph; Kiarie, James; Farquhar, Carey; Corey, Lawrence

2011-01-01

Recent in vitro studies suggest that acyclovir may directly inhibit HIV-1 replication and can select for a specific HIV-1 reverse transcriptase mutation (V75I) with concomitant loss of an anti-HIV-1 effect. We tested for HIV-1 genotypic resistance at reverse transcriptase codon 75 in plasma from 168 HIV-1–infected persons from Botswana, Kenya, Peru, and the United States taking daily acyclovir or valacyclovir for between 8 weeks and 24 months. No V75I cases were detected (95% confidence interval, 0%–2.2%). These prospective in vivo studies suggest that standard-dose acyclovir or valacyclovir does not select for HIV-1 resistance. PMID:21148504

Selection Dynamics in Transient Compartmentalization

NASA Astrophysics Data System (ADS)

Blokhuis, Alex; Lacoste, David; Nghe, Philippe; Peliti, Luca

2018-04-01

Transient compartments have been recently shown to be able to maintain functional replicators in the context of prebiotic studies. Here, we show that a broad class of selection dynamics is able to achieve this goal. We identify two key parameters, the relative amplification of nonactive replicators (parasites) and the size of compartments. These parameters account for competition and diversity, and the results are relevant to similar multilevel selection problems, such as those found in virus-host ecology and trait group selection.

How Darwinian is cultural evolution?

PubMed Central

Claidière, Nicolas; Scott-Phillips, Thomas C.; Sperber, Dan

2014-01-01

Darwin-inspired population thinking suggests approaching culture as a population of items of different types, whose relative frequencies may change over time. Three nested subtypes of populational models can be distinguished: evolutionary, selectional and replicative. Substantial progress has been made in the study of cultural evolution by modelling it within the selectional frame. This progress has involved idealizing away from phenomena that may be critical to an adequate understanding of culture and cultural evolution, particularly the constructive aspect of the mechanisms of cultural transmission. Taking these aspects into account, we describe cultural evolution in terms of cultural attraction, which is populational and evolutionary, but only selectional under certain circumstances. As such, in order to model cultural evolution, we must not simply adjust existing replicative or selectional models but we should rather generalize them, so that, just as replicator-based selection is one form that Darwinian selection can take, selection itself is one of several different forms that attraction can take. We present an elementary formalization of the idea of cultural attraction. PMID:24686939

How Darwinian is cultural evolution?

PubMed

Claidière, Nicolas; Scott-Phillips, Thomas C; Sperber, Dan

2014-05-19

Darwin-inspired population thinking suggests approaching culture as a population of items of different types, whose relative frequencies may change over time. Three nested subtypes of populational models can be distinguished: evolutionary, selectional and replicative. Substantial progress has been made in the study of cultural evolution by modelling it within the selectional frame. This progress has involved idealizing away from phenomena that may be critical to an adequate understanding of culture and cultural evolution, particularly the constructive aspect of the mechanisms of cultural transmission. Taking these aspects into account, we describe cultural evolution in terms of cultural attraction, which is populational and evolutionary, but only selectional under certain circumstances. As such, in order to model cultural evolution, we must not simply adjust existing replicative or selectional models but we should rather generalize them, so that, just as replicator-based selection is one form that Darwinian selection can take, selection itself is one of several different forms that attraction can take. We present an elementary formalization of the idea of cultural attraction.

RNA genetics

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

Domingo, E.; Holland, J.J.; Ahlquist, P.

1988-01-01

This book contains the proceedings on RNA genetics: RNA-directed virus replication Volume 1. Topics covered include: Replication of the poliovirus genome; Influenza viral RNA transcription and replication; and Relication of the reoviridal: Information derived from gene cloning and expression.

Identification of HIV-1 determinants for replication in vivo.

PubMed

Su, L; Kaneshima, H; Bonyhadi, M L; Lee, R; Auten, J; Wolf, A; Du, B; Rabin, L; Hahn, B H; Terwilliger, E; Mccune, J M

1997-01-06

Pathogenic organisms are frequently attenuated after long-term culture in vitro. The mechanisms of the attenuation process are not clear, but probably involve mutations of functions required for replication and pathogenicity in vivo. To identify these functions, a direct comparison must be made between attenuated genomes and those that remain pathogenic in vivo. In this study, we used the heterochimeric SCID-hu Thy/Liv mouse as an in vivo model to define human immunodeficiency virus type 1 (HIV-1) determinants which are uniquely required for replication in vivo. The Lai/IIIB isolate and its associated infectious molecular clones (e.g., HXB2) were found to infect T cell lines but failed to replicate in the SCID-hu Thy/Liv model. When a lab worker was accidentally infected by Lai/IIIB, however, HIV-1 was isolated only from infection of primary PBMC, and not from infection of T cell lines. We hypothesized that the lab worker was exposed to a heterogeneous viral stock which had been attenuated by passage in immortalized T cell lines. Either a rare family member from this stock was selected for in vivo replication or, alternatively, an attenuated genotype dominant in vitro may have reverted to become more infectious in vivo. To address this hypothesis, we have used the SCID-hu Thy/Liv model to study the replication of HXB2 and of HXB2 recombinant viruses with HIV-1 fragments isolated from the infected lab worker. HXB2 showed no or very low levels of replication in the Thy/Liv organ. Replacement of its subgenomic fragment encoding the envelope gene with a corresponding fragment from the lab worker isolate generated a recombinant virus (HXB2/LW) which replicated actively in SCID-hu mice. The NEF mutation in the HXB2 genome is still present in HXB2/LW. Thus, the LW sequences encode HIV-1 determinants which enhance HIV replication in vivo in a NEF-independent mechanism. The specific determinants have been mapped to the V1-V3 regions of the HIV-1 genome. Six unique mutations in the V3 loop region of HXB2/LW have been identified which contribute to the increased replication in vivo.

Fidelity of DNA Replication in Normal and Malignant Human Brest Cells.

DTIC Science & Technology

1995-08-31

cellular DNA replication machinery, we have initiated experiments that utilize a multiprotein DNA replication complex (MRC) isolated from breast cancer...gene in an in vitro DNA replication assay. By utilizing the target gene in a bacterial mutant selection assay we have begun to determine the...frequency with which mutational sequence errors occur as a result of the in vitro DNA replication mediated by the breast cancer cell MRC and the normal breast

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

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.

The scenario on the origin of translation in the RNA world: in principle of replication parsimony

PubMed Central

2010-01-01

Background It is now believed that in the origin of life, proteins should have been "invented" in an RNA world. However, due to the complexity of a possible RNA-based proto-translation system, this evolving process seems quite complicated and the associated scenario remains very blurry. Considering that RNA can bind amino acids with specificity, it has been reasonably supposed that initial peptides might have been synthesized on "RNA templates" containing multiple amino acid binding sites. This "Direct RNA Template (DRT)" mechanism is attractive because it should be the simplest mechanism for RNA to synthesize peptides, thus very likely to have been adopted initially in the RNA world. Then, how this mechanism could develop into a proto-translation system mechanism is an interesting problem. Presentation of the hypothesis Here an explanation to this problem is shown considering the principle of "replication parsimony" --- genetic information tends to be utilized in a parsimonious way under selection pressure, due to its replication cost (e.g., in the RNA world, nucleotides and ribozymes for RNA replication). Because a DRT would be quite long even for a short peptide, its replication cost would be great. Thus the diversity and the length of functional peptides synthesized by the DRT mechanism would be seriously limited. Adaptors (proto-tRNAs) would arise to allow a DRT's complementary strand (called "C-DRT" here) to direct the synthesis of the same peptide synthesized by the DRT itself. Because the C-DRT is a necessary part in the DRT's replication, fewer turns of the DRT's replication would be needed to synthesize definite copies of the functional peptide, thus saving the replication cost. Acting through adaptors, C-DRTs could transform into much shorter templates (called "proto-mRNAs" here) and substitute the role of DRTs, thus significantly saving the replication cost. A proto-rRNA corresponding to the small subunit rRNA would then emerge to aid the binding of proto-tRNAs and proto-mRNAs, allowing the reduction of base pairs between them (ultimately resulting in the triplet anticodon/codon pair), thus further saving the replication cost. In this context, the replication cost saved would allow the appearance of more and longer functional peptides and, finally, proteins. The hypothesis could be called "DRT-RP" ("RP" for "replication parsimony"). Testing the hypothesis The scenario described here is open for experimental work at some key scenes, including the compact DRT mechanism, the development of adaptors from aa-aptamers, the synthesis of peptides by proto-tRNAs and proto-mRNAs without the participation of proto-rRNAs, etc. Interestingly, a recent computer simulation study has demonstrated the plausibility of one of the evolving processes driven by replication parsimony in the scenario. Implication of the hypothesis An RNA-based proto-translation system could arise gradually from the DRT mechanism according to the principle of "replication parsimony" --- to save the replication cost of RNA templates for functional peptides. A surprising side deduction along the logic of the hypothesis is that complex, biosynthetic amino acids might have entered the genetic code earlier than simple, prebiotic amino acids, which is opposite to the common sense. Overall, the present discussion clarifies the blurry scenario concerning the origin of translation with a major clue, which shows vividly how life could "manage" to exploit potential chemical resources in nature, eventually in an efficient way over evolution. Reviewers This article was reviewed by Eugene V. Koonin, Juergen Brosius, and Arcady Mushegian. PMID:21110883

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

PubMed Central

Douglas, Max E.

2016-01-01

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

Aggregate and individual replication probability within an explicit model of the research process.

PubMed

Miller, Jeff; Schwarz, Wolf

2011-09-01

We study a model of the research process in which the true effect size, the replication jitter due to changes in experimental procedure, and the statistical error of effect size measurement are all normally distributed random variables. Within this model, we analyze the probability of successfully replicating an initial experimental result by obtaining either a statistically significant result in the same direction or any effect in that direction. We analyze both the probability of successfully replicating a particular experimental effect (i.e., the individual replication probability) and the average probability of successful replication across different studies within some research context (i.e., the aggregate replication probability), and we identify the conditions under which the latter can be approximated using the formulas of Killeen (2005a, 2007). We show how both of these probabilities depend on parameters of the research context that would rarely be known in practice. In addition, we show that the statistical uncertainty associated with the size of an initial observed effect would often prevent accurate estimation of the desired individual replication probability even if these research context parameters were known exactly. We conclude that accurate estimates of replication probability are generally unattainable.

The chemical end-ligation of homopyrimidine oligodeoxyribonucleotides within a DNA triple helix.

PubMed

Li, T; Weinstein, D S; Nicolaou, K

1997-03-01

Triple-helical nucleic acids, first reported in the late 1950s, are receiving attention for their possible involvement in controlling gene expression. Certain sequences of DNA are believed to form local triple-helical structures (H-form DNA), although this has not been directly observed in vivo. Studies carried out in our laboratories have suggested that self-replicating oligonucleotides could have been involved in chemical evolution via triple-helical intermediates. In addition to self-replication mechanisms, elucidating processes for the nonenzymatic elongation of biologically relevant polymers remains an important challenge in understanding the origin of life. To this end, we have studied a novel ligation of oligodeoxyribonucleotides that lie within a triple helix. The chemical end-ligation of homopyrimidine oligodeoxyribonucleotides on a triple helix is reported. This selective process, induced by cyanoimidazole, is facilitated by a template effect of the DNA aggregate and occurs between the 3' end (hydroxyl) of the third minor-groove-bound strand and the 5' end (phosphate) of the antiparallel oligopyrimidine strand. Double-helical homopurine/homopyrimidine DNA can serve as a template for the elongation of oligonucleotides in a manner that has not been described previously. The end-ligation of homopyrimidine oligomers, a nonenzymatic process, proceeds via a requisite triple-helical intermediate and constitutes an efficient and selective method for the template-directed elongation of nucleic acids. Such a process could conceivably have been involved in the elongation of primordial information-bearing biopolymers.

Selective skepticism: American and Chinese children's reasoning about evaluative academic feedback.

PubMed

Heyman, Gail D; Fu, Genyue; Lee, Kang

2013-03-01

Children's reasoning about the credibility of positive and negative evaluations of academic performance was examined. Across 2 studies, 7- and 10-year-olds from the United States and China (N = 334) judged the credibility of academic evaluations that were directed toward an unfamiliar peer. In Study 1, participants from China responded that criticism should be accepted to a greater extent than did participants from the United States, and children from both countries demonstrated a selective skepticism effect by treating negative feedback more skeptically than positive feedback. Study 2 replicated the selective skepticism effect among children from both countries and ruled out the possibility that it can be explained as a rational analysis of perceived base rates. The results suggest that children are selective in their trust of evaluative feedback and that their credibility judgments may be influenced by the desirability of the information that is being conveyed or its anticipated consequences.

Correlations and path analysis among agronomic and technological traits of upland cotton.

PubMed

Farias, F J C; Carvalho, L P; Silva Filho, J L; Teodoro, P E

2016-08-12

To date, path analysis has been used with the aim of breeding different cultures. However, for cotton, there have been few studies using this analysis, and all of these have used fiber productivity as the primary dependent variable. Therefore, the aim of the present study was to identify agronomic and technological properties that can be used as criteria for direct and indirect phenotypes in selecting cotton genotypes with better fibers. We evaluated 16 upland cotton genotypes in eight trials conducted during the harvest 2008/2009 in the State of Mato Grosso, using a randomized block design with four replicates. The evaluated traits were: plant height, average boll weight, percentage of fiber, cotton seed yield, fiber length, uniformity of fiber, short fiber index, fiber strength, elongation, maturity of the fibers, micronaire, reflectance, and the degree of yellowing. Phenotypic correlations between the traits and cotton fiber yield (main dependent variable) were unfolded in direct and indirect effects through path analysis. Fiber strength, uniformity of fiber, and reflectance were found to influence fiber length, and therefore, these traits are recommended for both direct and indirect selection of cotton genotypes.

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

Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides

NASA Astrophysics Data System (ADS)

Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun, Daisy; Zamecnik, Paul C.

1988-08-01

Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.

Commercial Building Partnerships Replication and Diffusion

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

Antonopoulos, Chrissi A.; Dillon, Heather E.; Baechler, Michael C.

2013-09-16

This study presents findings from survey and interview data investigating replication efforts of Commercial Building Partnership (CBP) partners that worked directly with the Pacific Northwest National Laboratory (PNNL). PNNL partnered directly with 12 organizations on new and retrofit construction projects, which represented approximately 28 percent of the entire U.S. Department of Energy (DOE) CBP program. Through a feedback survey mechanism, along with personal interviews, PNNL gathered quantitative and qualitative data relating to replication efforts by each organization. These data were analyzed to provide insight into two primary research areas: 1) CBP partners’ replication efforts of technologies and approaches used inmore » the CBP project to the rest of the organization’s building portfolio (including replication verification), and, 2) the market potential for technology diffusion into the total U.S. commercial building stock, as a direct result of the CBP program. The first area of this research focused specifically on replication efforts underway or planned by each CBP program participant. Factors that impact replication include motivation, organizational structure and objectives firms have for implementation of energy efficient technologies. Comparing these factors between different CBP partners revealed patterns in motivation for constructing energy efficient buildings, along with better insight into market trends for green building practices. The second area of this research develops a diffusion of innovations model to analyze potential broad market impacts of the CBP program on the commercial building industry in the United States.« less

Straightforward and precise approach to replicate complex hierarchical structures from plant surfaces onto soft matter polymer

PubMed Central

Speck, Thomas; Bohn, Holger F.

2018-01-01

The surfaces of plant leaves are rarely smooth and often possess a species-specific micro- and/or nano-structuring. These structures usually influence the surface functionality of the leaves such as wettability, optical properties, friction and adhesion in insect–plant interactions. This work presents a simple, convenient, inexpensive and precise two-step micro-replication technique to transfer surface microstructures of plant leaves onto highly transparent soft polymer material. Leaves of three different plants with variable size (0.5–100 µm), shape and complexity (hierarchical levels) of their surface microstructures were selected as model bio-templates. A thermoset epoxy resin was used at ambient conditions to produce negative moulds directly from fresh plant leaves. An alkaline chemical treatment was established to remove the entirety of the leaf material from the cured negative epoxy mould when necessary, i.e. for highly complex hierarchical structures. Obtained moulds were filled up afterwards with low viscosity silicone elastomer (PDMS) to obtain positive surface replicas. Comparative scanning electron microscopy investigations (original plant leaves and replicated polymeric surfaces) reveal the high precision and versatility of this replication technique. This technique has promising future application for the development of bioinspired functional surfaces. Additionally, the fabricated polymer replicas provide a model to systematically investigate the structural key points of surface functionalities. PMID:29765666

Replication timing and nuclear structure.

PubMed

Fu, Haiqing; Baris, Adrian; Aladjem, Mirit I

2018-06-01

DNA replication proceeds along spatially and temporally coordinated patterns within the nucleus, thus protecting the genome during the synthesis of new genetic material. While we have been able to visualize replication patterns on DNA fibers for 50 years, recent developments and discoveries have provided a greater insight into how DNA replication is controlled. In this review, we highlight many of these discoveries. Of great interest are the physiological role of the replication timing program, cis and trans-acting factors that modulate replication timing and the effects of chromatin structure on the replication timing program. We also discuss future directions in the study of replication timing. Published by Elsevier Ltd.

Selecting Fully-Modified XNA Aptamers Using Synthetic Genetics.

PubMed

Taylor, Alexander I; Holliger, Philipp

2018-06-01

This unit describes the application of "synthetic genetics," i.e., the replication of xeno nucleic acids (XNAs), artificial analogs of DNA and RNA bearing alternative backbone or sugar congeners, to the directed evolution of synthetic oligonucleotide ligands (XNA aptamers) specific for target proteins or nucleic acid motifs, using a cross-chemistry selective exponential enrichment (X-SELEX) approach. Protocols are described for synthesis of diverse-sequence XNA repertoires (typically 10 14 molecules) using DNA templates, isolation and panning for functional XNA sequences using targets immobilized on solid phase or gel shift induced by target binding in solution, and XNA reverse transcription to allow cDNA amplification or sequencing. The method may be generally applied to select fully-modified XNA aptamers specific for a wide range of target molecules. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

T7 replisome directly overcomes DNA damage

NASA Astrophysics Data System (ADS)

Sun, Bo; Pandey, Manjula; Inman, James T.; Yang, Yi; Kashlev, Mikhail; Patel, Smita S.; Wang, Michelle D.

2015-12-01

Cells and viruses possess several known `restart' pathways to overcome lesions during DNA replication. However, these `bypass' pathways leave a gap in replicated DNA or require recruitment of accessory proteins, resulting in significant delays to fork movement or even cell division arrest. Using single-molecule and ensemble methods, we demonstrate that the bacteriophage T7 replisome is able to directly replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion. We show that when a replisome encounters the lesion, a substantial fraction of DNA polymerase (DNAP) and helicase stay together at the lesion, the replisome does not dissociate and the helicase does not move forward on its own. The DNAP is able to directly replicate through the lesion by working in conjunction with helicase through specific helicase-DNAP interactions. These observations suggest that the T7 replisome is fundamentally permissive of DNA lesions via pathways that do not require fork adjustment or replisome reassembly.

A prebiotic template-directed peptide synthesis based on amyloids.

PubMed

Rout, Saroj K; Friedmann, Michael P; Riek, Roland; Greenwald, Jason

2018-01-16

The prebiotic replication of information-coding molecules is a central problem concerning life's origins. Here, we report that amyloids composed of short peptides can direct the sequence-selective, regioselective and stereoselective condensation of amino acids. The addition of activated DL-arginine and DL-phenylalanine to the peptide RFRFR-NH 2 in the presence of the complementary template peptide Ac-FEFEFEFE-NH 2 yields the isotactic product FRFRFRFR-NH 2 , 1 of 64 possible triple addition products, under conditions in which the absence of template yields only single and double additions of mixed stereochemistry. The templating mechanism appears to be general in that a different amyloid formed by (Orn)V(Orn)V(Orn)V(Orn)V-NH 2 and Ac-VDVDVDVDV-NH 2 is regioselective and stereoselective for N-terminal, L-amino-acid addition while the ornithine-valine peptide alone yields predominantly sidechain condensation products with little stereoselectivity. Furthermore, the templating reaction is stable over a wide range of pH (5.6-8.6), salt concentration (0-4 M NaCl), and temperature (25-90 °C), making the amyloid an attractive model for a prebiotic peptide replicating system.

Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins

PubMed Central

Samson, Rachel Y.; Abeyrathne, Priyanka D.; Bell, Stephen D.

2015-01-01

Summary Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaeal Orc1-1 fulfills both Orc1 and Cdc6 functions by binding to a replication origin and directly recruiting MCM helicase. We identify the interaction interface between these proteins and reveal how ATP binding by Orc1-1 modulates recruitment of MCM. Additionally, we provide evidence that an open-ring form of the archaeal MCM homohexamer is loaded at origins. PMID:26725007

Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication.

PubMed

Aye, Seaim Lwin; Fujiwara, Kei; Ueki, Asuka; Doi, Nobuhide

2018-05-05

Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5' to 3' exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol. Copyright © 2018 Elsevier Inc. All rights reserved.

Whole-Genome Resequencing of Experimental Populations Reveals Polygenic Basis of Egg-Size Variation in Drosophila melanogaster

PubMed Central

Jha, Aashish R.; Miles, Cecelia M.; Lippert, Nodia R.; Brown, Christopher D.; White, Kevin P.; Kreitman, Martin

2015-01-01

Complete genome resequencing of populations holds great promise in deconstructing complex polygenic traits to elucidate molecular and developmental mechanisms of adaptation. Egg size is a classic adaptive trait in insects, birds, and other taxa, but its highly polygenic architecture has prevented high-resolution genetic analysis. We used replicated experimental evolution in Drosophila melanogaster and whole-genome sequencing to identify consistent signatures of polygenic egg-size adaptation. A generalized linear-mixed model revealed reproducible allele frequency differences between replicated experimental populations selected for large and small egg volumes at approximately 4,000 single nucleotide polymorphisms (SNPs). Several hundred distinct genomic regions contain clusters of these SNPs and have lower heterozygosity than the genomic background, consistent with selection acting on polymorphisms in these regions. These SNPs are also enriched among genes expressed in Drosophila ovaries and many of these genes have well-defined functions in Drosophila oogenesis. Additional genes regulating egg development, growth, and cell size show evidence of directional selection as genes regulating these biological processes are enriched for highly differentiated SNPs. Genetic crosses performed with a subset of candidate genes demonstrated that these genes influence egg size, at least in the large genetic background. These findings confirm the highly polygenic architecture of this adaptive trait, and suggest the involvement of many novel candidate genes in regulating egg size. PMID:26044351

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

PubMed

Douglas, Max E; Diffley, John F X

2016-03-11

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

Common Chemical Inductors of Replication Stress:  Focus on Cell-Based Studies.

PubMed

Vesela, Eva; Chroma, Katarina; Turi, Zsofia; Mistrik, Martin

2017-02-21

DNA replication is a highly demanding process regarding the energy and material supply and must be precisely regulated, involving multiple cellular feedbacks. The slowing down or stalling of DNA synthesis and/or replication forks is referred to as replication stress (RS). Owing to the complexity and requirements of replication, a plethora of factors may interfere and challenge the genome stability, cell survival or affect the whole organism. This review outlines chemical compounds that are known inducers of RS and commonly used in laboratory research. These compounds act on replication by direct interaction with DNA causing DNA crosslinks and bulky lesions (cisplatin), chemical interference with the metabolism of deoxyribonucleotide triphosphates (hydroxyurea), direct inhibition of the activity of replicative DNA polymerases (aphidicolin) and interference with enzymes dealing with topological DNA stress (camptothecin, etoposide). As a variety of mechanisms can induce RS, the responses of mammalian cells also vary. Here, we review the activity and mechanism of action of these compounds based on recent knowledge, accompanied by examples of induced phenotypes, cellular readouts and commonly used doses.

Common Chemical Inductors of Replication Stress: Focus on Cell-Based Studies

PubMed Central

Vesela, Eva; Chroma, Katarina; Turi, Zsofia; Mistrik, Martin

2017-01-01

DNA replication is a highly demanding process regarding the energy and material supply and must be precisely regulated, involving multiple cellular feedbacks. The slowing down or stalling of DNA synthesis and/or replication forks is referred to as replication stress (RS). Owing to the complexity and requirements of replication, a plethora of factors may interfere and challenge the genome stability, cell survival or affect the whole organism. This review outlines chemical compounds that are known inducers of RS and commonly used in laboratory research. These compounds act on replication by direct interaction with DNA causing DNA crosslinks and bulky lesions (cisplatin), chemical interference with the metabolism of deoxyribonucleotide triphosphates (hydroxyurea), direct inhibition of the activity of replicative DNA polymerases (aphidicolin) and interference with enzymes dealing with topological DNA stress (camptothecin, etoposide). As a variety of mechanisms can induce RS, the responses of mammalian cells also vary. Here, we review the activity and mechanism of action of these compounds based on recent knowledge, accompanied by examples of induced phenotypes, cellular readouts and commonly used doses. PMID:28230817

Metabolically Coupled Replicator Systems: Overview of an RNA-world model concept of prebiotic evolution on mineral surfaces.

PubMed

Czárán, Tamás; Könnyű, Balázs; Szathmáry, Eörs

2015-09-21

Metabolically Coupled Replicator Systems (MCRS) are a family of models implementing a simple, physico-chemically and ecologically feasible scenario for the first steps of chemical evolution towards life. Evolution in an abiotically produced RNA-population sets in as soon as any one of the RNA molecules become autocatalytic by engaging in template directed self-replication from activated monomers, and starts increasing exponentially. Competition for the finite external supply of monomers ignites selection favouring RNA molecules with catalytic activity helping self-replication by any possible means. One way of providing such autocatalytic help is to become a replicase ribozyme. An additional way is through increasing monomer supply by contributing to monomer synthesis from external resources, i.e., by evolving metabolic enzyme activity. Retroevolution may build up an increasingly autotrophic, cooperating community of metabolic ribozymes running an increasingly complicated and ever more efficient metabolism. Maintaining such a cooperating community of metabolic replicators raises two serious ecological problems: one is keeping the system coexistent in spite of the different replicabilities of the cooperating replicators; the other is constraining parasitism, i.e., keeping "cheaters" in check. Surface-bound MCRS provide an automatic solution to both problems: coexistence and parasite resistance are the consequences of assuming the local nature of metabolic interactions. In this review we present an overview of results published in previous articles, showing that these effects are, indeed, robust in different MCRS implementations, by considering different environmental setups and realistic chemical details in a few different models. We argue that the MCRS model framework naturally offers a suitable starting point for the future modelling of membrane evolution and extending the theory to cover the emergence of the first protocell in a self-consistent manner. The coevolution of metabolic, genetic and membrane functions is hypothesized to follow the progressive sequestration scenario, the conceptual blueprint for the earliest steps of protocell evolution. Copyright © 2015 Elsevier Ltd. All rights reserved.

Replication of grazing incidence optics

NASA Technical Reports Server (NTRS)

Ulmer, Melville P.

1986-01-01

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

Replication protein A: directing traffic at the intersection of replication and repair.

PubMed

Oakley, Greg G; Patrick, Steve M

2010-06-01

Since the initial discovery of replication protein A (RPA) as a DNA replication factor, much progress has been made on elucidating critical roles for RPA in other DNA metabolic pathways. RPA has been shown to be required for DNA replication, DNA repair, DNA recombination, and the DNA damage response pathway with roles in checkpoint activation. This review summarizes the current understanding of RPA structure, phosphorylation and protein-protein interactions in mediating these DNA metabolic processes.

Lack of evolvability in self-sustaining autocatalytic networks constraints metabolism-first scenarios for the origin of life.

PubMed

Vasas, Vera; Szathmáry, Eörs; Santos, Mauro

2010-01-26

A basic property of life is its capacity to experience Darwinian evolution. The replicator concept is at the core of genetics-first theories of the origin of life, which suggest that self-replicating oligonucleotides or their similar ancestors may have been the first "living" systems and may have led to the evolution of an RNA world. But problems with the nonenzymatic synthesis of biopolymers and the origin of template replication have spurred the alternative metabolism-first scenario, where self-reproducing and evolving proto-metabolic networks are assumed to have predated self-replicating genes. Recent theoretical work shows that "compositional genomes" (i.e., the counts of different molecular species in an assembly) are able to propagate compositional information and can provide a setup on which natural selection acts. Accordingly, if we stick to the notion of replicator as an entity that passes on its structure largely intact in successive replications, those macromolecular aggregates could be dubbed "ensemble replicators" (composomes) and quite different from the more familiar genes and memes. In sharp contrast with template-dependent replication dynamics, we demonstrate here that replication of compositional information is so inaccurate that fitter compositional genomes cannot be maintained by selection and, therefore, the system lacks evolvability (i.e., it cannot substantially depart from the asymptotic steady-state solution already built-in in the dynamical equations). We conclude that this fundamental limitation of ensemble replicators cautions against metabolism-first theories of the origin of life, although ancient metabolic systems could have provided a stable habitat within which polymer replicators later evolved.

Analysis of Select Herpes Simplex Virus 1 (HSV-1) Proteins for Restriction of Human Immunodeficiency Virus Type 1 (HIV-1): HSV-1 gM Protein Potently Restricts HIV-1 by Preventing Intracellular Transport and Processing of Env gp160.

PubMed

Polpitiya Arachchige, Sachith; Henke, Wyatt; Pramanik, Ankita; Kalamvoki, Maria; Stephens, Edward B

2018-01-15

Virus-encoded proteins that impair or shut down specific host cell functions during replication can be used as probes to identify potential proteins/pathways used in the replication of viruses from other families. We screened nine proteins from herpes simplex virus 1 (HSV-1) for the ability to enhance or restrict human immunodeficiency virus type 1 (HIV-1) replication. We show that several HSV-1 proteins (glycoprotein M [gM], US3, and UL24) potently restricted the replication of HIV-1. Unlike UL24 and US3, which reduced viral protein synthesis, we observed that gM restriction of HIV-1 occurred through interference with the processing and transport of gp160, resulting in a significantly reduced level of mature gp120/gp41 released from cells. Finally, we show that an HSV-1 gM mutant lacking the majority of the C-terminal domain (HA-gM[Δ345-473]) restricted neither gp160 processing nor the release of infectious virus. These studies identify proteins from heterologous viruses that can restrict viruses through novel pathways. IMPORTANCE HIV-1 infection of humans results in AIDS, characterized by the loss of CD4 + T cells and increased susceptibility to opportunistic infections. Both HIV-1 and HSV-1 can infect astrocytes and microglia of the central nervous system (CNS). Thus, the identification of HSV-1 proteins that directly restrict HIV-1 or interfere with pathways required for HIV-1 replication could lead to novel antiretroviral strategies. The results of this study show that select viral proteins from HSV-1 can potently restrict HIV-1. Further, our results indicate that the gM protein of HSV-1 restricts HIV-1 through a novel pathway by interfering with the processing of gp160 and its incorporation into virus maturing from the cell. Copyright © 2018 American Society for Microbiology.

Cytosine methylation inhibits replication of African cassava mosaic virus by two distinct mechanisms.

PubMed Central

Ermak, G; Paszkowski, U; Wohlmuth, M; Scheid, O M; Paszkowski, J

1993-01-01

Extrachromosomally replicating viral DNA is usually free of cytosine methylation and viral templates methylated in vitro are poor substrates when used in replication assays. We have investigated the mechanism of inhibition of viral replication by DNA methylation using as a model the DNA A of African cassava mosaic virus. We have constructed two component helper systems which allow for separation of the transcriptional inhibition of viral genes necessary for replication from replication inhibition due to altered interaction between the replication complex and methylated viral DNA. Our results suggest that methylation-mediated reduction of viral replication is due to both repression mechanisms and that this provides two independent selection pressures for the maintenance of methylation-free replicons in infected cells. Images PMID:7688453

Insights to primitive replication derived from structures of small oligonucleotides

NASA Technical Reports Server (NTRS)

Smith, G. K.; Fox, G. E.

1995-01-01

Available information on the structure of small oligonucleotides is surveyed. It is observed that even small oligomers typically exhibit defined structures over a wide range of pH and temperature. These structures rely on a plethora of non-standard base-base interactions in addition to the traditional Watson-Crick pairings. Stable duplexes, though typically antiparallel, can be parallel or staggered and perfect complementarity is not essential. These results imply that primitive template directed reactions do not require high fidelity. Hence, the extensive use of Watson-Crick complementarity in genes rather than being a direct consequence of the primitive condensation process, may instead reflect subsequent selection based on the advantage of accuracy in maintaining the primitive genetic machinery once it arose.

Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

PubMed

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

2018-01-01

Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell endomembrane system to produce a membranous replication organelle (RO). The underlying mechanisms are far from being elucidated fully. In this report, we provide evidence that HCV RNA replication depends on functional lipid transport along the endosomal-lysosomal pathway that is mediated by several lipid transfer proteins, such as the Niemann-Pick type C1 (NPC1) protein. Pharmacological inhibition of NPC1 function reduced viral replication, impaired the transport of cholesterol to the viral replication organelle, and altered organelle morphology. Besides NPC1, our study reports the importance of additional endosomal and lysosomal lipid transfer proteins required for viral replication, thus contributing to our understanding of how HCV manipulates their function in order to generate a membranous replication organelle. These results might have implications for the biogenesis of replication organelles of other positive-strand RNA viruses. Copyright © 2017 American Society for Microbiology.

Human Papillomavirus E6E7-Mediated Adenovirus Cell Killing: Selectivity of Mutant Adenovirus Replication in Organotypic Cultures of Human Keratinocytes

PubMed Central

Balagué, Cristina; Noya, Francisco; Alemany, Ramon; Chow, Louise T.; Curiel, David T.

2001-01-01

Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells. PMID:11462032

Selection of recombinant MVA by rescue of the essential D4R gene.

PubMed

Ricci, Patricia S; Schäfer, Birgit; Kreil, Thomas R; Falkner, Falko G; Holzer, Georg W

2011-12-12

Modified vaccinia virus Ankara (MVA) has become a promising vaccine vector due to its immunogenicity and its proven safety in humans. As a general approach for stringent and rapid selection of recombinant MVA, we assessed marker rescue of the essential viral D4R gene in an engineered deletion mutant that is fully replication defective in wild-type cells. Recombinant, replicating virus was obtained by re-introduction of the deleted viral gene as a dominant selection marker into the deletion mutant.

Selective attention and avoidance of pain-related stimuli: a dot-probe evaluation in a pain-free population.

PubMed

Roelofs, Jeffrey; Peters, Madelon L; van der Zijden, Marianne; Thielen, Frans G J M; Vlaeyen, Johan W S

2003-08-01

The present study investigated selective attention and avoidance of pain-related stimuli by applying a dot-probe paradigm to healthy university students. The study consisted of 2 successive experiments. The first experiment, a direct replication of a previous study, failed to find evidence for the presence of attentional bias toward pain-related words in highly fearful individuals compared to those who were low in pain-related fear. A second experiment was set up to examine whether avoidance of pain stimuli was influenced by presentation time of word pairs and to investigate the effects of gender on processing pain-related stimuli. The results from the second experiment showed that presentation time of words, fear of pain scores, and gender in isolation or in interaction with each other did not significantly influence attention to and avoidance of pain-related stimuli. Implications of the results are discussed, and directions for future research are provided.

Genetic parameters and selection of soybean lines based on selection indexes.

PubMed

Teixeira, F G; Hamawaki, O T; Nogueira, A P O; Hamawaki, R L; Jorge, G L; Hamawaki, C L; Machado, B Q V; Santana, A J O

2017-09-21

Defining selection criteria is important to obtain promising genotypes in a breeding program. The objective of this study was to estimate genetic parameters for agronomic traits and to perform soybean line selection using selection indices. The experiment was conducted at an experimental area located at Capim Branco farm, belonging to the Federal University of Uberlândia. A total of 37 soybean genotypes were evaluated in randomized complete block design with three replicates, in which twelve agronomic traits were evaluated. Analysis of variance, the Scott-Knott test at the 1 and 5% level of probability, and selection index analyses were performed. There was genetic variability for all agronomic traits, with medium to high levels of genotype determination coefficient. Twelve lines with a total cycle up to 110 days were observed and grouped with the cultivars MSOY 6101 and UFUS 7910. Three lines, UFUS FG 03, UFUS FG 20, and UFUS FG 31, were highlighted regarding grain yield with higher values than the national average of 3072 kg/ha. The direct selection enabled the highest trait individual gains. The Williams (1962) index and the Smith (1936) and Hazel (1943) index presented the highest selection gain for the grain yield character. The genotype-ideotype distance index and the index of the sum of ranks of Mulamba and Mock (1978) presented higher values of total selection gain. The lines UFUS FG 12, UFUS FG 14, UFUS FG 18, UFUS FG 25, and UFUS FG 31 were distinguished as superior genotypes by direct selection methods and selection indexes.

Using Replication Projects in Teaching Research Methods

ERIC Educational Resources Information Center

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

2014-01-01

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

Transient compartmentalization of RNA replicators prevents extinction due to parasites.

PubMed

Matsumura, Shigeyoshi; Kun, Ádám; Ryckelynck, Michael; Coldren, Faith; Szilágyi, András; Jossinet, Fabrice; Rick, Christian; Nghe, Philippe; Szathmáry, Eörs; Griffiths, Andrew D

2016-12-09

The appearance of molecular replicators (molecules that can be copied) was probably a critical step in the origin of life. However, parasitic replicators would take over and would have prevented life from taking off unless the replicators were compartmentalized in reproducing protocells. Paradoxically, control of protocell reproduction would seem to require evolved replicators. We show here that a simpler population structure, based on cycles of transient compartmentalization (TC) and mixing of RNA replicators, is sufficient to prevent takeover by parasitic mutants. TC tends to select for ensembles of replicators that replicate at a similar rate, including a diversity of parasites that could serve as a source of opportunistic functionality. Thus, TC in natural, abiological compartments could have allowed life to take hold. Copyright © 2016, American Association for the Advancement of Science.

Characterizing rare-event property distributions via replicate molecular dynamics simulations of proteins.

PubMed

Krishnan, Ranjani; Walton, Emily B; Van Vliet, Krystyn J

2009-11-01

As computational resources increase, molecular dynamics simulations of biomolecules are becoming an increasingly informative complement to experimental studies. In particular, it has now become feasible to use multiple initial molecular configurations to generate an ensemble of replicate production-run simulations that allows for more complete characterization of rare events such as ligand-receptor unbinding. However, there are currently no explicit guidelines for selecting an ensemble of initial configurations for replicate simulations. Here, we use clustering analysis and steered molecular dynamics simulations to demonstrate that the configurational changes accessible in molecular dynamics simulations of biomolecules do not necessarily correlate with observed rare-event properties. This informs selection of a representative set of initial configurations. We also employ statistical analysis to identify the minimum number of replicate simulations required to sufficiently sample a given biomolecular property distribution. Together, these results suggest a general procedure for generating an ensemble of replicate simulations that will maximize accurate characterization of rare-event property distributions in biomolecules.

Biases in research: risk factors for non-replicability in psychotherapy and pharmacotherapy research.

PubMed

Leichsenring, F; Abbass, A; Hilsenroth, M J; Leweke, F; Luyten, P; Keefe, J R; Midgley, N; Rabung, S; Salzer, S; Steinert, C

2017-04-01

Replicability of findings is an essential prerequisite of research. For both basic and clinical research, however, low replicability of findings has recently been reported. Replicability may be affected by research biases not sufficiently controlled for by the existing research standards. Several biases such as researcher allegiance or selective reporting are well-known for affecting results. For psychotherapy and pharmacotherapy research, specific additional biases may affect outcome (e.g. therapist allegiance, therapist effects or impairments in treatment implementation). For meta-analyses further specific biases are relevant. In psychotherapy and pharmacotherapy research these biases have not yet been systematically discussed in the context of replicability. Using a list of 13 biases as a starting point, we discuss each bias's impact on replicability. We illustrate each bias by selective findings of recent research, showing that (1) several biases are not yet sufficiently controlled for by the presently applied research standards, (2) these biases have a pernicious effect on replicability of findings. For the sake of research credibility, it is critical to avoid these biases in future research. To control for biases and to improve replicability, we propose to systematically implement several measures in psychotherapy and pharmacotherapy research, such as adversarial collaboration (inviting academic rivals to collaborate), reviewing study design prior to knowing the results, triple-blind data analysis (including subjects, investigators and data managers/statisticians), data analysis by other research teams (crowdsourcing), and, last not least, updating reporting standards such as CONSORT or the Template for Intervention Description and Replication (TIDieR).

The origin of replicators and reproducers

PubMed Central

Szathmáry, Eörs

2006-01-01

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

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.

Community-wide plasmid gene mobilization and selection

PubMed Central

Sentchilo, Vladimir; Mayer, Antonia P; Guy, Lionel; Miyazaki, Ryo; Green Tringe, Susannah; Barry, Kerrie; Malfatti, Stephanie; Goessmann, Alexander; Robinson-Rechavi, Marc; van der Meer, Jan R

2013-01-01

Plasmids have long been recognized as an important driver of DNA exchange and genetic innovation in prokaryotes. The success of plasmids has been attributed to their independent replication from the host's chromosome and their frequent self-transfer. It is thought that plasmids accumulate, rearrange and distribute nonessential genes, which may provide an advantage for host proliferation under selective conditions. In order to test this hypothesis independently of biases from culture selection, we study the plasmid metagenome from microbial communities in two activated sludge systems, one of which receives mostly household and the other chemical industry wastewater. We find that plasmids from activated sludge microbial communities carry among the largest proportion of unknown gene pools so far detected in metagenomic DNA, confirming their presumed role of DNA innovators. At a system level both plasmid metagenomes were dominated by functions associated with replication and transposition, and contained a wide variety of antibiotic and heavy metal resistances. Plasmid families were very different in the two metagenomes and grouped in deep-branching new families compared with known plasmid replicons. A number of abundant plasmid replicons could be completely assembled directly from the metagenome, providing insight in plasmid composition without culturing bias. Functionally, the two metagenomes strongly differed in several ways, including a greater abundance of genes for carbohydrate metabolism in the industrial and of general defense factors in the household activated sludge plasmid metagenome. This suggests that plasmids not only contribute to the adaptation of single individual prokaryotic species, but of the prokaryotic community as a whole under local selective conditions. PMID:23407308

Canopy Temperature and Vegetation Indices from High-Throughput Phenotyping Improve Accuracy of Pedigree and Genomic Selection for Grain Yield in Wheat

PubMed Central

Rutkoski, Jessica; Poland, Jesse; Mondal, Suchismita; Autrique, Enrique; Pérez, Lorena González; Crossa, José; Reynolds, Matthew; Singh, Ravi

2016-01-01

Genomic selection can be applied prior to phenotyping, enabling shorter breeding cycles and greater rates of genetic gain relative to phenotypic selection. Traits measured using high-throughput phenotyping based on proximal or remote sensing could be useful for improving pedigree and genomic prediction model accuracies for traits not yet possible to phenotype directly. We tested if using aerial measurements of canopy temperature, and green and red normalized difference vegetation index as secondary traits in pedigree and genomic best linear unbiased prediction models could increase accuracy for grain yield in wheat, Triticum aestivum L., using 557 lines in five environments. Secondary traits on training and test sets, and grain yield on the training set were modeled as multivariate, and compared to univariate models with grain yield on the training set only. Cross validation accuracies were estimated within and across-environment, with and without replication, and with and without correcting for days to heading. We observed that, within environment, with unreplicated secondary trait data, and without correcting for days to heading, secondary traits increased accuracies for grain yield by 56% in pedigree, and 70% in genomic prediction models, on average. Secondary traits increased accuracy slightly more when replicated, and considerably less when models corrected for days to heading. In across-environment prediction, trends were similar but less consistent. These results show that secondary traits measured in high-throughput could be used in pedigree and genomic prediction to improve accuracy. This approach could improve selection in wheat during early stages if validated in early-generation breeding plots. PMID:27402362

Replication and meiotic transmission of yeast ribosomal RNA genes.

PubMed

Brewer, B J; Zakian, V A; Fangman, W L

1980-11-01

The yeast Saccharomyces cerevisiae has approximately 120 genes for the ribosomal RNAs (rDNA) which are organized in tandem within chromosomal DNA. These multiple-copy genes are homogeneous in sequence but can undergo changes in copy number and topology. To determine if these changes reflect unusual features of rDNA metabolism, we have examined both the replication of rDNA in the mitotic cell cycle and the inheritance of rDNA during meiosis. The results indicate that rDNA behaves identically to chromosomal DNA: each rDNA unit is replicated once during the S phase of each cell cycle and each unit is conserved through meiosis. Therefore, the flexibility in copy number and topology of rDNA does not arise from the selective replication of units in each S phase nor by the selective inheritance of units in meiosis.

BK Polyomavirus Replication in Renal Tubular Epithelial Cells Is Inhibited by Sirolimus, but Activated by Tacrolimus Through a Pathway Involving FKBP-12.

PubMed

Hirsch, H H; Yakhontova, K; Lu, M; Manzetti, J

2016-03-01

BK polyomavirus (BKPyV) replication causes nephropathy and premature kidney transplant failure. Insufficient BKPyV-specific T cell control is regarded as a key mechanism, but direct effects of immunosuppressive drugs on BKPyV replication might play an additional role. We compared the effects of mammalian target of rapamycin (mTOR)- and calcineurin-inhibitors on BKPyV replication in primary human renal tubular epithelial cells. Sirolimus impaired BKPyV replication with a 90% inhibitory concentration of 4 ng/mL by interfering with mTOR-SP6-kinase activation. Sirolimus inhibition was rapid and effective up to 24 h postinfection during viral early gene expression, but not thereafter, during viral late gene expression. The mTORC-1 kinase inhibitor torin-1 showed a similar inhibition profile, supporting the notion that early steps of BKPyV replication depend on mTOR activity. Cyclosporine A also inhibited BKPyV replication, while tacrolimus activated BKPyV replication and reversed sirolimus inhibition. FK binding protein 12kda (FKBP-12) siRNA knockdown abrogated sirolimus inhibition and increased BKPyV replication similar to adding tacrolimus. Thus, sirolimus and tacrolimus exert opposite effects on BKPyV replication in renal tubular epithelial cells by a mechanism involving FKBP-12 as common target. Immunosuppressive drugs may therefore contribute directly to the risk of BKPyV replication and nephropathy besides suppressing T cell functions. The data provide rationales for clinical trials aiming at reducing the risk of BKPyV replication and disease in kidney transplantation. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

Models of Protocellular Structure, Function and Evolution

NASA Technical Reports Server (NTRS)

New, Michael H.; Pohorille, Andrew; Szostak, Jack W.; Keefe, Tony; Lanyi, Janos K.

2001-01-01

In the absence of any record of protocells, the most direct way to test our understanding of the origin of cellular life is to construct laboratory models that capture important features of protocellular systems. Such efforts are currently underway in a collaborative project between NASA-Ames, Harvard Medical School and University of California. They are accompanied by computational studies aimed at explaining self-organization of simple molecules into ordered structures. The centerpiece of this project is a method for the in vitro evolution of protein enzymes toward arbitrary catalytic targets. A similar approach has already been developed for nucleic acids in which a small number of functional molecules are selected from a large, random population of candidates. The selected molecules are next vastly multiplied using the polymerase chain reaction. A mutagenic approach, in which the sequences of selected molecules are randomly altered, can yield further improvements in performance or alterations of specificities. Unfortunately, the catalytic potential of nucleic acids is rather limited. Proteins are more catalytically capable but cannot be directly amplified. In the new technique, this problem is circumvented by covalently linking each protein of the initial, diverse, pool to the RNA sequence that codes for it. Then, selection is performed on the proteins, but the nucleic acids are replicated. Additional information is contained in the original extended abstract.

Fast and cheap fabrication of molding tools for polymer replication

NASA Astrophysics Data System (ADS)

Richter, Christiane; Kirschner, Nadine; Worgull, Matthias; Rapp, Bastian E.

2017-02-01

Polymer replication is a prerequisite for low-cost microstructure components for consumer and end user market. The production of cost-effective microstructure in polymers requires metal molding tools which are often fabricated by direct structuring methods like milling or laser machining both of which are time-consuming and cost-intensive. We present an alternative fabrication method based on replication processes which allows the cheap ( 50 €) and fast ( 12 h) replication of complex microstructures into metal. The process comprises three steps: 1. Generation of the microstructure in a photoresist via lithography. 2. Casting of the structure into a high-temperature silicone which serves as original mold for creation of the metal molding tool. 3. Melting of an eutectic alloy of Sn, Ag and Cu under light pressure directly inside of the silicone within an oven. After cooling to room temperature the metal molding tool can be used for polymer replication into conventional thermoplastic polymers. As a first example we structured polymethylmethacrylate (PMMA) foils with a thickness of 1 mm via hot embossing and feature sizes of 100 μm could be replicated with high fidelity.

Inhibition of aldolase A blocks biogenesis of ATP and attenuates Japanese encephalitis virus production.

PubMed

Tien, Chih-Feng; Cheng, Shih-Ching; Ho, Yen-Peng; Chen, Yi-Shiuan; Hsu, Jung-Hsin; Chang, Ruey-Yi

2014-01-10

Viral replication depends on host proteins to supply energy and replication accessories for the sufficient production of viral progeny. In this study, we identified fructose-bisphosphate aldolase A as a binding partner of Japanese encephalitis virus (JEV) untranslated regions (UTRs) on the antigenome via RNA affinity capture and mass spectrometry. Direct interaction of aldolase A with JEV RNAs was confirmed by gel mobility shift assay and colocalization with active replication of double-stranded RNA in JEV-infected cells. Infection of JEV caused an increase in aldolase A expression of up to 33%. Knocking down aldolase A reduced viral translation, genome replication, and viral production significantly. Furthermore, JEV infection consumed 50% of cellular ATP, and the ATP level decreased by 70% in the aldolase A-knockdown cells. Overexpression of aldolase A in aldolase A-knockdown cells increased ATP levels significantly. Taken together, these results indicate that JEV replication requires aldolase A and consumes ATP. This is the first report of direct involvement of a host metabolic enzyme, aldolase A protein, in JEV replication. Copyright © 2013 Elsevier Inc. All rights reserved.

Probing of molecular replication and accumulation in shallow heat gradients through numerical simulations.

PubMed

Keil, Lorenz; Hartmann, Michael; Lanzmich, Simon; Braun, Dieter

2016-07-27

How can living matter arise from dead matter? All known living systems are built around information stored in RNA and DNA. To protect this information against molecular degradation and diffusion, the second law of thermodynamics imposes the need for a non-equilibrium driving force. Following a series of successful experiments using thermal gradients, we have shown that heat gradients across sub-millimetre pores can drive accumulation, replication, and selection of ever longer molecules, implementing all the necessary parts for Darwinian evolution. For these lab experiments to proceed with ample speed, however, the temperature gradients have to be quite steep, reaching up to 30 K per 100 μm. Here we use computer simulations based on experimental data to show that 2000-fold shallower temperature gradients - down to 100 K over one metre - can still drive the accumulation of protobiomolecules. This finding opens the door for various environments to potentially host the origins of life: volcanic, water-vapour, or hydrothermal settings. Following the trajectories of single molecules in simulation, we also find that they are subjected to frequent temperature oscillations inside these pores, facilitating e.g. template-directed replication mechanisms. The tilting of the pore configuration is the central strategy to achieve replication in a shallow temperature gradient. Our results suggest that shallow thermal gradients across porous rocks could have facilitated the formation of evolutionary machines, significantly increasing the number of potential sites for the origin of life on young rocky planets.

DNA Replication Origins and Fork Progression at Mammalian Telomeres

PubMed Central

Higa, Mitsunori; Fujita, Masatoshi; Yoshida, Kazumasa

2017-01-01

Telomeres are essential chromosomal regions that prevent critical shortening of linear chromosomes and genomic instability in eukaryotic cells. The bulk of telomeric DNA is replicated by semi-conservative DNA replication in the same way as the rest of the genome. However, recent findings revealed that replication of telomeric repeats is a potential cause of chromosomal instability, because DNA replication through telomeres is challenged by the repetitive telomeric sequences and specific structures that hamper the replication fork. In this review, we summarize current understanding of the mechanisms by which telomeres are faithfully and safely replicated in mammalian cells. Various telomere-associated proteins ensure efficient telomere replication at different steps, such as licensing of replication origins, passage of replication forks, proper fork restart after replication stress, and dissolution of post-replicative structures. In particular, shelterin proteins have central roles in the control of telomere replication. Through physical interactions, accessory proteins are recruited to maintain telomere integrity during DNA replication. Dormant replication origins and/or homology-directed repair may rescue inappropriate fork stalling or collapse that can cause defects in telomere structure and functions. PMID:28350373

Sex differences, sexual selection, and ageing: an experimental evolution approach.

PubMed

Maklakov, Alexei A; Bonduriansky, Russell; Brooks, Robert C

2009-10-01

Life-history (LH) theory predicts that selection will optimize the trade-off between reproduction and somatic maintenance. Reproductive ageing and finite life span are direct consequences of such optimization. Sexual selection and conflict profoundly affect the reproductive strategies of the sexes and thus can play an important role in the evolution of life span and ageing. In theory, sexual selection can favor the evolution of either faster or slower ageing, but the evidence is equivocal. We used a novel selection experiment to investigate the potential of sexual selection to influence the adaptive evolution of age-specific LH traits. We selected replicate populations of the seed beetle Callosobruchus maculatus for age at reproduction ("Young" and "Old") either with or without sexual selection. We found that LH selection resulted in the evolution of age-specific reproduction and mortality but these changes were largely unaffected by sexual selection. Sexual selection depressed net reproductive performance and failed to promote adaptation. Nonetheless, the evolution of several traits differed between males and females. These data challenge the importance of current sexual selection in promoting rapid adaptation to environmental change but support the hypothesis that sex differences in LH-a historical signature of sexual selection-are key in shaping trait responses to novel selection.

Fertilization selection on egg and jelly-coat size in the sand dollar Dendraster excentricus.

PubMed

Levitan, D R; Irvine, S D

2001-12-01

Organisms with external fertilization are often sperm limited, and in echinoids, larger eggs have a higher probability of fertilization than smaller eggs. This difference is thought to be a result of the more frequent sperm-egg collisions experienced by larger targets. Here we report how two components of egg target size, the egg cell and jelly coat, contributed to fertilization success in a selection experiment. We used a cross-sectional analysis of correlated characters to estimate the selection gradients on egg and jelly-coat size in five replicate male pairs of the sand dollar Dendraster excentricus. Results indicated that eggs with larger cells and jelly coats were preferentially fertilized under sperm limitation in the laboratory. The selection gradients were an average of 922% steeper for egg than for jelly-coat size. The standardized selection gradients for egg and jelly-coat size were similar. Our results suggest that fertilization selection can act on both egg-cell and jelly-coat size but that an increase in egg-cell volume is much more likely to increase fertilization success than an equal change in jelly-coat volume. The strengths of the selection gradients were inversely related to the correlation of egg traits across replicate egg clutches. This result suggests the importance of replication in studies of selection of correlated characters.

A novel computational method to simulate non-enzymatic self-replication. [Abstract only

NASA Technical Reports Server (NTRS)

Navarro-Gonzalez, Rafael; Reggia, James A.; Wu, Jayoung; Chou, Hui-Hsien

1994-01-01

Non-enzymatic, template-directed synthesis of oligonucleotides has been extensively studied in the laboratory as a model to understand the kind of chemical processes that might have contributed to the origin of life on Earth. Several oligonucleotides have been shown to catalyze the synthesis of their complements from activated mononucleotides; however, a restricted number of them have been found to self-replicate. Recently we developed an efficient modified cellular automata method that supports the study of self-replicating oligonucleotides. With this method the oligonucleotide molecules are represented as active cells imbedded in a two-dimensional array of inactive cells symbolizing the environment. Random movements and probability-governed chemical reactions occurring in a cellular space can effectively simulate the experimental behavior observed in self-directed replication of oligonucleotides.

DNA replication stress: from molecular mechanisms to human disease.

PubMed

Muñoz, Sergio; Méndez, Juan

2017-02-01

The genome of proliferating cells must be precisely duplicated in each cell division cycle. Chromosomal replication entails risks such as the possibility of introducing breaks and/or mutations in the genome. Hence, DNA replication requires the coordinated action of multiple proteins and regulatory factors, whose deregulation causes severe developmental diseases and predisposes to cancer. In recent years, the concept of "replicative stress" (RS) has attracted much attention as it impinges directly on genomic stability and offers a promising new avenue to design anticancer therapies. In this review, we summarize recent progress in three areas: (1) endogenous and exogenous factors that contribute to RS, (2) molecular mechanisms that mediate the cellular responses to RS, and (3) the large list of diseases that are directly or indirectly linked to RS.

Mutualists and antagonists drive among-population variation in selection and evolution of floral display in a perennial herb

PubMed Central

Ågren, Jon; Hellström, Frida; Toräng, Per; Ehrlén, Johan

2013-01-01

Spatial variation in the direction of selection drives the evolution of adaptive differentiation. However, few experimental studies have examined the relative importance of different environmental factors for variation in selection and evolutionary trajectories in natural populations. Here, we combine 8 y of observational data and field experiments to assess the relative importance of mutualistic and antagonistic interactions for spatial variation in selection and short-term evolution of a genetically based floral display dimorphism in the short-lived perennial herb Primula farinosa. Natural populations of this species include two floral morphs: long-scaped plants that present their flowers well above the ground and short-scaped plants with flowers positioned close to the ground. The direction and magnitude of selection on scape morph varied among populations, and so did the frequency of the short morph (median 19%, range 0–100%; n = 69 populations). A field experiment replicated at four sites demonstrated that variation in the strength of interactions with grazers and pollinators were responsible for among-population differences in relative fitness of the two morphs. Selection exerted by grazers favored the short-scaped morph, whereas pollinator-mediated selection favored the long-scaped morph. Moreover, variation in selection among natural populations was associated with differences in morph frequency change, and the experimental removal of grazers at nine sites significantly reduced the frequency of the short-scaped morph over 8 y. The results demonstrate that spatial variation in intensity of grazing and pollination produces a selection mosaic, and that changes in biotic interactions can trigger rapid genetic changes in natural plant populations. PMID:24145439

Mutualists and antagonists drive among-population variation in selection and evolution of floral display in a perennial herb.

PubMed

Agren, Jon; Hellström, Frida; Toräng, Per; Ehrlén, Johan

2013-11-05

Spatial variation in the direction of selection drives the evolution of adaptive differentiation. However, few experimental studies have examined the relative importance of different environmental factors for variation in selection and evolutionary trajectories in natural populations. Here, we combine 8 y of observational data and field experiments to assess the relative importance of mutualistic and antagonistic interactions for spatial variation in selection and short-term evolution of a genetically based floral display dimorphism in the short-lived perennial herb Primula farinosa. Natural populations of this species include two floral morphs: long-scaped plants that present their flowers well above the ground and short-scaped plants with flowers positioned close to the ground. The direction and magnitude of selection on scape morph varied among populations, and so did the frequency of the short morph (median 19%, range 0-100%; n = 69 populations). A field experiment replicated at four sites demonstrated that variation in the strength of interactions with grazers and pollinators were responsible for among-population differences in relative fitness of the two morphs. Selection exerted by grazers favored the short-scaped morph, whereas pollinator-mediated selection favored the long-scaped morph. Moreover, variation in selection among natural populations was associated with differences in morph frequency change, and the experimental removal of grazers at nine sites significantly reduced the frequency of the short-scaped morph over 8 y. The results demonstrate that spatial variation in intensity of grazing and pollination produces a selection mosaic, and that changes in biotic interactions can trigger rapid genetic changes in natural plant populations.

Comparison of roll-to-roll replication approaches for microfluidic and optical functions in lab-on-a-chip diagnostic devices

NASA Astrophysics Data System (ADS)

Brecher, Christian; Baum, Christoph; Bastuck, Thomas

2015-03-01

Economically advantageous microfabrication technologies for lab-on-a-chip diagnostic devices substituting commonly used glass etching or injection molding processes are one of the key enablers for the emerging market of microfluidic devices. On-site detection in fields of life sciences, point of care diagnostics and environmental analysis requires compact, disposable and highly functionalized systems. Roll-to-roll production as a high volume process has become the emerging fabrication technology for integrated, complex high technology products within recent years (e.g. fuel cells). Differently functionalized polymer films enable researchers to create a new generation of lab-on-a-chip devices by combining electronic, microfluidic and optical functions in multilayer architecture. For replication of microfluidic and optical functions via roll-to-roll production process competitive approaches are available. One of them is to imprint fluidic channels and optical structures of micro- or nanometer scale from embossing rollers into ultraviolet (UV) curable lacquers on polymer substrates. Depending on dimension, shape and quantity of those structures there are alternative manufacturing technologies for the embossing roller. Ultra-precise diamond turning, electroforming or casting polymer materials are used either for direct structuring or manufacturing of roller sleeves. Mastering methods are selected for application considering replication quality required and structure complexity. Criteria for the replication quality are surface roughness and contour accuracy. Structure complexity is evaluated by shapes producible (e.g. linear, circular) and aspect ratio. Costs for the mastering process and structure lifetime are major cost factors. The alternative replication approaches are introduced and analyzed corresponding to the criteria presented. Advantages and drawbacks of each technology are discussed and exemplary applications are presented.

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

Development of a fiber coating based on molecular sol-gel imprinting technology for selective solid-phase micro extraction of caffeine from human serum and determination by gas chromatography/mass spectrometry.

PubMed

Rajabi Khorrami, Afshin; Rashidpur, Amene

2012-05-21

In this work, a molecular sol-gel imprinting approach has been introduced to produce a fiber coating for selective direct immersion solid-phase microextraction (SPME) of caffeine. The polymerization mixture was composed of vinyl trimethoxysilane and methacrylic acid as vinyl sol-gel precursor and functional monomer, respectively. Caffeine was used as template molecule during polymerization process. The prepared fibers could be coupled directly to gas chromatography/mass spectrometry (GC/MS) and used for trace analysis of caffeine in a complex sample such as human serum. The parameters influencing SPME such as time, temperature and stirring speed were optimized. The prepared coating showed good selectivity towards caffeine in the presence of some structurally related compounds. Also, it offered high imprinting capability in comparison to bare fiber and non-imprinted coating. Linear range for caffeine detection was 1-80 μg mL(-1) and the limit of detection was 0.1 μg mL(-1). The intra-day and inter-day precisions of the peak areas for five replicates were 10 and 16%, respectively. Copyright © 2012 Elsevier B.V. All rights reserved.

Hepatitis C virus resistance to the new direct-acting antivirals.

PubMed

Esposito, Isabella; Trinks, Julieta; Soriano, Vicente

2016-10-01

The treatment of hepatitis C virus (HCV) infection has dramatically improved in recent years with the widespread use of interferon-free combination regimens. Despite the high sustained virological response (SVR) rates (over 90%) obtained with direct-acting antivirals (DAAs), drug resistance has emerged as a potential challenge. The high replication rate of HCV and the low fidelity of its RNA polymerase result in a high degree of genetic variability in the HCV population, which ultimately explains the rapid selection of drug resistance associated variants (RAVs). Results from clinical trials and real-world experience have both provided important information on the rate and clinical significance of RAVs. They can be present in treatment-naive patients as natural polymorphisms although more frequently they are selected upon treatment failure. In patients engaged in high-risk behaviors, RAVs can be transmitted. Although DAA failures generally occur in less than 10% of treated chronic hepatitis C patients, selection of drug resistance is the rule in most cases. HCV re-treatment options are available, but first-line therapeutic strategies should be optimized to efficiently prevent DAA failure due to baseline HCV resistance. Considerable progress is being made and next-generation DAAs are coming with pangenotypic activity and higher resistance barrier.

Mechanisms of DNA replication termination.

PubMed

Dewar, James M; Walter, Johannes C

2017-08-01

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

Identification of BMS-200475 as a potent and selective inhibitor of hepatitis B virus.

PubMed Central

Innaimo, S F; Seifer, M; Bisacchi, G S; Standring, D N; Zahler, R; Colonno, R J

1997-01-01

BMS-200475 is a novel carbocyclic 2'-deoxyguanosine analog found to possess potent and selective anti-hepatitis B virus (anti-HBV) activity. BMS-200475 is distinguished from guanosine by replacement of the natural furanose oxygen on the sugar moiety with an exo carbon-carbon double bond. In the HepG2 stably transfected cell line 2.2.15, BMS-200475 had a 50% effective concentration (EC50) of 3.75 nM against HBV, as determined by analysis of secreted HBV DNA. Structurally related compounds with adenine, iodouracil, or thymine base substitutions were significantly less potent or were inactive. Direct comparison of the antiviral activities of BMS-200475 with those of a variety of other nucleoside analogs, including lamivudine (EC50 = 116.26 nM), demonstrated the clearly superior in vitro potency of BMS-200475 in 2.2.15 cells. Intracellular HBV replicative intermediates were uniformly reduced when cells were treated with BMS-200475, but rebounded after treatment was terminated. The concentration of BMS-200475 causing 50% cytotoxicity in 2.2.15 cell cultures was 30 microM, approximately 8,000-fold greater than the concentration required to inhibit HBV replication in the same cell line. Treatment with BMS-200475 resulted in no apparent inhibitory effects on mitochondrial DNA content. PMID:9210663

The word class effect in the picture–word interference paradigm

PubMed Central

Janssen, Niels; Melinger, Alissa; Mahon, Bradford Z.; Finkbeiner, Matthew; Caramazza, Alfonso

2010-01-01

The word class effect in the picture–word interference paradigm is a highly influential finding that has provided some of the most compelling support for word class constraints on lexical selection. However, methodological concerns called for a replication of the most convincing of those effects. Experiment 1 was a direct replication of Pechmann and Zerbst (2002; Experiment 4). Participants named pictures of objects in the context of noun and adverb distractors. Naming took place in bare noun and sentence frame contexts. A word class effect emerged in both bare noun and sentence frame naming conditions, suggesting a semantic origin of the effect. In Experiment 2, participants named objects in the context of noun and verb distractors whose word class relationship to the target and imageability were orthogonally manipulated. As before, naming took place in bare noun and sentence frame naming contexts. In both naming contexts, distractor imageability but not word class affected picture naming latencies. These findings confirm the sensitivity of the picture–word interference paradigm to distractor imageability and suggest the paradigm is not sensitive to distractor word class. The results undermine the use of the word class effect in the picture–word interference paradigm as supportive of word class constraints during lexical selection. PMID:19998070

Elimination of A-type inclusion formation enhances cowpox virus replication in mice: implications for orthopoxvirus evolution.

PubMed

Kastenmayer, Robin J; Maruri-Avidal, Liliana; Americo, Jeffrey L; Earl, Patricia L; Weisberg, Andrea S; Moss, Bernard

2014-03-01

Some orthopoxviruses including cowpox virus embed virus particles in dense bodies, comprised of the A-type inclusion (ATI) protein, which may provide long-term environmental protection. This strategy could be beneficial if the host population is sparse or spread is inefficient or indirect. However, the formation of ATI may be neutral or disadvantageous for orthopoxviruses that rely on direct respiratory spread. Disrupted ATI open reading frames in orthopoxviruses such as variola virus, the agent of smallpox, and monkeypox virus suggests that loss of this feature provided positive selection. To test this hypothesis, we constructed cowpox virus mutants with deletion of the ATI gene or another gene required for embedding virions. The ATI deletion mutant caused greater weight loss and higher replication in the respiratory tract than control viruses, supporting our hypothesis. Deletion of the gene for embedding virions had a lesser effect, possibly due to known additional functions of the encoded protein. Published by Elsevier Inc.

Whole-Genome Resequencing of Experimental Populations Reveals Polygenic Basis of Egg-Size Variation in Drosophila melanogaster.

PubMed

Jha, Aashish R; Miles, Cecelia M; Lippert, Nodia R; Brown, Christopher D; White, Kevin P; Kreitman, Martin

2015-10-01

Complete genome resequencing of populations holds great promise in deconstructing complex polygenic traits to elucidate molecular and developmental mechanisms of adaptation. Egg size is a classic adaptive trait in insects, birds, and other taxa, but its highly polygenic architecture has prevented high-resolution genetic analysis. We used replicated experimental evolution in Drosophila melanogaster and whole-genome sequencing to identify consistent signatures of polygenic egg-size adaptation. A generalized linear-mixed model revealed reproducible allele frequency differences between replicated experimental populations selected for large and small egg volumes at approximately 4,000 single nucleotide polymorphisms (SNPs). Several hundred distinct genomic regions contain clusters of these SNPs and have lower heterozygosity than the genomic background, consistent with selection acting on polymorphisms in these regions. These SNPs are also enriched among genes expressed in Drosophila ovaries and many of these genes have well-defined functions in Drosophila oogenesis. Additional genes regulating egg development, growth, and cell size show evidence of directional selection as genes regulating these biological processes are enriched for highly differentiated SNPs. Genetic crosses performed with a subset of candidate genes demonstrated that these genes influence egg size, at least in the large genetic background. These findings confirm the highly polygenic architecture of this adaptive trait, and suggest the involvement of many novel candidate genes in regulating egg size. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The cost of copy number in a selfish genetic element: the 2-μm plasmid of Saccharomyces cerevisiae.

PubMed

Harrison, Ellie; Koufopanou, V; Burt, A; MacLean, R C

2012-11-01

Many autonomously replicating genetic elements exist as multiple copies within the cell. The copy number of these elements is often assumed to have important fitness consequences for both element and host, yet the forces shaping its evolution are not well understood. The 2 μm is a multicopy plasmid of Saccharomyces yeasts, encoding just four genes that are solely involved in plasmid replication. One simple model for the fitness relationship between yeasts and 2 μm is that plasmid copy number evolves as a trade-off between selection for increased vertical transmission, favouring high copy number, and selection for decreased virulence, favouring low copy number. To test this model, we experimentally manipulated the copy number of the plasmid and directly measured the fitness cost, in terms of growth rate reduction, associated with high plasmid copy number. We find that the fitness burden imposed by the 2 μm increases with plasmid copy number, such that each copy imposes a fitness burden of 0.17% (± 0.008%), greatly exceeding the cost expected for it to be stably maintained in yeast populations. Our results demonstrate the crucial importance of copy number in the evolution of yeast per 2 μm associations and pave the way for future studies examining how selection can shape the cost of multicopy elements. © 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.

Versatile Cosmid Vectors for the Isolation, Expression, and Rescue of Gene Sequences: Studies with the Human α -globin Gene Cluster

NASA Astrophysics Data System (ADS)

Lau, Yun-Fai; Kan, Yuet Wai

1983-09-01

We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers-SV2-gpt, SV2-DHFR, and SV2-neo-in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human α -globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of α -globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult α -globin genes were more actively expressed than the embryonic zeta -globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.

Understanding how replication processes can maintain systems away from equilibrium using Algorithmic Information Theory.

PubMed

Devine, Sean D

2016-02-01

Replication can be envisaged as a computational process that is able to generate and maintain order far-from-equilibrium. Replication processes, can self-regulate, as the drive to replicate can counter degradation processes that impact on a system. The capability of replicated structures to access high quality energy and eject disorder allows Landauer's principle, in conjunction with Algorithmic Information Theory, to quantify the entropy requirements to maintain a system far-from-equilibrium. Using Landauer's principle, where destabilising processes, operating under the second law of thermodynamics, change the information content or the algorithmic entropy of a system by ΔH bits, replication processes can access order, eject disorder, and counter the change without outside interventions. Both diversity in replicated structures, and the coupling of different replicated systems, increase the ability of the system (or systems) to self-regulate in a changing environment as adaptation processes select those structures that use resources more efficiently. At the level of the structure, as selection processes minimise the information loss, the irreversibility is minimised. While each structure that emerges can be said to be more entropically efficient, as such replicating structures proliferate, the dissipation of the system as a whole is higher than would be the case for inert or simpler structures. While a detailed application to most real systems would be difficult, the approach may well be useful in understanding incremental changes to real systems and provide broad descriptions of system behaviour. Copyright © 2016 The Author. Published by Elsevier Ireland Ltd.. All rights reserved.

Integration of Five Health Behaviour Models: Common Strengths and Unique Contributions to Understanding Condom Use

PubMed Central

Reid, Allecia E.; Aiken, Leona S.

2011-01-01

The purpose of this research was to select from the health belief model (HBM), theories of reasoned action (TRA) and planned behaviour (TPB), information-motivation-behavioural skills model (IMB), and social cognitive theory (SCT) the strongest longitudinal predictors of women’s condom use and to combine these constructs into a single integrated model of condom use. The integrated model was evaluated for prediction of condom use among young women who had steady versus casual partners. At Time 1, all constructs of the five models and condom use were assessed in an initial and a replication sample (n= 193, n= 161). Condom use reassessed 8 weeks later (Time 2) served as the main outcome. Information from IMB, perceived susceptibility, benefits, and barriers from HBM, self-efficacy and self-evaluative expectancies from SCT, and partner norm and attitudes from TPB served as indirect or direct predictors of condom use. All paths replicated across samples. Direct predictors of behaviour varied with relationship status: self-efficacy significantly predicted condom use for women with casual partners, while attitude and partner norm predicted for those with steady partners. Integrated psychosocial models, rich in constructs and relationships drawn from multiple theories of behaviour, may provide a more complete characterization of health protective behaviour. PMID:21678166

Mechanisms and regulation of DNA replication initiation in eukaryotes

PubMed Central

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

2017-01-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a given cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the Origin Recognition Complex (ORC), and subsequent activation of the helicase by incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms. PMID:28094588

Mechanisms and regulation of DNA replication initiation in eukaryotes.

PubMed

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

2017-04-01

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

Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation

PubMed Central

Devlin, Rebecca; Marques, Catarina A; Paape, Daniel; Prorocic, Marko; Zurita-Leal, Andrea C; Campbell, Samantha J; Lapsley, Craig; Dickens, Nicholas; McCulloch, Richard

2016-01-01

Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating – a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility. DOI: http://dx.doi.org/10.7554/eLife.12765.001 PMID:27228154

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

PubMed

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

2014-02-15

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

Genetic Interaction Mapping in Schizosaccharomyces pombe Using the Pombe Epistasis Mapper (PEM) System and a ROTOR HDA Colony Replicating Robot in a 1536 Array Format.

PubMed

Roguev, Assen; Xu, Jiewei; Krogan, Nevan

2018-02-01

This protocol describes an optimized high-throughput procedure for generating double deletion mutants in Schizosaccharomyces pombe using the colony replicating robot ROTOR HDA and the PEM (pombe epistasis mapper) system. The method is based on generating high-density colony arrays (1536 colonies per agar plate) and passaging them through a series of antidiploid and mating-type selection (ADS-MTS) and double-mutant selection (DMS) steps. Detailed program parameters for each individual replication step are provided. Using this procedure, batches of 25 or more screens can be routinely performed. © 2018 Cold Spring Harbor Laboratory Press.

Communicative genes in the evolution of empathy and altruism.

PubMed

Buck, Ross

2011-11-01

This paper discusses spontaneous communication and its implications for understanding empathy and altruism. The question of the possibility of "true" altruism-giving up one's genetic potential in favor of the genetic potential of another-is a fundamental issue common to the biological, behavioral, and social sciences. Darwin regarded "social instincts and sympathies" to be critical to the social order, but the possibility of biologically-based prosocial motives and emotions was questioned when selection was interpreted as operating at the level of the gene. In the selfish gene hypothesis, Dawkins argued that the unit of evolutionary selection must be an active, germ-line replicator: a unit whose activities determine whether copies of it are made across evolutionary timescales. He argued that the only active replicator existing across evolutionary timescales is the gene, so that the "selfish gene" is a replicator motivated only to make copies of itself. The communicative gene hypothesis notes that genes function by communicating, and the phenotype communication involves not only the individual sending and receiving abilities of the individual genes involved, but also the relationship between them relative to other genes. Therefore the selection of communication as phenotype involves the selection of individual genes and also their relationship. Relationships become replicators, and are selected across evolutionary timescales including social relationships (e.g., sex, nurturance, dominance-submission). An interesting implication of this view: apparent altruism has been interpreted by selfish gene theorists as due to kin selection and reciprocity, in which the survival of kin and comrade indirectly favor the genetic potential of the altruist. From the viewpoint of the communicative gene hypothesis, rather than underlying altruism, kin selection and reciprocity are ways of restricting altruism to kin and comrade: they are mechanisms not of altruism but of xenophobia.

Universal Sequence Replication, Reversible Polymerization and Early Functional Biopolymers: A Model for the Initiation of Prebiotic Sequence Evolution

PubMed Central

Walker, Sara Imari; Grover, Martha A.; Hud, Nicholas V.

2012-01-01

Many models for the origin of life have focused on understanding how evolution can drive the refinement of a preexisting enzyme, such as the evolution of efficient replicase activity. Here we present a model for what was, arguably, an even earlier stage of chemical evolution, when polymer sequence diversity was generated and sustained before, and during, the onset of functional selection. The model includes regular environmental cycles (e.g. hydration-dehydration cycles) that drive polymers between times of replication and functional activity, which coincide with times of different monomer and polymer diffusivity. Template-directed replication of informational polymers, which takes place during the dehydration stage of each cycle, is considered to be sequence-independent. New sequences are generated by spontaneous polymer formation, and all sequences compete for a finite monomer resource that is recycled via reversible polymerization. Kinetic Monte Carlo simulations demonstrate that this proposed prebiotic scenario provides a robust mechanism for the exploration of sequence space. Introduction of a polymer sequence with monomer synthetase activity illustrates that functional sequences can become established in a preexisting pool of otherwise non-functional sequences. Functional selection does not dominate system dynamics and sequence diversity remains high, permitting the emergence and spread of more than one functional sequence. It is also observed that polymers spontaneously form clusters in simulations where polymers diffuse more slowly than monomers, a feature that is reminiscent of a previous proposal that the earliest stages of life could have been defined by the collective evolution of a system-wide cooperation of polymer aggregates. Overall, the results presented demonstrate the merits of considering plausible prebiotic polymer chemistries and environments that would have allowed for the rapid turnover of monomer resources and for regularly varying monomer/polymer diffusivities. PMID:22493682

Emergence of viruses resistant to neutralization by V3-specific antibodies in experimental human immunodeficiency virus type 1 IIIB infection of chimpanzees.

PubMed Central

Nara, P L; Smit, L; Dunlop, N; Hatch, W; Merges, M; Waters, D; Kelliher, J; Gallo, R C; Fischinger, P J; Goudsmit, J

1990-01-01

Emergence in two chimpanzees of human immunodeficiency virus type 1 (HIV-1) IIIB variants resistant to neutralization by the preexisting antibody is described. Viruses isolated from the HIV-1 IIIB gp120-vaccinated and -challenged animal were more resistant to neutralization by the chimpanzee's own serum than viruses isolated from the naive infected animal, indicating immune pressure as the selective mechanism. However, all reisolated viruses were 16- to 256-fold more neutralization resistant than the inoculum virus to antibodies binding to the third variable domain (V3) of the HIV-1 external envelope. Early chimpanzee serum samples that neutralized the inoculum strain but not the reisolated viruses were found to bind an HIV-1 IIIB common nonapeptide (IQRGPGRAF) derived from the gp120 isolate-specific V3 domain shown to induce isolate-specific neutralization in other animals. Amplification of the V3 coding sequence by polymerase chain reaction and subsequent sequence analysis of the neutralization-resistant variants obtained from in vivo-infected animals indicated that early resistance to neutralization by an HIV-1 IIIB monoclonal antibody (0.5 beta) was conferred by changes outside the direct binding site for the selective neutralizing antibody. The reisolated neutralization-resistant isolates consisted of the lower-replication-competent virus subpopulations of the HIV-1 IIIB stock, as confirmed by biological and sequence analyses. In vitro passage of the HIV-1 IIIB stock through chimpanzee and human peripheral blood mononuclear cell cultures void of HIV-specific antibody resulted in homogenic amplification of the more-replication-competent subpopulation preexisting in the original viral stock, suggesting a role for the immune system in suppressing the more-replication-competent viruses. Images PMID:2370681

Modeling DNA Replication.

ERIC Educational Resources Information Center

Bennett, Joan

1998-01-01

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

Enquire within: cultural evolution and cognitive science.

PubMed

Heyes, Cecilia

2018-04-05

Cultural evolution and cognitive science need each other. Cultural evolution needs cognitive science to find out whether the conditions necessary for Darwinian evolution are met in the cultural domain. Cognitive science needs cultural evolution to explain the origins of distinctively human cognitive processes. Focusing on the first question, I argue that cultural evolutionists can get empirical traction on third-way cultural selection by rooting the distinction between replication and reconstruction, two modes of cultural inheritance, in the distinction between System 1 and System 2 cognitive processes. This move suggests that cultural epidemiologists are right in thinking that replication has higher fidelity than reconstruction, and replication processes are not genetic adaptations for culture, but wrong to assume that replication is rare. If replication is not rare, an important requirement for third-way cultural selection, one-shot fidelity , is likely to be met. However, there are other requirements, overlooked by dual-inheritance theorists when they conflate strong (Darwinian) and weak (choice) senses of 'cultural selection', including dumb choices and recurrent fidelity In a second excursion into cognitive science, I argue that these requirements can be met by metacognitive social learning strategies , and trace the origins of these distinctively human cognitive processes to cultural evolution. Like other forms of cultural learning, they are not cognitive instincts but cognitive gadgets.This article is part of the theme issue 'Bridging cultural gaps: interdisciplinary studies in human cultural evolution'. © 2018 The Author(s).

Selectionist and Evolutionary Approaches to Brain Function: A Critical Appraisal

PubMed Central

Fernando, Chrisantha; Szathmáry, Eörs; Husbands, Phil

2012-01-01

We consider approaches to brain dynamics and function that have been claimed to be Darwinian. These include Edelman’s theory of neuronal group selection, Changeux’s theory of synaptic selection and selective stabilization of pre-representations, Seung’s Darwinian synapse, Loewenstein’s synaptic melioration, Adam’s selfish synapse, and Calvin’s replicating activity patterns. Except for the last two, the proposed mechanisms are selectionist but not truly Darwinian, because no replicators with information transfer to copies and hereditary variation can be identified in them. All of them fit, however, a generalized selectionist framework conforming to the picture of Price’s covariance formulation, which deliberately was not specific even to selection in biology, and therefore does not imply an algorithmic picture of biological evolution. Bayesian models and reinforcement learning are formally in agreement with selection dynamics. A classification of search algorithms is shown to include Darwinian replicators (evolutionary units with multiplication, heredity, and variability) as the most powerful mechanism for search in a sparsely occupied search space. Examples are given of cases where parallel competitive search with information transfer among the units is more efficient than search without information transfer between units. Finally, we review our recent attempts to construct and analyze simple models of true Darwinian evolutionary units in the brain in terms of connectivity and activity copying of neuronal groups. Although none of the proposed neuronal replicators include miraculous mechanisms, their identification remains a challenge but also a great promise. PMID:22557963

How can we model selectively neutral density dependence in evolutionary games.

PubMed

Argasinski, Krzysztof; Kozłowski, Jan

2008-03-01

The problem of density dependence appears in all approaches to the modelling of population dynamics. It is pertinent to classic models (i.e., Lotka-Volterra's), and also population genetics and game theoretical models related to the replicator dynamics. There is no density dependence in the classic formulation of replicator dynamics, which means that population size may grow to infinity. Therefore the question arises: How is unlimited population growth suppressed in frequency-dependent models? Two categories of solutions can be found in the literature. In the first, replicator dynamics is independent of background fitness. In the second type of solution, a multiplicative suppression coefficient is used, as in a logistic equation. Both approaches have disadvantages. The first one is incompatible with the methods of life history theory and basic probabilistic intuitions. The logistic type of suppression of per capita growth rate stops trajectories of selection when population size reaches the maximal value (carrying capacity); hence this method does not satisfy selective neutrality. To overcome these difficulties, we must explicitly consider turn-over of individuals dependent on mortality rate. This new approach leads to two interesting predictions. First, the equilibrium value of population size is lower than carrying capacity and depends on the mortality rate. Second, although the phase portrait of selection trajectories is the same as in density-independent replicator dynamics, pace of selection slows down when population size approaches equilibrium, and then remains constant and dependent on the rate of turn-over of individuals.

Replication and Immunogenicity of Swine, Equine, and Avian H3 Subtype Influenza Viruses in Mice and Ferrets

PubMed Central

Baz, Mariana; Paskel, Myeisha; Matsuoka, Yumiko; Zengel, James; Cheng, Xing; Jin, Hong

2013-01-01

Since it is difficult to predict which influenza virus subtype will cause an influenza pandemic, it is important to prepare influenza virus vaccines against different subtypes and evaluate the safety and immunogenicity of candidate vaccines in preclinical and clinical studies prior to a pandemic. In addition to infecting humans, H3 influenza viruses commonly infect pigs, horses, and avian species. We selected 11 swine, equine, and avian H3 influenza viruses and evaluated their kinetics of replication and ability to induce a broadly cross-reactive antibody response in mice and ferrets. The swine and equine viruses replicated well in the upper respiratory tract of mice. With the exception of one avian virus that replicated poorly in the lower respiratory tract, all of the viruses replicated in mouse lungs. In ferrets, all of the viruses replicated well in the upper respiratory tract, but the equine viruses replicated poorly in the lungs. Extrapulmonary spread was not observed in either mice or ferrets. No single virus elicited antibodies that cross-reacted with viruses from all three animal sources. Avian and equine H3 viruses elicited broadly cross-reactive antibodies against heterologous viruses isolated from the same or other species, but the swine viruses did not. We selected an equine and an avian H3 influenza virus for further development as vaccines. PMID:23576512

The selective processing of emotional visual stimuli while detecting auditory targets: an ERP analysis.

PubMed

Schupp, Harald T; Stockburger, Jessica; Bublatzky, Florian; Junghöfer, Markus; Weike, Almut I; Hamm, Alfons O

2008-09-16

Event-related potential studies revealed an early posterior negativity (EPN) for emotional compared to neutral pictures. Exploring the emotion-attention relationship, a previous study observed that a primary visual discrimination task interfered with the emotional modulation of the EPN component. To specify the locus of interference, the present study assessed the fate of selective visual emotion processing while attention is directed towards the auditory modality. While simply viewing a rapid and continuous stream of pleasant, neutral, and unpleasant pictures in one experimental condition, processing demands of a concurrent auditory target discrimination task were systematically varied in three further experimental conditions. Participants successfully performed the auditory task as revealed by behavioral performance and selected event-related potential components. Replicating previous results, emotional pictures were associated with a larger posterior negativity compared to neutral pictures. Of main interest, increasing demands of the auditory task did not modulate the selective processing of emotional visual stimuli. With regard to the locus of interference, selective emotion processing as indexed by the EPN does not seem to reflect shared processing resources of visual and auditory modality.

Molecular flip–flops formed by overlapping Fis sites

PubMed Central

Hengen, Paul N.; Lyakhov, Ilya G.; Stewart, Lisa E.; Schneider, Thomas D.

2003-01-01

The DNA-binding protein Fis frequently uses pairs of sites 7 or 11 base pairs (bp) apart. Two overlapping Fis sites separated by 11 bp are found in the Escherichia coli origin of chromosomal replication. Only one of these sites is bound by Fis at a time, so the structure is a molecular flip–flop that could direct alternative firing of replication complexes in opposite directions. Alternatively, the flip–flop could represent part of an on–off switch for replication. Because they can be used to create precise switched states, molecular flip–flops could be used as the basis of a novel molecular computer. PMID:14602927

Molecular flip-flops formed by overlapping Fis sites.

PubMed

Hengen, Paul N; Lyakhov, Ilya G; Stewart, Lisa E; Schneider, Thomas D

2003-11-15

The DNA-binding protein Fis frequently uses pairs of sites 7 or 11 base pairs (bp) apart. Two overlapping Fis sites separated by 11 bp are found in the Escherichia coli origin of chromosomal replication. Only one of these sites is bound by Fis at a time, so the structure is a molecular flip-flop that could direct alternative firing of replication complexes in opposite directions. Alternatively, the flip-flop could represent part of an on-off switch for replication. Because they can be used to create precise switched states, molecular flip-flops could be used as the basis of a novel molecular computer.

Do males pay for sex? Sex-specific selection coefficients suggest not.

PubMed

Prokop, Zofia M; Prus, Monika A; Gaczorek, Tomasz S; Sychta, Karolina; Palka, Joanna K; Plesnar-Bielak, Agata; Skarboń, Magdalena

2017-03-01

Selection acting on males can reduce mutation load of sexual relative to asexual populations, thus mitigating the twofold cost of sex, provided that it seeks and destroys the same mutations as selection acting on females, but with higher efficiency. This could happen due to sexual selection-a potent evolutionary force that in most systems predominantly affects males. We used replicate populations of red flour beetles (Tribolium castaneum) to study sex-specific selection against deleterious mutations introduced with ionizing radiation. We found no evidence for selection being stronger in males than in females; in fact, we observed a nonsignificant trend in the opposite direction. This suggests that selection on males does not reduce mutation load below the level expected under the (hypothetical) scenario of asexual reproduction. Additionally, we employed a novel approach, based on a simple model, to quantify the relative contributions of sexual and offspring viability selection to the overall selection observed in males. We found them to be similar in magnitude; however, only the offspring viability component was statistically significant. In summary, we found no support for the hypothesis that selection on males in general, and sexual selection in particular, contributes to the evolutionary maintenance of sex. © 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.

Scoring schemes of palindrome clusters for more sensitive prediction of replication origins in herpesviruses

PubMed Central

Chew, David S. H.; Choi, Kwok Pui; Leung, Ming-Ying

2005-01-01

Many empirical studies show that there are unusual clusters of palindromes, closely spaced direct and inverted repeats around the replication origins of herpesviruses. In this paper, we introduce two new scoring schemes to quantify the spatial abundance of palindromes in a genomic sequence. Based on these scoring schemes, a computational method to predict the locations of replication origins is developed. When our predictions are compared with 39 known or annotated replication origins in 19 herpesviruses, close to 80% of the replication origins are located within 2% of the genome length. A list of predicted locations of replication origins in all the known herpesviruses with complete genome sequences is reported. PMID:16141192

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

A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells.

PubMed Central

Piechaczek, C; Fetzer, C; Baiker, A; Bode, J; Lipps, H J

1999-01-01

We have developed an episomal replicating expression vector in which the SV40 gene coding for the large T-antigen was replaced by chromosomal scaffold/matrix attached regions. Southern analysis as well as vector rescue experiments in CHO cells and in Escherichia coli demonstrate that the vector replicates episomally in CHO cells. It occurs in a very low copy number in the cells and is stably maintained over more than 100 generations without selection pressure. PMID:9862961

The Importance of Replication in Measurement Research: Using Curriculum-Based Measures with Postsecondary Students with Developmental Disabilities

ERIC Educational Resources Information Center

Hosp, John L.; Ford, Jeremy W.; Huddle, Sally M.; Hensley, Kiersten K.

2018-01-01

Replication is a foundation of the development of a knowledge base in an evidence-based field such as education. This study includes two direct replications of Hosp, Hensley, Huddle, and Ford which found evidence of criterion-related validity of curriculum-based measurement (CBM) for reading and mathematics with postsecondary students with…

Effect of Inhibition of Deoxyribonucleic Acid and Protein Synthesis on the Direction of Cell Wall Growth in Streptococcus faecalis

PubMed Central

Higgins, M. L.; Daneo-Moore, L.; Boothby, D.; Shockman, G. D.

1974-01-01

Selective inhibition of protein synthesis in Streptococcus faecalis (ATCC 9790) was accompanied by a rapid and severe inhibition of cell division and a reduction of enlargement of cellular surface area. Continued synthesis of cell wall polymers resulted in rapid thickening of the wall to an extent not seen in exponential-phase populations. Thus, the normal direction of wall growth was changed from a preferential feeding out of new wall surface to that of thickening existing cell surfaces. However, the overall manner in which the wall thickened, from nascent septa toward polar regions, was the same in both exponential-phase and inhibited populations. In contrast, selective inhibition of deoxyribonucleic acid (DNA) synthesis using mitomycin C was accompanied by an increase in cellular surface area and by division of about 80% of the cells in random populations. Little or no wall thickening was observed until the synthesis of macromolecules other than DNA was impaired and further cell division ceased. Concomitant inhibition of both DNA and protein synthesis inhibited cell division but permitted an increase in average cell volume. In such doubly inhibited cells, walls thickened less than in cells inhibited for protein synthesis only. On the basis of the results obtained, a model for cell surface enlargement and cell division is presented. The model proposes that: (i) each wall enlargement site is influenced by an individual chromosome replication cycle; (ii) during chromosome replication peripheral surface enlargement would be favored over thickening (or septation); (iii) a signal associated with chromosome termination would favor thickening (and septation) at the expense of surface enlargement; and (iv) a factor or signal related to protein synthesis would be required for one or more of the near terminal stages of cell division or cell separation, or both. Images PMID:4133352

Unveiling the mystery of mitochondrial DNA replication in yeasts.

PubMed

Chen, Xin Jie; Clark-Walker, George Desmond

2018-01-01

Conventional DNA replication is initiated from specific origins and requires the synthesis of RNA primers for both the leading and lagging strands. In contrast, the replication of yeast mitochondrial DNA is origin-independent. The replication of the leading strand is likely primed by recombinational structures and proceeded by a rolling circle mechanism. The coexistent linear and circular DNA conformers facilitate the recombination-based initiation. The replication of the lagging strand is poorly understood. Re-evaluation of published data suggests that the rolling circle may also provide structures for the synthesis of the lagging-strand by mechanisms such as template switching. Thus, the coupling of recombination with rolling circle replication and possibly, template switching, may have been selected as an economic replication mode to accommodate the reductive evolution of mitochondria. Such a replication mode spares the need for conventional replicative components, including those required for origin recognition/remodelling, RNA primer synthesis and lagging-strand processing. Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Constructor theory of life

PubMed Central

Marletto, Chiara

2015-01-01

Neo-Darwinian evolutionary theory explains how the appearance of purposive design in the adaptations of living organisms can have come about without their intentionally being designed. The explanation relies crucially on the possibility of certain physical processes: mainly, gene replication and natural selection. In this paper, I show that for those processes to be possible without the design of biological adaptations being encoded in the laws of physics, those laws must have certain other properties. The theory of what these properties are is not part of evolution theory proper, yet without it the neo-Darwinian theory does not fully achieve its purpose of explaining the appearance of design. To this end, I apply constructor theory's new mode of explanation to express exactly within physics the appearance of design, no-design laws, and the logic of self-reproduction and natural selection. I conclude that self-reproduction, replication and natural selection are possible under no-design laws, the only non-trivial condition being that they allow digital information to be physically instantiated. This has an exact characterization in the constructor theory of information. I also show that under no-design laws an accurate replicator requires the existence of a ‘vehicle’ constituting, together with the replicator, a self-reproducer. PMID:25589566

Response of Two Heat Shock Genes to Selection for Knockdown Heat Resistance in Drosophila Melanogaster

PubMed Central

McColl, G.; Hoffmann, A. A.; McKechnie, S. W.

1996-01-01

To identify genes involved in stress resistance and heat hardening, replicate lines of Drosophila melanogaster were selected for increased resistance to knockdown by a 39° heat stress. Two selective regimes were used, one with and one without prior hardening. Mean knockdown times were increased from ~5 min to >20 min after 18 generations. Initial realized heritabilities were as high as 10% for lines selected without hardening, and crosses between lines indicated simple additive gene effects for the selected phenotypes. To survey allelic variation and correlated selection responses in two candidate stress genes, hsr-omega and hsp68, we applied denaturing gradient gel electrophoresis to amplified DNA sequences from small regions of these genes. After eight generations of selection, allele frequencies at both loci showed correlated responses for selection following hardening, but not without hardening. The hardening process itself was associated with a hsp68 frequency change in the opposite direction to that associated with selection that followed hardening. These stress loci are closely linked on chromosome III, and the hardening selection established a disequilibrium, suggesting an epistatic effect on resistance. The data indicate that molecular variation in both hsr-omega and hsp68 contribute to natural heritable variation for hardened heat resistance. PMID:8844150

Insights into the Initiation of Eukaryotic DNA Replication.

PubMed

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

2015-01-01

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

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

Active RNA replication of hepatitis C virus downregulates CD81 expression.

PubMed

Ke, Po-Yuan; Chen, Steve S-L

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

Active RNA Replication of Hepatitis C Virus Downregulates CD81 Expression

PubMed Central

Ke, Po-Yuan; Chen, Steve S.-L.

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81. PMID:23349980

Selective Modification of Adenovirus Replication Can Be Achieved through Rational Mutagenesis of the Adenovirus Type 5 DNA Polymerase

PubMed Central

Capella, Cristina; Beltejar, Michael-John; Brown, Caitlin; Fong, Vincent; Daddacha, Waaqo; Kim, Baek

2012-01-01

Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates. PMID:22811532

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication.

PubMed

Herzig, Eytan; Voronin, Nickolay; Kucherenko, Nataly; Hizi, Amnon

2015-08-01

The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Quantifying Selection with Pool-Seq Time Series Data.

PubMed

Taus, Thomas; Futschik, Andreas; Schlötterer, Christian

2017-11-01

Allele frequency time series data constitute a powerful resource for unraveling mechanisms of adaptation, because the temporal dimension captures important information about evolutionary forces. In particular, Evolve and Resequence (E&R), the whole-genome sequencing of replicated experimentally evolving populations, is becoming increasingly popular. Based on computer simulations several studies proposed experimental parameters to optimize the identification of the selection targets. No such recommendations are available for the underlying parameters selection strength and dominance. Here, we introduce a highly accurate method to estimate selection parameters from replicated time series data, which is fast enough to be applied on a genome scale. Using this new method, we evaluate how experimental parameters can be optimized to obtain the most reliable estimates for selection parameters. We show that the effective population size (Ne) and the number of replicates have the largest impact. Because the number of time points and sequencing coverage had only a minor effect, we suggest that time series analysis is feasible without major increase in sequencing costs. We anticipate that time series analysis will become routine in E&R studies. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing.

PubMed

Senkevich, Tatiana G; Bruno, Daniel; Martens, Craig; Porcella, Stephen F; Wolf, Yuri I; Moss, Bernard

2015-09-01

Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNA-primed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.

ONYX-015: mechanisms of action and clinical potential of a replication-selective adenovirus

PubMed Central

Ries, S; Korn, W M

2002-01-01

Accumulated knowledge in the molecular processes of tumour development combined with the availability of genetically modified viruses resemble the basis for new promising cancer therapeutics. The main advantages of employing replication-competent viruses are achievement of tumour selective killing and amplification of their oncolytic potential within the tumour mass. In this review, we describe the development of ONYX-015, one of the first and most advanced replication-competent viruses for cancer therapy. We discuss the molecular biology of this therapeutic approach and the interesting results obtained with this virus in clinical trials. British Journal of Cancer (2002) 86, 5–11. DOI: 10.1038/sj/bjc/6600006 www.bjcancer.com © 2002 The Cancer Research Campaign PMID:11857003

The dynamics of genome replication using deep sequencing

PubMed Central

Müller, Carolin A.; Hawkins, Michelle; Retkute, Renata; Malla, Sunir; Wilson, Ray; Blythe, Martin J.; Nakato, Ryuichiro; Komata, Makiko; Shirahige, Katsuhiko; de Moura, Alessandro P.S.; Nieduszynski, Conrad A.

2014-01-01

Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology. PMID:24089142

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.

Identifying sites of replication initiation in yeast chromosomes: looking for origins in all the right places.

PubMed

van Brabant, A J; Hunt, S Y; Fangman, W L; Brewer, B J

1998-06-01

DNA fragments that contain an active origin of replication generate bubble-shaped replication intermediates with diverging forks. We describe two methods that use two-dimensional (2-D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes. The first method uses 2-D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2-D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.

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

Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

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

Ortega, Corrie; Anderson, Lindsey N.; Frando, Andrew

The transition between replication and non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenicity, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating, persistent populations is a priority for tuberculosis treatment, but only few drug targets in non-replicating Mtb are currently known. Here, we directly measure the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication by activity-based proteomics. We predict serine hydrolase activity for 78 proteins, including 27 proteins with previously unknown function, and identify 37 SHs that remain active even in the absence ofmore » replication, providing a set of candidate persistence targets. Non-replication was associated with large shifts in the activity of the majority of SHs. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.« less

Co-localization of polar replication fork barriers and rRNA transcription terminators in mouse rDNA.

PubMed

López-estraño, C; Schvartzman, J B; Krimer, D B; Hernández, P

1998-03-27

We investigated the replication of the region where transcription terminates in mouse rDNA. It contains a replication fork barrier (RFB) that behaves in a polar manner, arresting only replication forks moving in the direction opposite to transcription. This RFB consists of several closely spaced fork arrest sites that co-localize with the transcription terminator elements, known as Sal boxes. Sal boxes are the target for mTTF-I (murine transcription termination factor I). These results suggest that both termination of rRNA transcription and replication fork arrest may share cis-acting as well as trans-acting factors. Copyright 1998 Academic Press Limited.

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.

Suppression of the Escherichia coli dnaA46 mutation by changes in the activities of the pyruvate-acetate node links DNA replication regulation to central carbon metabolism.

PubMed

Tymecka-Mulik, Joanna; Boss, Lidia; Maciąg-Dorszyńska, Monika; Matias Rodrigues, João F; Gaffke, Lidia; Wosinski, Anna; Cech, Grzegorz M; Szalewska-Pałasz, Agnieszka; Węgrzyn, Grzegorz; Glinkowska, Monika

2017-01-01

To ensure faithful transmission of genetic material to progeny cells, DNA replication is tightly regulated, mainly at the initiation step. Escherichia coli cells regulate the frequency of initiation according to growth conditions. Results of the classical, as well as the latest studies, suggest that the DNA replication in E. coli starts at a predefined, constant cell volume per chromosome but the mechanisms coordinating DNA replication with cell growth are still not fully understood. Results of recent investigations have revealed a role of metabolic pathway proteins in the control of cell division and a direct link between metabolism and DNA replication has also been suggested both in Bacillus subtilis and E. coli cells. In this work we show that defects in the acetate overflow pathway suppress the temperature-sensitivity of a defective replication initiator-DnaA under acetogenic growth conditions. Transcriptomic and metabolic analyses imply that this suppression is correlated with pyruvate accumulation, resulting from alterations in the pyruvate dehydrogenase (PDH) activity. Consequently, deletion of genes encoding the pyruvate dehydrogenase subunits likewise resulted in suppression of the thermal-sensitive growth of the dnaA46 strain. We propose that the suppressor effect may be directly related to the PDH complex activity, providing a link between an enzyme of the central carbon metabolism and DNA replication.

Laser-induced heating integrated with a microfluidic platform for real-time DNA replication and detection

NASA Astrophysics Data System (ADS)

Hung, Min-Sheng; Ho, Chia-Chin; Chen, Chih-Pin

2016-08-01

This study developed a microfluidic platform for replicating and detecting DNA in real time by integrating a laser and a microfluidic device composed of polydimethylsiloxane. The design of the microchannels consisted of a laser-heating area and a detection area. An infrared laser was used as the heating source for DNA replication, and the laser power was adjusted to heat the solutions directly. In addition, strong biotin-avidin binding was used to capture and detect the replicated products. The biotin on one end was bound to avidin and anchored to the surface of the microchannels, whereas the biotin on the other end was bound to the quantum dots (Qdots). The results showed that the fluorescent intensity of the Qdots bound to the replicated products in the detection area increased with the number of thermal cycles created by the laser. When the number of thermal cycles was ≥10, the fluorescent intensity of the Qdots was directly detectable on the surface of the microchannels. The proposed method is more sensitive than detection methods entailing gel electrophoresis.

High-affinity DNA-binding Domains of Replication Protein A (RPA) Direct SMARCAL1-dependent Replication Fork Remodeling*

PubMed Central

Bhat, Kamakoti P.; Bétous, Rémy; Cortez, David

2015-01-01

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. PMID:25552480

High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.

PubMed

Bhat, Kamakoti P; Bétous, Rémy; Cortez, David

2015-02-13

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Studies in Historical Replication in Psychology I: Introduction

NASA Astrophysics Data System (ADS)

Tweney, Ryan D.

2008-05-01

This special issue reports a project in which the replication of historically meaningful studies was carried out by graduate students in a history of psychology course. In this introduction, I outline the nature of the project and its rationale, and briefly sketch the results. The subsequent five papers represent scholarly presentations of five selected replications written by students in the course. These are followed by a commentary on the project by an educational psychologist.

Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication.

PubMed

Feng, Wenyi; Collingwood, David; Boeck, Max E; Fox, Lindsay A; Alvino, Gina M; Fangman, Walton L; Raghuraman, Mosur K; Brewer, Bonita J

2006-02-01

During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.

Phagemid vectors for phage display: properties, characteristics and construction.

PubMed

Qi, Huan; Lu, Haiqin; Qiu, Hua-Ji; Petrenko, Valery; Liu, Aihua

2012-03-30

Phagemids are filamentous-phage-derived vectors containing the replication origin of a plasmid. Phagemids usually encode no or only one kind of coat proteins. Other structural and functional proteins necessary to accomplish the life cycle of phagemid are provided by the helper phage. In addition, other elements such as molecular tags and selective markers are introduced into the phagemids to facilitate the subsequent operations, such as gene manipulation and protein purification. This review summarizes the elements of the phagemids and their corresponding functions. Finally, the possible trends and future direction to improve the characteristics of the phagemids are highlighted. Copyright © 2012 Elsevier Ltd. All rights reserved.

GENDER DIFFERENCES IN PERCEPTIONS OF SEXUAL INTENT: A QUALITATIVE REVIEW AND INTEGRATION

PubMed Central

Lindgren, Kristen P.; Parkhill, Michele R.; George, William H.; Hendershot, Christian S.

2009-01-01

Men appear to interpret people’s behaviors more sexually than do women. This finding, which has been replicated in scores of studies using a variety of methodological approaches, has been linked to important social concerns, including sexual assault and sexual harassment. This article provides a critical review of the published literature on gender differences in sexual intent perception, using selective examples to illustrate and summarize the field’s major constructs, methodologies, and empirical findings. Theoretical explanations for gender differences in sexual intent perceptions are reviewed. Finally, we highlight the field’s remaining issues and make several recommendations for future research directions. PMID:19763282

DNA replication origins—where do we begin?

PubMed Central

Prioleau, Marie-Noëlle; MacAlpine, David M.

2016-01-01

For more than three decades, investigators have sought to identify the precise locations where DNA replication initiates in mammalian genomes. The development of molecular and biochemical approaches to identify start sites of DNA replication (origins) based on the presence of defining and characteristic replication intermediates at specific loci led to the identification of only a handful of mammalian replication origins. The limited number of identified origins prevented a comprehensive and exhaustive search for conserved genomic features that were capable of specifying origins of DNA replication. More recently, the adaptation of origin-mapping assays to genome-wide approaches has led to the identification of tens of thousands of replication origins throughout mammalian genomes, providing an unprecedented opportunity to identify both genetic and epigenetic features that define and regulate their distribution and utilization. Here we summarize recent advances in our understanding of how primary sequence, chromatin environment, and nuclear architecture contribute to the dynamic selection and activation of replication origins across diverse cell types and developmental stages. PMID:27542827

Gender Bias in Leader Selection.

ERIC Educational Resources Information Center

Teaching of Psychology, 1995

1995-01-01

Describes a classroom exercise showing students how stereotypes can result in sex-biased leader selection. Finds that task-oriented competitive instructions produce a disproportionate number of selected male leaders. Includes procedures for replicating and evaluating the exercise. (CFR)

Event-based Plausibility Immediately Influences On-line Language Comprehension

PubMed Central

Matsuki, Kazunaga; Chow, Tracy; Hare, Mary; Elman, Jeffrey L.; Scheepers, Christoph; McRae, Ken

2011-01-01

In some theories of sentence comprehension, linguistically-relevant lexical knowledge such as selectional restrictions is privileged in terms of the time-course of its access and influence. We examined whether event knowledge computed by combining multiple concepts can rapidly influence language understanding even in the absence of selectional restriction violations. Specifically, we investigated whether instruments can combine with actions to influence comprehension of ensuing patients. Instrument-verb-patient triplets were created in a norming study designed to tap directly into event knowledge. In self-paced reading (Experiment 1), participants were faster to read patient nouns such as hair when they were typical of the instrument-action pair (Donna used the shampoo to wash vs. the hose to wash). Experiment 2 showed that these results were not due to direct instrument-patient relations. Experiment 3 replicated Experiment 1 using eyetracking, with effects of event typicality observed in first fixation and gaze durations on the patient noun. This research demonstrates that conceptual event-based expectations are computed and used rapidly and dynamically during on-line language comprehension. We discuss relationships among plausibility and predictability, as well as their implications. We conclude that selectional restrictions may be best considered as event-based conceptual knowledge, rather than lexical-grammatical knowledge. PMID:21517222

Exogenous selection rather than cytonuclear incompatibilities shapes asymmetrical fitness of reciprocal Arabidopsis hybrids.

PubMed

Muir, Graham; Ruiz-Duarte, Paola; Hohmann, Nora; Mable, Barbara K; Novikova, Polina; Schmickl, Roswitha; Guggisberg, Alessia; Koch, Marcus A

2015-04-01

Reciprocal crosses between species often display an asymmetry in the fitness of F1 hybrids. This pattern, referred to as isolation asymmetry or Darwin's corollary to Haldane's rule, is a general feature of reproductive isolation in plants, yet factors determining its magnitude and direction remain unclear. We evaluated reciprocal species crosses between two naturally hybridizing diploid species of Arabidopsis to assess the degree of isolation asymmetry at different postmating life stages. We found that pollen from Arabidopsis arenosa will usually fertilize ovules from Arabidopsis lyrata; the reverse receptivity being less complete. Maternal A. lyrata parents set more F1 hybrid seed, but germinate at lower frequency, reversing the asymmetry. As predicted by theory, A. lyrata (the maternal parent with lower seed viability in crosses) exhibited accelerated chloroplast evolution, indicating that cytonuclear incompatibilities may play a role in reproductive isolation. However, this direction of asymmetrical reproductive isolation is not replicated in natural suture zones, where delayed hybrid breakdown of fertility at later developmental stages, or later-acting selection against A. arenosa maternal hybrids (unrelated to hybrid fertility, e.g., substrate adaptation) may be responsible for an excess of A. lyrata maternal hybrids. Exogenous selection rather than cytonuclear incompatibilities thus shapes the asymmetrical postmating isolation in nature.

Resistance and cross-resistance profile of the diaryltriazine NNRTI and candidate microbicide UAMC01398.

PubMed

Ariën, Kevin K; Venkatraj, Muthusamy; Michiels, Johan; Joossens, Jurgen; Vereecken, Katleen; Van der Veken, Pieter; Heeres, Jan; De Winter, Hans; Heyndrickx, Leo; Augustyns, Koen; Vanham, Guido

2016-05-01

The resistance development, cross-resistance to other NNRTIs and the impact of resistance on viral replicative fitness were studied for the new and potent NNRTI UAMC01398. Resistance was selected by dose escalation and by single high-dose selection against a comprehensive panel of NNRTIs used as therapeutics and NNRTIs under investigation for pre-exposure prophylaxis of sexual HIV transmission. A panel of 27 site-directed mutants with single mutations or combinations of mutations involved in reverse transcriptase (RT) inhibitor-mediated resistance was developed and used to confirm resistance to UAMC01398. Cross-resistance to other NNRTIs was assessed, as well as susceptibility of UAMC01398-resistant HIV to diarylpyrimidine-resistant viruses. Finally, the impact of UAMC01398 resistance on HIV replicative fitness was studied. We showed that UAMC01398 has potent activity against dapivirine-resistant HIV, that at least four mutations in the RT are required in concert for resistance and that the resistance profile is similar to rilpivirine, both genotypically and phenotypically. Resistance development to UAMC01398 is associated with a severe fitness cost. These data, together with the enhanced safety profile and good solubility in aqueous gels, make UAMC01398 an excellent candidate for HIV topical prevention. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Genome-wide Scan of 29,141 African Americans Finds No Evidence of Directional Selection since Admixture

PubMed Central

Bhatia, Gaurav; Tandon, Arti; Patterson, Nick; Aldrich, Melinda C.; Ambrosone, Christine B.; Amos, Christopher; Bandera, Elisa V.; Berndt, Sonja I.; Bernstein, Leslie; Blot, William J.; Bock, Cathryn H.; Caporaso, Neil; Casey, Graham; Deming, Sandra L.; Diver, W. Ryan; Gapstur, Susan M.; Gillanders, Elizabeth M.; Harris, Curtis C.; Henderson, Brian E.; Ingles, Sue A.; Isaacs, William; De Jager, Phillip L.; John, Esther M.; Kittles, Rick A.; Larkin, Emma; McNeill, Lorna H.; Millikan, Robert C.; Murphy, Adam; Neslund-Dudas, Christine; Nyante, Sarah; Press, Michael F.; Rodriguez-Gil, Jorge L.; Rybicki, Benjamin A.; Schwartz, Ann G.; Signorello, Lisa B.; Spitz, Margaret; Strom, Sara S.; Tucker, Margaret A.; Wiencke, John K.; Witte, John S.; Wu, Xifeng; Yamamura, Yuko; Zanetti, Krista A.; Zheng, Wei; Ziegler, Regina G.; Chanock, Stephen J.; Haiman, Christopher A.; Reich, David; Price, Alkes L.

2014-01-01

The extent of recent selection in admixed populations is currently an unresolved question. We scanned the genomes of 29,141 African Americans and failed to find any genome-wide-significant deviations in local ancestry, indicating no evidence of selection influencing ancestry after admixture. A recent analysis of data from 1,890 African Americans reported that there was evidence of selection in African Americans after their ancestors left Africa, both before and after admixture. Selection after admixture was reported on the basis of deviations in local ancestry, and selection before admixture was reported on the basis of allele-frequency differences between African Americans and African populations. The local-ancestry deviations reported by the previous study did not replicate in our very large sample, and we show that such deviations were expected purely by chance, given the number of hypotheses tested. We further show that the previous study’s conclusion of selection in African Americans before admixture is also subject to doubt. This is because the FST statistics they used were inflated and because true signals of unusual allele-frequency differences between African Americans and African populations would be best explained by selection that occurred in Africa prior to migration to the Americas. PMID:25242497

Functional impairment of cytomegalovirus specific CD8 T cells predicts high-level replication after renal transplantation.

PubMed

Mattes, F M; Vargas, A; Kopycinski, J; Hainsworth, E G; Sweny, P; Nebbia, G; Bazeos, A; Lowdell, M; Klenerman, P; Phillips, R E; Griffiths, P D; Emery, V C

2008-05-01

Human cytomegalovirus (HCMV) remains an important cause of morbidity after allotransplantation, causing a range of direct effects including hepatitis, pneumonitis, enteritis and retinitis. A dominant risk factor for HCMV disease is high level viral replication in blood but it remains unexplained why only a subset of patients develop such diseases. In this detailed study of 25 renal transplant recipients, we show that functional impairment of HCMV specific CD8 T cells in the production of interferon gamma was associated with a 14-fold increased risk of progression to high level replication. The CD8 T-cell impairment persisted during the period of high level replication and was more prominent in patients above 40 years of age (odds ratio = 1.37, p = 0.01) and was also evident in dialysis patients. Threshold levels of functional impairment were associated with an increased risk of future HCMV replication and there was a direct relationship between the functional capacity of HCMV ppUL83 CD8 T cells and HCMV load (R(2)= 0.83). These results help to explain why a subset of seropositive individuals develop HCMV replication and are at risk of end-organ disease and may facilitate the early identification of individuals who would benefit from targeted anti-HCMV therapy after renal transplantation.

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

Genome complexity, robustness and genetic interactions in digital organisms

NASA Astrophysics Data System (ADS)

Lenski, Richard E.; Ofria, Charles; Collier, Travis C.; Adami, Christoph

1999-08-01

Digital organisms are computer programs that self-replicate, mutate and adapt by natural selection. They offer an opportunity to test generalizations about living systems that may extend beyond the organic life that biologists usually study. Here we have generated two classes of digital organism: simple programs selected solely for rapid replication, and complex programs selected to perform mathematical operations that accelerate replication through a set of defined `metabolic' rewards. To examine the differences in their genetic architecture, we introduced millions of single and multiple mutations into each organism and measured the effects on the organism's fitness. The complex organisms are more robust than the simple ones with respect to the average effects of single mutations. Interactions among mutations are common and usually yield higher fitness than predicted from the component mutations assuming multiplicative effects; such interactions are especially important in the complex organisms. Frequent interactions among mutations have also been seen in bacteria, fungi and fruitflies. Our findings support the view that interactions are a general feature of genetic systems.

Genome complexity, robustness and genetic interactions in digital organisms.

PubMed

Lenski, R E; Ofria, C; Collier, T C; Adami, C

1999-08-12

Digital organisms are computer programs that self-replicate, mutate and adapt by natural selection. They offer an opportunity to test generalizations about living systems that may extend beyond the organic life that biologists usually study. Here we have generated two classes of digital organism: simple programs selected solely for rapid replication, and complex programs selected to perform mathematical operations that accelerate replication through a set of defined 'metabolic' rewards. To examine the differences in their genetic architecture, we introduced millions of single and multiple mutations into each organism and measured the effects on the organism's fitness. The complex organisms are more robust than the simple ones with respect to the average effects of single mutations. Interactions among mutations are common and usually yield higher fitness than predicted from the component mutations assuming multiplicative effects; such interactions are especially important in the complex organisms. Frequent interactions among mutations have also been seen in bacteria, fungi and fruitflies. Our findings support the view that interactions are a general feature of genetic systems.

Metacognitive evaluation in the avoidance of demand.

PubMed

Dunn, Timothy L; Lutes, David J C; Risko, Evan F

2016-09-01

In the current set of experiments our goal was to test the hypothesis that individuals avoid courses of action based on a kind of metacognitive evaluation of demand in a Demand Selection Task (DST). Individuals in Experiment 1 completed a DST utilizing visual stimuli known to yield a dissociation between performance and perceived demand. Patterns of demand avoidance followed that of perceived demand. Experiment 2 provided a replication of the aforementioned results, in addition to demonstrating a second dissociation between a peripheral physiological measure of demand (i.e., blink rates) and demand avoidance. Experiment 3 directly tested the assumption that individuals make use of a general metacognitive evaluation of task demand during selections. A DST was utilized in a forced-choice paradigm that required individuals to either select the most effortful, time demanding, or least accurate of 2 choices. Patterns of selections were similar across all rating dimensions, lending credit to this notion. Findings are discussed within a metacognitive framework of demand avoidance and contrasted to current theories. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

Action video games and improved attentional control: Disentangling selection- and response-based processes.

PubMed

Chisholm, Joseph D; Kingstone, Alan

2015-10-01

Research has demonstrated that experience with action video games is associated with improvements in a host of cognitive tasks. Evidence from paradigms that assess aspects of attention has suggested that action video game players (AVGPs) possess greater control over the allocation of attentional resources than do non-video-game players (NVGPs). Using a compound search task that teased apart selection- and response-based processes (Duncan, 1985), we required participants to perform an oculomotor capture task in which they made saccades to a uniquely colored target (selection-based process) and then produced a manual directional response based on information within the target (response-based process). We replicated the finding that AVGPs are less susceptible to attentional distraction and, critically, revealed that AVGPs outperform NVGPs on both selection-based and response-based processes. These results not only are consistent with the improved-attentional-control account of AVGP benefits, but they suggest that the benefit of action video game playing extends across the full breadth of attention-mediated stimulus-response processes that impact human performance.

Dissociating the two faces of selective memory retrieval.

PubMed

Dobler, Ina M; Bäuml, Karl-Heinz T

2012-07-01

Research in the past four decades has repeatedly shown that selective retrieval of some (non-target) memories can impair subsequent retrieval of other (target) information, a finding known as retrieval-induced forgetting. More recently, however, there is evidence that selective retrieval can both impair and enhance recall of related memories (K-H. T. Bäuml & Samenieh, 2010). To identify possible experimental dissociations between the detrimental and the beneficial effects of memory retrieval, we examined retrieval dynamics in listwise directed forgetting, varying the delay between preceding non-target and subsequent target recall. When target recall immediately followed non-target recall, we replicated the prior work and found detrimental effects of memory retrieval on to-be-remembered items but beneficial effects on to-be-forgotten items. In contrast, when a delay was introduced between non-target and target recall, the detrimental effects were present but the beneficial effects were absent. The results demonstrate a first experimental dissociation between the two effects of memory retrieval. They are consistent with a recent two-factor account of the two faces of selective memory retrieval.

Human CST has independent functions during telomere duplex replication and C-strand fill-in

PubMed Central

Wang, Feng; Stewart, Jason A.; Kasbek, Christopher; Zhao, Yong; Wright, Woodring E.; Price, Carolyn M.

2012-01-01

Summary Human CST (CTC1-STN1-TEN1) is an RPA-like complex that is needed for efficient replication through the telomere duplex and genome-wide replication restart after fork stalling. Here we show that STN1/CST has a second function in telomere replication during G-overhang maturation. Analysis of overhang structure after STN1 depletion revealed normal kinetics for telomerase-mediated extension in S-phase but a delay in subsequent overhang shortening. This delay resulted from a defect in C-strand fill-in. Short telomeres exhibited the fill-in defect but normal telomere duplex replication, indicating that STN1/CST functions independently in these processes. Our work also indicates that the requirement for STN1/CST in telomere duplex replication correlates with increasing telomere length and replication stress. Our results provide the first direct evidence that STN1/CST participates in C-strand fill-in. They also demonstrate that STN1/CST participates in two mechanistically separate steps during telomere replication and identify CST as a novel replication factor that solves diverse replication-associated problems. PMID:23142664

Replication profile of Saccharomyces cerevisiae chromosome VI.

PubMed

Friedman, K L; Brewer, B J; Fangman, W L

1997-11-01

An understanding of the replication programme at the genome level will require the identification and characterization of origins of replication through large, contiguous regions of DNA. As a step toward this goal, origin efficiencies and replication times were determined for 10 ARSs spanning most of the 270 kilobase (kb) chromosome VI of Saccharomyces cerevisiae. Chromosome VI shows a wide variation in the percentage of cell cycles in which different replication origins are utilized. Most of the origins are activated in only a fraction of cells, suggesting that the pattern of origin usage on chromosome VI varies greatly within the cell population. The replication times of fragments containing chromosome VI origins show a temporal pattern that has been recognized on other chromosomes--the telomeres replicate late in S phase, while the central region of the chromosome replicates early. As demonstrated here for chromosome VI, analysis of the direction of replication fork movement along a chromosome and determination of replication time by measuring a period of hemimethylation may provide an efficient means of surveying origin activity over large regions of the genome.

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.

Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner.

PubMed

Loutre, Romuald; Heckel, Anne-Marie; Jeandard, Damien; Tarassov, Ivan; Entelis, Nina

2018-01-01

Mutations in mitochondrial DNA are an important source of severe and incurable human diseases. The vast majority of these mutations are heteroplasmic, meaning that mutant and wild-type genomes are present simultaneously in the same cell. Only a very high proportion of mutant mitochondrial DNA (heteroplasmy level) leads to pathological consequences. We previously demonstrated that mitochondrial targeting of small RNAs designed to anneal with mutant mtDNA can decrease the heteroplasmy level by specific inhibition of mutant mtDNA replication, thus representing a potential therapy. We have also shown that 5S ribosomal RNA, partially imported into human mitochondria, can be used as a vector to deliver anti-replicative oligoribonucleotides into human mitochondria. So far, the efficiency of cellular expression of recombinant 5S rRNA molecules bearing therapeutic insertions remained very low. In the present study, we designed new versions of anti-replicative recombinant 5S rRNA targeting a large deletion in mitochondrial DNA which causes the KSS syndrome, analyzed their specific annealing to KSS mitochondrial DNA and demonstrated their import into mitochondria of cultured human cells. To obtain an increased level of the recombinant 5S rRNA stable expression, we created transmitochondrial cybrid cell line bearing a site for Flp-recombinase and used this system for the recombinase-mediated integration of genes coding for the anti-replicative recombinant 5S rRNAs into nuclear genome. We demonstrated that stable expression of anti-replicative 5S rRNA versions in human transmitochondrial cybrid cells can induce a shift in heteroplasmy level of KSS mutation in mtDNA. This shift was directly dependent on the level of the recombinant 5S rRNA expression and the sequence of the anti-replicative insertion. Quantification of mtDNA copy number in transfected cells revealed the absence of a non-specific effect on wild type mtDNA replication, indicating that the decreased proportion between mutant and wild type mtDNA molecules is not a consequence of a random repopulation of depleted pool of mtDNA genomes. The heteroplasmy change could be also modulated by cell growth conditions, namely increased by cells culturing in a carbohydrate-free medium, thus forcing them to use oxidative phosphorylation and providing a selective advantage for cells with improved respiration capacities. We discuss the advantages and limitations of this approach and propose further development of the anti-replicative strategy based on the RNA import into human mitochondria.

Correlation and path coefficient analysis of quantitative characters in spine gourd (Momordica dioica Roxb.).

PubMed

Aliya, F; Begum, H; Reddy, M T; Sivaraj, N; Pandravada, S R; Narshimulu, G

2014-05-01

Fifty genotypes of spine gourd (Momordica dioica Roxb.) were evaluated in a randomized block design with two replications at the Vegetable Research Station, Rajendranagar, Hyderabad, Andhra Pradesh, India during kharif, 2012. Correlation and path coefficient analysis were carried out to study the character association and contribution, respectively for twelve quantitative characters namely vine length (m), number of stems per plant, days to first female flower appearance, first female flowering node, days to first fruit harvest, days to last fruit harvest, fruiting period (days), fruit length (cm), fruit width (cm), fruit weight (g), number of fruits per plant and fruit yield per plant (kg) for identification of the potential selection indices. Correlation and path coefficient analyses revealed that fruiting period and number of fruits per plant not only had positively significant correlation with fruit yield but also had positively high direct effect on it and are regarded as the main determinants of fruit yield. Days to first fruit harvest had positively moderate direct effect on fruit yield and its association was negatively significant, days to last fruit harvest had negatively high direct effect on fruit yield and its association was significant positively, hence restricted simultaneous selection can be made for days to first fruit harvest and days to last fruit harvest. The improvement in fruit yield can be effective if selection is based on days to first fruit harvest, days to last fruit harvest, fruiting period and number of fruits per plant.

CyDNA: Synthesis and Replication of Highly Cy-Dye Substituted DNA by an Evolved Polymerase

PubMed Central

2010-01-01

DNA not only transmits genetic information but can also serve as a versatile supramolecular scaffold. Here we describe a strategy for the synthesis and replication of DNA displaying hundreds of substituents using directed evolution of polymerase function by short-patch compartmentalized self-replication (spCSR) and the widely used fluorescent dye labeled deoxinucleotide triphosphates Cy3-dCTP and Cy5-dCTP as substrates. In just two rounds of spCSR selection, we have isolated a polymerase that allows the PCR amplification of double stranded DNA fragments up to 1kb, in which all dC bases are substituted by its fluorescent dye-labeled equivalent Cy3- or Cy5-dC. The resulting “CyDNA” displays hundreds of aromatic heterocycles on the outside of the DNA helix and is brightly colored and highly fluorescent. CyDNA also exhibits significantly altered physicochemical properties compared to standard B-form DNA, including loss of silica and intercalating dye binding, resistance to cleavage by some endonucleases, an up to 40% increased apparent diameter as judged by atomic force microscopy and organic phase partitioning during phenol extraction. CyDNA also displays very bright fluorescence enabling significant signal gains in microarray and microfluidic applications. CyDNA represents a step toward a long-term goal of the encoded synthesis of DNA-based polymers of programmable and evolvable sequence and properties. PMID:20235594

A Rapid Screen for Host-Encoded miRNAs with Inhibitory Effects against Ebola Virus Using a Transcription- and Replication-Competent Virus-Like Particle System.

PubMed

Wang, Zhongyi; Li, Jiaming; Fu, Yingying; Zhao, Zongzheng; Zhang, Chunmao; Li, Nan; Li, Jingjing; Cheng, Hongliang; Jin, Xiaojun; Lu, Bing; Guo, Zhendong; Qian, Jun; Liu, Linna

2018-05-16

MicroRNAs (miRNAs) may become efficient antiviral agents against the Ebola virus (EBOV) targeting viral genomic RNAs or transcripts. We previously conducted a genome-wide search for differentially expressed miRNAs during viral replication and transcription. In this study, we established a rapid screen for miRNAs with inhibitory effects against EBOV using a tetracistronic transcription- and replication-competent virus-like particle (trVLP) system. This system uses a minigenome comprising an EBOV leader region, luciferase reporter, VP40, GP, VP24, EBOV trailer region, and three noncoding regions from the EBOV genome and can be used to model the life cycle of EBOV under biosafety level (BSL) 2 conditions. Informatic analysis was performed to select up-regulated miRNAs targeting the coding regions of the minigenome with the highest binding energy to perform inhibitory effect screening. Among these miRNAs, miR-150-3p had the most significant inhibitory effect. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and double fluorescence reporter experiments demonstrated that miR-150-3p inhibited the reproduction of trVLPs via the regulation of GP and VP40 expression by directly targeting the coding regions of GP and VP40. This novel, rapid, and convenient screening method will efficiently facilitate the exploration of miRNAs against EBOV under BSL-2 conditions.

CyDNA: synthesis and replication of highly Cy-dye substituted DNA by an evolved polymerase.

PubMed

Ramsay, Nicola; Jemth, Ann-Sofie; Brown, Anthony; Crampton, Neal; Dear, Paul; Holliger, Philipp

2010-04-14

DNA not only transmits genetic information but can also serve as a versatile supramolecular scaffold. Here we describe a strategy for the synthesis and replication of DNA displaying hundreds of substituents using directed evolution of polymerase function by short-patch compartmentalized self-replication (spCSR) and the widely used fluorescent dye labeled deoxinucleotide triphosphates Cy3-dCTP and Cy5-dCTP as substrates. In just two rounds of spCSR selection, we have isolated a polymerase that allows the PCR amplification of double stranded DNA fragments up to 1kb, in which all dC bases are substituted by its fluorescent dye-labeled equivalent Cy3- or Cy5-dC. The resulting "CyDNA" displays hundreds of aromatic heterocycles on the outside of the DNA helix and is brightly colored and highly fluorescent. CyDNA also exhibits significantly altered physicochemical properties compared to standard B-form DNA, including loss of silica and intercalating dye binding, resistance to cleavage by some endonucleases, an up to 40% increased apparent diameter as judged by atomic force microscopy and organic phase partitioning during phenol extraction. CyDNA also displays very bright fluorescence enabling significant signal gains in microarray and microfluidic applications. CyDNA represents a step toward a long-term goal of the encoded synthesis of DNA-based polymers of programmable and evolvable sequence and properties.

Selfish drive can trump function when animal mitochondrial genomes compete.

PubMed

Ma, Hansong; O'Farrell, Patrick H

2016-07-01

Mitochondrial genomes compete for transmission from mother to progeny. We explored this competition by introducing a second genome into Drosophila melanogaster to follow transmission. Competitions between closely related genomes favored those functional in electron transport, resulting in a host-beneficial purifying selection. In contrast, matchups between distantly related genomes often favored those with negligible, negative or lethal consequences, indicating selfish selection. Exhibiting powerful selfish selection, a genome carrying a detrimental mutation displaced a complementing genome, leading to population death after several generations. In a different pairing, opposing selfish and purifying selection counterbalanced to give stable transmission of two genomes. Sequencing of recombinant mitochondrial genomes showed that the noncoding region, containing origins of replication, governs selfish transmission. Uniparental inheritance prevents encounters between distantly related genomes. Nonetheless, in each maternal lineage, constant competition among sibling genomes selects for super-replicators. We suggest that this relentless competition drives positive selection, promoting change in the sequences influencing transmission.

Selfish drive can trump function when animal mitochondrial genomes compete

PubMed Central

Ma, Hansong; O’Farrell, Patrick H.

2016-01-01

Mitochondrial genomes compete for transmission from mother to progeny. We explored this competition by introducing a second genome into Drosophila melanogaster to follow transmission. Competitions between closely related genomes favored those functional in electron transport, resulting in a host-beneficial purifying selection1. Contrastingly, matchups between distant genomes often favored those with negligible, negative or lethal consequences, indicating selfish selection. Exhibiting powerful selfish selection, a genome carrying a detrimental mutation displaced a complementing genome leading to population death after several generations. In a different pairing, opposing selfish and purifying selection counterbalanced to give stable transmission of two genomes. Sequencing of recombinant mitochondrial genomes revealed that the non-coding region, containing origins of replication, governs selfish transmission. Uniparental inheritance prevents encounters between distantly related genomes. Nonetheless, within each maternal lineage, constant competition among sibling genomes selects for super-replicators. We suggest that this relentless competition drives positive selection promoting change in the sequences influencing transmission. PMID:27270106

Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm.

PubMed

Hernández-Pichardo, J E; Ducolomb, Y; Romo, S; Kjelland, M E; Fierro, R; Casillas, F; Betancourt, M

2016-01-01

In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up (SU) or swim up + zona pellucida (SU + ZP) binding. Experiment 1, 4-20 replicates with total 821 in vitro matured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation (precense of one PN). Treatments showed similar results (54, 47, 42 %, respectively) but statistically differents ( P  < 0.05) than mechanical activated oocytes in sham, ICSI and sham injection (13, 25, 32 %, respectively) (10-17 replicates; n  = 429). Experiment 2: Twelve ejaculates and 28 straws of semen were used (11-19 replicates). Sperm were selected by SU in BSA-TCM 199-H medium. A total of 2,294 fresh sperm and 2,760 from frozen-thawed semen were analyzed after SU or SU + ZP binding. Fresh sperm selected by SU showed acrosome reaction (AR) of 59 %, the sperm selected by SU + ZP binding increased AR to 91 %. In comparison, the AR of frozen-thawed sperm using SU or SU + ZP binding was 77 and 86 %, respectively ( P  < 0.05). Experiment 3: fertilization in 200 mechanical activativated oocytes (17 replicates) was 4 %, but fertilization increased in ethanol activated oocytes after ICSI (12-28 %) (5-6 replicates). When fresh sperm only selected by SU were injected to 123 oocytes, a fertilization rate (28 %) was achieved; in sperm selected by SU + ZP was 25 % (73 oocytes). In comparison, in frozen-thawed sperm selected by SU, fertilization was 13 % (70 oocytes), whereas sperm from SU + ZP binding displayed 12 % (51 oocytes) ( P  > 0.05). Chemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU + ZP increased AR/A and AR/D rates in comparison with SU in fresh and frozen-thawed sperm. According to this, in terms of fertilization rates, chemical activation after ICSI increased oocyte PN formation compared to mechanical activation. Also, fresh sperm treated with SU and SU + ZP were significantly different than frozen-thawed sperm, but between sperm treatments no significant differences were obtained.

Data entry and error embedding system

NASA Technical Reports Server (NTRS)

Woo, Daniel N. (Inventor); Woo, Jr., John (Inventor)

1998-01-01

A data entry and error embedding system in which, first, a document is bitmapped and recorded in a first memory. Then, it is displayed, and portions of it to be replicated by data entry are underlayed by a window, into which window replicated data is entered in location and size such that it is juxtaposed just below that which is replicated, enhancing the accuracy of replication. Second, with this format in place, selected portions of the replicated data are altered by the insertion of character or word substitutions, thus the embedding of errors. Finally, a proofreader would endeavor to correct the error embedded data and a record of his or her changes recorded. In this manner, the skill level of the proofreader and accuracy of the data are computed.

The Function of Herpes Simplex Virus Genes: A Primer for Genetic Engineering of Novel Vectors

NASA Astrophysics Data System (ADS)

Roizman, Bernard

1996-10-01

Herpes simplex virus vectors are being developed for delivery and expression of human genes to the central nervous system, selective destruction of cancer cells, and as carriers for genes encoding antigens that induce protective immunity against infectious agents. Vectors constructed to meet these objectives must differ from wild-type virus with respect to host range, reactivation from latency, and expression of viral genes. The vectors currently being developed are (i) helper free amplicons, (ii) replication defective viruses, and (iii) genetically engineered replication competent viruses with restricted host range. Whereas the former two types of vectors require stable, continuous cell lines expressing viral genes for their replication, the replication competent viruses will replicate on approved primary human cell strains.

Conflict Resolution in the Genome: How Transcription and Replication Make It Work.

PubMed

Hamperl, Stephan; Cimprich, Karlene A

2016-12-01

The complex machineries involved in replication and transcription translocate along the same DNA template, often in opposing directions and at different rates. These processes routinely interfere with each other in prokaryotes, and mounting evidence now suggests that RNA polymerase complexes also encounter replication forks in higher eukaryotes. Indeed, cells rely on numerous mechanisms to avoid, tolerate, and resolve such transcription-replication conflicts, and the absence of these mechanisms can lead to catastrophic effects on genome stability and cell viability. In this article, we review the cellular responses to transcription-replication conflicts and highlight how these inevitable encounters shape the genome and impact diverse cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanisms of Hepatitis C Viral Resistance to Direct Acting Antivirals.

PubMed

Ahmed, Asma; Felmlee, Daniel J

2015-12-18

There has been a remarkable transformation in the treatment of chronic hepatitis C in recent years with the development of direct acting antiviral agents targeting virus encoded proteins important for viral replication including NS3/4A, NS5A and NS5B. These agents have shown high sustained viral response (SVR) rates of more than 90% in phase 2 and phase 3 clinical trials; however, this is slightly lower in real-life cohorts. Hepatitis C virus resistant variants are seen in most patients who do not achieve SVR due to selection and outgrowth of resistant hepatitis C virus variants within a given host. These resistance associated mutations depend on the class of direct-acting antiviral drugs used and also vary between hepatitis C virus genotypes and subtypes. The understanding of these mutations has a clear clinical implication in terms of choice and combination of drugs used. In this review, we describe mechanism of action of currently available drugs and summarize clinically relevant resistance data.

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.

Transient dominant host-range selection using Chinese hamster ovary cells to generate marker-free recombinant viral vectors from vaccinia virus.

PubMed

Liu, Liang; Cooper, Tamara; Eldi, Preethi; Garcia-Valtanen, Pablo; Diener, Kerrilyn R; Howley, Paul M; Hayball, John D

2017-04-01

Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Replicative ability is restored by co-insertion of the host-range genes, providing for dominant selection of the recombinant viruses. Here, we describe a new method for the construction of rVACVs using the cowpox CP77 protein and unmodified VACV as the starting material. Our selection system will expand the range of tools available for positive selection of rVACV during vector construction, and it is substantially more high-fidelity than approaches based on selection for drug resistance.

Targeting eukaryotic Rab proteins: a smart strategy for chlamydial survival and replication.

PubMed

Damiani, María Teresa; Gambarte Tudela, Julián; Capmany, Anahí

2014-09-01

Chlamydia, an obligate intracellular bacterium which passes its entire lifecycle within a membrane-bound vacuole called the inclusion, has evolved a variety of unique strategies to establish an advantageous intracellular niche for survival. This review highlights the mechanisms by which Chlamydia subverts vesicular transport in host cells, particularly by hijacking the master controllers of eukaryotic trafficking, the Rab proteins. A subset of Rabs and Rab interacting proteins that control the recycling pathway or the biosynthetic route are selectively recruited to the chlamydial inclusion membrane. By interfering with Rab-controlled transport steps, this intracellular pathogen not only prevents its own degradation in the phagocytic pathway, but also creates a favourable intracellular environment for growth and replication. Chlamydia, a highly adapted and successful intracellular pathogen, has several redundant strategies to re-direct vesicles emerging from biosynthetic compartments that carry host molecules essential for bacterial development. Although current knowledge is limited, the latest findings have shed light on the role of Rab proteins in the course of chlamydial infections and could open novel opportunities for anti-chlamydial therapy. © 2014 John Wiley & Sons Ltd.

Inhibition of adenovirus multiplication by short interfering RNAs directly or indirectly targeting the viral DNA replication machinery.

PubMed

Kneidinger, Doris; Ibrišimović, Mirza; Lion, Thomas; Klein, Reinhard

2012-06-01

Human adenoviruses are a common threat to immunocompromised patients, e.g., HIV-positive individuals or solid-organ and, in particular, allogeneic stem cell transplant recipients. Antiviral drugs have a limited effect on adenoviruses, and existing treatment modalities often fail to prevent fatal outcome. Silencing of viral genes by short interfering RNAs (siRNAs) holds a great promise in the treatment of viral infections. The aim of the present study was to identify adenoviral candidate targets for RNA interference-mediated inhibition of adenoviral replication. We investigated the impact of silencing of a set of early, middle, and late viral genes on the replication of adenovirus 5 in vitro. Adenovirus replication was inhibited by siRNAs directed against the adenoviral E1A, DNA polymerase, preterminal protein (pTP), IVa2, hexon, and protease genes. Silencing of early and middle genes was more effective in inhibiting adenovirus multiplication than was silencing of late genes. A siRNA directed against the viral DNA polymerase mRNA decreased viral genome copy numbers and infectious virus progeny by several orders of magnitude. Since silencing of any of the early genes directly or indirectly affected viral DNA synthesis, our data suggest that reducing viral genome copy numbers is a more promising strategy for the treatment of adenoviral infections than is reducing the numbers of proteins necessary for capsid generation. Thus, adenoviral DNA replication was identified as a key target for RNAi-mediated inhibition of adenovirus multiplication. In addition, the E1A transcripts emerged as a second important target, because its knockdown markedly improved the viability of cells at late stages of infection. Copyright © 2012 Elsevier B.V. All rights reserved.

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

Replication landscape of the human genome

PubMed Central

Petryk, Nataliya; Kahli, Malik; d'Aubenton-Carafa, Yves; Jaszczyszyn, Yan; Shen, Yimin; Silvain, Maud; Thermes, Claude; Chen, Chun-Long; Hyrien, Olivier

2016-01-01

Despite intense investigation, human replication origins and termini remain elusive. Existing data have shown strong discrepancies. Here we sequenced highly purified Okazaki fragments from two cell types and, for the first time, quantitated replication fork directionality and delineated initiation and termination zones genome-wide. Replication initiates stochastically, primarily within non-transcribed, broad (up to 150 kb) zones that often abut transcribed genes, and terminates dispersively between them. Replication fork progression is significantly co-oriented with the transcription. Initiation and termination zones are frequently contiguous, sometimes separated by regions of unidirectional replication. Initiation zones are enriched in open chromatin and enhancer marks, even when not flanked by genes, and often border ‘topologically associating domains' (TADs). Initiation zones are enriched in origin recognition complex (ORC)-binding sites and better align to origins previously mapped using bubble-trap than λ-exonuclease. This novel panorama of replication reveals how chromatin and transcription modulate the initiation process to create cell-type-specific replication programs. PMID:26751768

Effects of Hand Proximity and Movement Direction in Spatial and Temporal Gap Discrimination.

PubMed

Wiemers, Michael; Fischer, Martin H

2016-01-01

Previous research on the interplay between static manual postures and visual attention revealed enhanced visual selection near the hands (near-hand effect). During active movements there is also superior visual performance when moving toward compared to away from the stimulus (direction effect). The "modulated visual pathways" hypothesis argues that differential involvement of magno- and parvocellular visual processing streams causes the near-hand effect. The key finding supporting this hypothesis is an increase in temporal and a reduction in spatial processing in near-hand space (Gozli et al., 2012). Since this hypothesis has, so far, only been tested with static hand postures, we provide a conceptual replication of Gozli et al.'s (2012) result with moving hands, thus also probing the generality of the direction effect. Participants performed temporal or spatial gap discriminations while their right hand was moving below the display. In contrast to Gozli et al. (2012), temporal gap discrimination was superior at intermediate and not near hand proximity. In spatial gap discrimination, a direction effect without hand proximity effect suggests that pragmatic attentional maps overshadowed temporal/spatial processing biases for far/near-hand space.

Cytologic Effects of Air Force Chemicals

DTIC Science & Technology

1980-11-01

Studies of DNA replication and repair in cell cultures have shown that hydrazine, although highly toxic to cells, does not damage DNA and thus...interfere directly with DNA replication in Chinese hamster ovary cells grown in vitro, nor does it affect DNA repair synthesis in CCL-185 human lung cells...vitro with chemicals and monitoring their effect on DNA replication and repair. This method has been used to show that the alkylating agents MMS and 4

Biological species is the only possible form of existence for higher organisms: the evolutionary meaning of sexual reproduction

PubMed Central

2010-01-01

Consistent holistic view of sexual species as the highest form of biological existence is presented. The Weismann's idea that sex and recombination provide the variation for the natural selection to act upon is dominated in most discussions of the biological meaning of the sexual reproduction. Here, the idea is substantiated that the main advantage of sex is the opposite: the ability to counteract not only extinction but further evolution as well. Living systems live long owing to their ability to reproduce themselves with a high fidelity. Simple organisms (like bacteria) reach the continued existence due to the high fidelity of individual genome replication. In organisms with a large genome and complex development, the achievable fidelity of DNA replication is not enough for the precise reproduction of the genome. Such species must be capable of surviving and must remain unchanged in spite of the continuous changes of their genes. This problem has no solution in the frame of asexual ("homeogenomic") lineages. They would rapidly degrade and become extinct or blurred out in the course of the reckless evolution. The core outcome of the transition to sexual reproduction was the creation of multiorganismic entity - biological species. Individual organisms forfeited their ability to reproduce autonomously. It implies that individual organisms forfeited their ability to substantive evolution. They evolve as a part of the biological species. In case of obligatory sexuality, there is no such a thing as synchronic multi-level selection. Natural selection cannot select anything that is not a unit of reproduction. Hierarchy in biology implies the functional predestination of the parts for the sake of the whole. A crucial feature of the sexual reproduction is the formation of genomes of individual organisms by random picking them over from the continuously shuffled gene pool instead of the direct replication of the ancestor's genome. A clear anti-evolutionary consequence of the sexuality is evident from the fact that the genotypes of the individuals with an enhanced competitiveness are not transmitted to the next generation. Instead, after mating with "ordinary" individuals, these genotypes scatter and rearrange in new gene combinations, thus preventing the winner from exploiting the success. Reviewers This article was reviewed by Pierre Antoine Pontarotti, Michael T. Ghiselin (nominated by Dr. Juergen Brosius) and Emanuel Tannenboum (nominated by Dr. Doron Lancet) PMID:20307287

Antibody-mediated targeting of replication-competent retroviral vectors.

PubMed

Tai, Chien-Kuo; Logg, Christopher R; Park, Jinha M; Anderson, W French; Press, Michael F; Kasahara, Noriyuki

2003-05-20

Replication-competent murine leukemia virus (MLV) vectors can be engineered to achieve high efficiency gene transfer to solid tumors in vivo and tumor-restricted replication, however their safety can be further enhanced by redirecting tropism of the virus envelope. We have therefore tested the targeting capability and replicative stability of ecotropic and amphotropic replication-competent retrovirus (RCR) vectors containing two tandem repeats from the immunoglobulin G-binding domain of Staphylococcal protein A inserted into the proline-rich "hinge" region of the envelope, which enables modular use of antibodies of various specificities for vector targeting. The modified envelopes were efficiently expressed and incorporated into virions, were capable of capturing monoclonal anti-HER2 antibodies, and mediated efficient binding of the virus-antibody complex to HER2-positive target cells. While infectivity was markedly reduced by pseudotyping with targeted envelopes alone, coexpression of wild-type envelope rescued efficient cellular entry. Both ecotropic and amphotropic RCR vector/anti-HER2 antibody complexes achieved significant enhancement of transduction on murine target cells overexpressing HER2, which could be competed by preincubation with excess free antibodies. Interestingly, HER2-expressing human breast cancer cells did not show enhancement of transduction despite efficient antibody-mediated cell surface binding, suggesting that target cell-specific parameters markedly affect the efficiency of post-binding entry processes. Serial replication of targeted vectors resulted in selection of Z domain deletion variants, but reduction of the overall size of the vector genome enhanced its stability. Application of antibody-mediated targeting to the initial localization of replication-competent virus vectors to tumor sites will thus require optimized target selection and vector design.

Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR.

PubMed

Jahn, Michael; Vorpahl, Carsten; Hübschmann, Thomas; Harms, Hauke; Müller, Susann

2016-12-19

Plasmids are widely used for molecular cloning or production of proteins in laboratory and industrial settings. Constant modification has brought forth countless plasmid vectors whose characteristics in terms of average plasmid copy number (PCN) and stability are rarely known. The crucial factor determining the PCN is the replication system; most replication systems in use today belong to a small number of different classes and are available through repositories like the Standard European Vector Architecture (SEVA). In this study, the PCN was determined in a set of seven SEVA-based expression plasmids only differing in the replication system. The average PCN for all constructs was determined by Droplet Digital PCR and ranged between 2 and 40 per chromosome in the host organism Escherichia coli. Furthermore, a plasmid-encoded EGFP reporter protein served as a means to assess variability in reporter gene expression on the single cell level. Only cells with one type of plasmid (RSF1010 replication system) showed a high degree of heterogeneity with a clear bimodal distribution of EGFP intensity while the others showed a normal distribution. The heterogeneous RSF1010-carrying cell population and one normally distributed population (ColE1 replication system) were further analyzed by sorting cells of sub-populations selected according to EGFP intensity. For both plasmids, low and highly fluorescent sub-populations showed a remarkable difference in PCN, ranging from 9.2 to 123.4 for ColE1 and from 0.5 to 11.8 for RSF1010, respectively. The average PCN determined here for a set of standardized plasmids was generally at the lower end of previously reported ranges and not related to the degree of heterogeneity. Further characterization of a heterogeneous and a homogeneous population demonstrated considerable differences in the PCN of sub-populations. We therefore present direct molecular evidence that the average PCN does not represent the true number of plasmid molecules in individual cells.

Methods for sampling geographically mobile female traders in an East African market setting

PubMed Central

Achiro, Lillian; Kwena, Zachary A.; McFarland, Willi; Neilands, Torsten B.; Cohen, Craig R.; Bukusi, Elizabeth A.; Camlin, Carol S.

2018-01-01

Background The role of migration in the spread of HIV in sub-Saharan Africa is well-documented. Yet migration and HIV research have often focused on HIV risks to male migrants and their partners, or migrants overall, often failing to measure the risks to women via their direct involvement in migration. Inconsistent measures of mobility, gender biases in those measures, and limited data sources for sex-specific population-based estimates of mobility have contributed to a paucity of research on the HIV prevention and care needs of migrant and highly mobile women. This study addresses an urgent need for novel methods for developing probability-based, systematic samples of highly mobile women, focusing on a population of female traders operating out of one of the largest open air markets in East Africa. Our method involves three stages: 1.) identification and mapping of all market stall locations using Global Positioning System (GPS) coordinates; 2.) using female market vendor stall GPS coordinates to build the sampling frame using replicates; and 3.) using maps and GPS data for recruitment of study participants. Results The location of 6,390 vendor stalls were mapped using GPS. Of these, 4,064 stalls occupied by women (63.6%) were used to draw four replicates of 128 stalls each, and a fifth replicate of 15 pre-selected random alternates for a total of 527 stalls assigned to one of five replicates. Staff visited 323 stalls from the first three replicates and from these successfully recruited 306 female vendors into the study for a participation rate of 94.7%. Mobilization strategies and involving traders association representatives in participant recruitment were critical to the study’s success. Conclusion The study’s high participation rate suggests that this geospatial sampling method holds promise for development of probability-based samples in other settings that serve as transport hubs for highly mobile populations. PMID:29324780

Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle

PubMed Central

Serre, Stéphanie B. N.; Jensen, Sanne B.; Ghanem, Lubna; Humes, Daryl G.; Ramirez, Santseharay; Li, Yi-Ping; Krarup, Henrik; Bukh, Jens

2016-01-01

Hepatitis C virus (HCV) NS3 protease inhibitors (PIs) are important components of novel HCV therapy regimens. Studies of PI resistance initially focused on genotype 1. Therefore, knowledge about the determinants of PI resistance for the highly prevalent genotypes 2 to 6 remains limited. Using Huh7.5 cell culture-infectious HCV recombinants with genotype 1 to 6 NS3 protease, we identified protease positions 54, 155, and 156 as hot spots for the selection of resistance substitutions under treatment with the first licensed PIs, telaprevir and boceprevir. Treatment of a genotype 2 isolate with the newer PIs vaniprevir, faldaprevir, simeprevir, grazoprevir, paritaprevir, and deldeprevir identified positions 156 and 168 as hot spots for resistance; the Y56H substitution emerged for three newer PIs. Substitution selection also depended on the specific recombinant. The substitutions identified conferred cross-resistance to several PIs; however, most substitutions selected under telaprevir or boceprevir treatment conferred less resistance to certain newer PIs. In a single-cycle production assay, across genotypes, PI treatment primarily decreased viral replication, which was rescued by PI resistance substitutions. The substitutions identified resulted in differential effects on viral fitness, depending on the original recombinant and the substitution. Across genotypes, fitness impairment induced by resistance substitutions was due primarily to decreased replication. Most combinations of substitutions that were identified increased resistance or fitness. Combinations of resistance substitutions with fitness-compensating substitutions either rescued replication or compensated for decreased replication by increasing assembly. This comprehensive study provides insight into the selection patterns and effects of PI resistance substitutions for HCV genotypes 1 to 6 in the context of the infectious viral life cycle, which is of interest for clinical and virological HCV research. PMID:27021330

Inhibitors Selective for Mycobacterial Versus Human Proteasomes

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

Lin, G.; Li, D; Sorio de Carvalho, L

Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-onemore » compounds kill non-replicating M.?tuberculosis and act as selective suicide-substrate inhibitors of the M.?tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue.« less

Hormones and the Evolution of Complex Traits: Insights from Artificial Selection on Behavior

PubMed Central

Garland, Theodore; Zhao, Meng; Saltzman, Wendy

2016-01-01

Although behavior may often be a fairly direct target of natural or sexual selection, it cannot evolve without changes in subordinate traits that cause or permit its expression. In principle, changes in endocrine function could be a common mechanism underlying behavioral evolution because they are well positioned to mediate integrated responses to behavioral selection. More specifically, hormones can influence both motivational (e.g., brain) and performance (e.g., muscles) components of behavior simultaneously and in a coordinated fashion. If the endocrine system is often “used” as a general mechanism to effect responses to selection, then correlated responses in other aspects of behavior, life history, and organismal performance (e.g., locomotor abilities) should commonly occur because any cell with appropriate receptors could be affected. Ways in which behavior coadapts with other aspects of the phenotype can be studied directly through artificial selection and experimental evolution. Several studies have targeted rodent behavior for selective breeding and reported changes in other aspects of behavior, life history, and lower-level effectors of these organismal traits, including endocrine function. One example involves selection for high levels of voluntary wheel running, one aspect of physical activity, in four replicate High Runner (HR) lines of mice. Circulating levels of several hormones (including insulin, testosterone, thyroxine, triiodothyronine) have been characterized, three of which—corticosterone, leptin, and adiponectin—differ between HR and control lines, depending on sex, age, and generation. Potential changes in circulating levels of other behaviorally and metabolically relevant hormones, as well as in other components of the endocrine system (e.g., receptors), have yet to be examined. Overall, results to date identify promising avenues for further studies on the endocrine basis of activity levels. PMID:27252193

Direct and conceptual replications of the taxometric analysis of type a behavior.

PubMed

Wilmot, Michael P; Haslam, Nick; Tian, Jingyuan; Ones, Deniz S

2018-05-17

We present direct and conceptual replications of the influential taxometric analysis of Type A Behavior (TAB; Strube, 1989), which reported evidence for the latent typology of the construct. Study 1, the direct replication (N = 2,373), duplicated sampling and methodological procedures of the original study, but results showed that the item indicators used in the original study lacked sufficient validity to unambiguously determine latent structure. Using improved factorial subscale indicators to further test the question, multiple taxometric procedures, in combination with parallel analyses of simulated data, failed to replicate the original typological finding. Study 2, the conceptual replication, tested the latent structure of the wider construct of TAB using the sample from the Caerphilly Prospective Study (N = 2,254), which contains responses to the three most widely used self-report measures of TAB: the Jenkins Activity Survey, Bortner scale, and Framingham scale. Factorial subscale indicators were derived from the measures and submitted to multiple taxometric procedures. Results of Study 2 converged with those of Study 1, providing clear evidence of latent dimensional structure. Overall, results suggest there is no evidence for the type in TAB. Findings imply that theoretical models of TAB, assessment practices, and data analytic procedures that assume a typology should be replaced by dimensional models, factorial subscale measures, and corresponding statistical approaches. Specific subscale measures that tap multiple Big Five trait domains, and show evidence of predictive utility, are also recommended. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation.

PubMed

Lian, Hui-Yong; Robertson, E Douglas; Hiraga, Shin-ichiro; Alvino, Gina M; Collingwood, David; McCune, Heather J; Sridhar, Akila; Brewer, Bonita J; Raghuraman, M K; Donaldson, Anne D

2011-05-15

DNA replication in Saccharomyces cerevisiae proceeds according to a temporal program. We have investigated the role of the telomere-binding Ku complex in specifying late replication of telomere-proximal sequences. Genome-wide analysis shows that regions extending up to 80 kb from telomeres replicate abnormally early in a yku70 mutant. We find that Ku does not appear to regulate replication time by binding replication origins directly, nor is its effect on telomere replication timing mediated by histone tail acetylation. We show that Ku instead regulates replication timing through its effect on telomere length, because deletion of the telomerase regulator Pif1 largely reverses the short telomere defect of a yku70 mutant and simultaneously rescues its replication timing defect. Consistent with this conclusion, deleting the genome integrity component Elg1 partially rescued both length and replication timing of yku70 telomeres. Telomere length-mediated control of replication timing requires the TG(1-3) repeat-counting component Rif1, because a rif1 mutant replicates telomeric regions early, despite having extended TG(1-3) tracts. Overall, our results suggest that the effect of Ku on telomere replication timing results from its impact on TG(1-3) repeat length and support a model in which Rif1 measures telomere repeat length to ensure that telomere replication timing is correctly programmed.

Self-replicating systems: A systems engineering approach

NASA Technical Reports Server (NTRS)

Vontiesenhausen, G.; Darbro, W. A.

1980-01-01

A first approach to conceptualize self-replicating systems was developed from past and present abstract theories. The engineering elements of self-replicating systems are defined in terms of a basic reference system. A number of options are investigated. The growth characteristics and their problems are analyzed, the mathematics of various exponential growth options are outlined, and the problems of universal parts production and systems closure are discussed. Selected areas of further study are defined and a 20 year development and demonstration program is presented.

The hunt for origins of DNA replication in multicellular eukaryotes

PubMed Central

Urban, John M.; Foulk, Michael S.; Casella, Cinzia

2015-01-01

Origins of DNA replication (ORIs) occur at defined regions in the genome. Although DNA sequence defines the position of ORIs in budding yeast, the factors for ORI specification remain elusive in metazoa. Several methods have been used recently to map ORIs in metazoan genomes with the hope that features for ORI specification might emerge. These methods are reviewed here with analysis of their advantages and shortcomings. The various factors that may influence ORI selection for initiation of DNA replication are discussed. PMID:25926981

Analysis of DNA replication associated chromatin decondensation: in vivo assay for understanding chromatin remodeling mechanisms of selected proteins.

PubMed

Borysov, Sergiy; Bryant, Victoria L; Alexandrow, Mark G

2015-01-01

Of critical importance to many of the events underlying transcriptional control of gene expression are modifications to core and linker histones that regulate the accessibility of trans-acting factors to the DNA substrate within the context of chromatin. Likewise, control over the initiation of DNA replication, as well as the ability of the replication machinery to proceed during elongation through the multiple levels of chromatin condensation that are likely to be encountered, is known to involve the creation of chromatin accessibility. In the latter case, chromatin access will likely need to be a transient event so as to prevent total genomic unraveling of the chromatin that would be deleterious to cells. While there are many molecular and biochemical approaches in use to study histone changes and their relationship to transcription and chromatin accessibility, few techniques exist that allow a molecular dissection of the events underlying DNA replication control as it pertains to chromatin changes and accessibility. Here, we outline a novel experimental strategy for addressing the ability of specific proteins to induce large-scale chromatin unfolding (decondensation) in vivo upon site-specific targeting to an engineered locus. Our laboratory has used this powerful system in novel ways to directly address the ability of DNA replication proteins to create chromatin accessibility, and have incorporated modifications to the basic approach that allow for a molecular genetic analysis of the mechanisms and associated factors involved in causing chromatin decondensation by a protein of interest. Alternative approaches involving co-expression of other proteins (competitors or stimulators), concurrent drug treatments, and analysis of co-localizing histone modifications are also addressed, all of which are illustrative of the utility of this experimental system for extending basic findings to physiologically relevant mechanisms. Although used by our group to analyze mechanisms underlying DNA replication associated chromatin accessibility, this unique and powerful experimental system has the propensity to be a valuable tool for understanding chromatin remodeling mechanisms orchestrated by other cellular processes such as DNA repair, recombination, mitotic chromosome condensation, or other chromosome dynamics involving chromatin alterations and accessibility.

DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells

NASA Astrophysics Data System (ADS)

Sokol, Anna M.; Cruet-Hennequart, Séverine; Pasero, Philippe; Carty, Michael P.

2013-11-01

Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.

ATAD2 is an epigenetic reader of newly synthesized histone marks during DNA replication.

PubMed

Koo, Seong Joo; Fernández-Montalván, Amaury E; Badock, Volker; Ott, Christopher J; Holton, Simon J; von Ahsen, Oliver; Toedling, Joern; Vittori, Sarah; Bradner, James E; Gorjánácz, Mátyás

2016-10-25

ATAD2 (ATPase family AAA domain-containing protein 2) is a chromatin regulator harboring an AAA+ ATPase domain and a bromodomain, previously proposed to function as an oncogenic transcription co-factor. Here we suggest that ATAD2 is also required for DNA replication. ATAD2 is co-expressed with genes involved in DNA replication in various cancer types and predominantly expressed in S phase cells where it localized on nascent chromatin (replication sites). Our extensive biochemical and cellular analyses revealed that ATAD2 is recruited to replication sites through a direct interaction with di-acetylated histone H4 at K5 and K12, indicative of newly synthesized histones during replication-coupled chromatin reassembly. Similar to ATAD2-depletion, ectopic expression of ATAD2 mutants that are deficient in binding to these di-acetylation marks resulted in reduced DNA replication and impaired loading of PCNA onto chromatin, suggesting relevance of ATAD2 in DNA replication. Taken together, our data show a novel function of ATAD2 in cancer and for the first time identify a reader of newly synthesized histone di-acetylation-marks during replication.

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.

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

PubMed

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

2016-04-01

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

Resistance to pyridine-based inhibitor KF116 reveals an unexpected role of integrase in HIV-1 Gag-Pol polyprotein proteolytic processing.

PubMed

Hoyte, Ashley C; Jamin, Augusta V; Koneru, Pratibha C; Kobe, Matthew J; Larue, Ross C; Fuchs, James R; Engelman, Alan N; Kvaratskhelia, Mamuka

2017-12-01

The pyridine-based multimerization selective HIV-1 integrase (IN) inhibitors (MINIs) are a distinct subclass of allosteric IN inhibitors. MINIs potently inhibit HIV-1 replication during virion maturation by inducing hyper- or aberrant IN multimerization but are largely ineffective during the early steps of viral replication. Here, we investigated the mechanism for the evolution of a triple IN substitution (T124N/V165I/T174I) that emerges in cell culture with a representative MINI, KF116. We show that HIV-1 NL4-3(IN T124N/V165I/T174I) confers marked (>2000-fold) resistance to KF116. Two IN substitutions (T124N/T174I) directly weaken inhibitor binding at the dimer interface of the catalytic core domain but at the same time markedly impair HIV-1 replication capacity. Unexpectedly, T124N/T174I IN substitutions inhibited proteolytic processing of HIV-1 polyproteins Gag and Gag-Pol, resulting in immature virions. Strikingly, the addition of the third IN substitution (V165I) restored polyprotein processing, virus particle maturation, and significant levels of replication capacity. These results reveal an unanticipated role of IN for polyprotein proteolytic processing during virion morphogenesis. The complex evolutionary pathway for the emergence of resistant viruses, which includes the need for the compensatory V165I IN substitution, highlights a relatively high genetic barrier exerted by MINI KF116. Additionally, we have solved the X-ray structure of the drug-resistant catalytic core domain protein, which provides means for rational development of second-generation MINIs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3' untranslated region: function in replication and influence of RNA secondary structure.

PubMed

Gerresheim, Gesche K; Dünnes, Nadia; Nieder-Röhrmann, Anika; Shalamova, Lyudmila A; Fricke, Markus; Hofacker, Ivo; Höner Zu Siederdissen, Christian; Marz, Manja; Niepmann, Michael

2017-02-01

We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3' untranslated region (3'UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3'UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3'UTR target site inhibits translation directed by the HCV 5'UTR. Thus, the miR-122 target sites in the 3'-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.

DNA replication origins-where do we begin?

PubMed

Prioleau, Marie-Noëlle; MacAlpine, David M

2016-08-01

For more than three decades, investigators have sought to identify the precise locations where DNA replication initiates in mammalian genomes. The development of molecular and biochemical approaches to identify start sites of DNA replication (origins) based on the presence of defining and characteristic replication intermediates at specific loci led to the identification of only a handful of mammalian replication origins. The limited number of identified origins prevented a comprehensive and exhaustive search for conserved genomic features that were capable of specifying origins of DNA replication. More recently, the adaptation of origin-mapping assays to genome-wide approaches has led to the identification of tens of thousands of replication origins throughout mammalian genomes, providing an unprecedented opportunity to identify both genetic and epigenetic features that define and regulate their distribution and utilization. Here we summarize recent advances in our understanding of how primary sequence, chromatin environment, and nuclear architecture contribute to the dynamic selection and activation of replication origins across diverse cell types and developmental stages. © 2016 Prioleau and MacAlpine; Published by Cold Spring Harbor Laboratory Press.

Hydrodynamic Mediation of Killifish Predation on Infaunal Polychaetes

NASA Astrophysics Data System (ADS)

Hentschel, B. T.; Hayman, N. T.; Anderson, T. W.

2016-02-01

To explore predator-prey interactions between California killifish (Fundulus parvipinnis) and spionid polychaetes (Polydora cornuta and Streblospio benedicti) in tidal creeks, we conducted a laboratory flume experiment to quantify whether killifish prey-patch selectivity varies with flow speed. The flume included a 300-cm2 area of defaunated sediment within which we centrally positioned 24 P. cornuta, 24 S. benedicti, or no worms as a prey-free control. We videotaped groups of three killifish for 50 min at one of six unidirectional flow speeds (3, 6, 9, 12, 15, or 18 cm/s measured 1.5 cm above bottom) and recorded their bite rate anywhere in the sediment area vs. bites directed at the central prey patch (98 cm2). Each flow speed and prey treatment was replicated by four independent flume runs (i.e., 72 total flume runs). The percentage of bites directed at the central patch varied significantly with flow speed and worm presence. With defaunated sediment only, 33% of bites were directed at the central patch at all flow speeds, consistent with a null model of non-selective foraging. When either worm species inhabited the central patch, 65% of bites were directed at the central patch at 3 and 6 cm/s, and patch selectivity declined linearly as flow increased. Despite differences in morphology and behavior, the two prey species elicited similar foraging activity by killifish. We pooled the P. cornuta and S. benedicti treatments to determine the flow speed at which prey-patch selectivity by killifish became statistically indistinguishable from non-selective biting in the absence of prey. At flow speeds of 3, 6, and 9 cm/s, the percentage of bites located in the 98-cm2 patch was significantly greater when live worms were present compared to the prey-free controls. At 12, 15, and 18 cm/s, there was not a significant difference between the control patches and those containing live worms, indicating 12 cm/s is a flow threshold above which killifish cannot selectively forage on dense patches of infauna.

REML/BLUP and sequential path analysis in estimating genotypic values and interrelationships among simple maize grain yield-related traits.

PubMed

Olivoto, T; Nardino, M; Carvalho, I R; Follmann, D N; Ferrari, M; Szareski, V J; de Pelegrin, A J; de Souza, V Q

2017-03-22

Methodologies using restricted maximum likelihood/best linear unbiased prediction (REML/BLUP) in combination with sequential path analysis in maize are still limited in the literature. Therefore, the aims of this study were: i) to use REML/BLUP-based procedures in order to estimate variance components, genetic parameters, and genotypic values of simple maize hybrids, and ii) to fit stepwise regressions considering genotypic values to form a path diagram with multi-order predictors and minimum multicollinearity that explains the relationships of cause and effect among grain yield-related traits. Fifteen commercial simple maize hybrids were evaluated in multi-environment trials in a randomized complete block design with four replications. The environmental variance (78.80%) and genotype-vs-environment variance (20.83%) accounted for more than 99% of the phenotypic variance of grain yield, which difficult the direct selection of breeders for this trait. The sequential path analysis model allowed the selection of traits with high explanatory power and minimum multicollinearity, resulting in models with elevated fit (R 2 > 0.9 and ε < 0.3). The number of kernels per ear (NKE) and thousand-kernel weight (TKW) are the traits with the largest direct effects on grain yield (r = 0.66 and 0.73, respectively). The high accuracy of selection (0.86 and 0.89) associated with the high heritability of the average (0.732 and 0.794) for NKE and TKW, respectively, indicated good reliability and prospects of success in the indirect selection of hybrids with high-yield potential through these traits. The negative direct effect of NKE on TKW (r = -0.856), however, must be considered. The joint use of mixed models and sequential path analysis is effective in the evaluation of maize-breeding trials.

Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase.

PubMed

Watashi, Koichi; Ishii, Naoto; Hijikata, Makoto; Inoue, Daisuke; Murata, Takayuki; Miyanari, Yusuke; Shimotohno, Kunitada

2005-07-01

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus (HCV) genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.

Treating hepatitis C: can you teach old dogs new tricks?

PubMed

Rice, Charles M; You, Shihyun

2005-12-01

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.

A Host Susceptibility Gene, DR1, Facilitates Influenza A Virus Replication by Suppressing Host Innate Immunity and Enhancing Viral RNA Replication

PubMed Central

Hsu, Shih-Feng; Su, Wen-Chi; Jeng, King-Song

2015-01-01

ABSTRACT Influenza A virus (IAV) depends on cellular factors to complete its replication cycle; thus, investigation of the factors utilized by IAV may facilitate antiviral drug development. To this end, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNA interference (RNAi) screen. Knockdown (KD) of DR1 resulted in reductions of viral RNA and protein production, demonstrating that DR1 acts as a positive host factor in IAV replication. Genome-wide transcriptomic analysis showed that there was a strong induction of interferon-stimulated gene (ISG) expression after prolonged DR1 KD. We found that beta interferon (IFN-β) was induced by DR1 KD, thereby activating the JAK-STAT pathway to turn on ISG expression, which led to a strong inhibition of IAV replication. This result suggests that DR1 in normal cells suppresses IFN induction, probably to prevent undesired cytokine production, but that this suppression may create a milieu that favors IAV replication once cells are infected. Furthermore, biochemical assays of viral RNA replication showed that DR1 KD suppressed viral RNA replication. We also showed that DR1 associated with all three subunits of the viral RNA-dependent RNA polymerase (RdRp) complex, indicating that DR1 may interact with individual components of the viral RdRp complex to enhance viral RNA replication. Thus, DR1 may be considered a novel host susceptibility gene for IAV replication via a dual mechanism, not only suppressing the host defense to indirectly favor IAV replication but also directly facilitating viral RNA replication. IMPORTANCE Investigations of virus-host interactions involved in influenza A virus (IAV) replication are important for understanding viral pathogenesis and host defenses, which may manipulate influenza virus infection or prevent the emergence of drug resistance caused by a high error rate during viral RNA replication. For this purpose, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNAi screen as a positive regulator in IAV replication. In the current studies, we showed that DR1 suppressed the gene expression of a large set of host innate immunity genes, which indirectly facilitated IAV replication in the event of IAV infection. Besides this scenario, DR1 also directly enhanced the viral RdRp activity, likely through associating with individual components of the viral RdRp complex. Thus, DR1 represents a novel host susceptibility gene for IAV replication via multiple functions, not only suppressing the host defense but also enhancing viral RNA replication. DR1 may be a potential target for drug development against influenza virus infection. PMID:25589657

Chromatin Structure and Replication Origins: Determinants Of Chromosome Replication And Nuclear Organization

PubMed Central

Smith, Owen K.; Aladjem, Mirit I.

2014-01-01

The DNA replication program is, in part, determined by the epigenetic landscape that governs local chromosome architecture and directs chromosome duplication. Replication must coordinate with other biochemical processes occurring concomitantly on chromatin, such as transcription and remodeling, to insure accurate duplication of both genetic and epigenetic features and to preserve genomic stability. The importance of genome architecture and chromatin looping in coordinating cellular processes on chromatin is illustrated by two recent sets of discoveries. First, chromatin-associated proteins that are not part of the core replication machinery were shown to affect the timing of DNA replication. These chromatin-associated proteins could be working in concert, or perhaps in competition, with the transcriptional machinery and with chromatin modifiers to determine the spatial and temporal organization of replication initiation events. Second, epigenetic interactions are mediated by DNA sequences that determine chromosomal replication. In this review we summarize recent findings and current models linking spatial and temporal regulation of the replication program with epigenetic signaling. We discuss these issues in the context of the genome’s three-dimensional structure with an emphasis on events occurring during the initiation of DNA replication. PMID:24905010

Analysis of host response to bacterial infection using error model based gene expression microarray experiments

PubMed Central

Stekel, Dov J.; Sarti, Donatella; Trevino, Victor; Zhang, Lihong; Salmon, Mike; Buckley, Chris D.; Stevens, Mark; Pallen, Mark J.; Penn, Charles; Falciani, Francesco

2005-01-01

A key step in the analysis of microarray data is the selection of genes that are differentially expressed. Ideally, such experiments should be properly replicated in order to infer both technical and biological variability, and the data should be subjected to rigorous hypothesis tests to identify the differentially expressed genes. However, in microarray experiments involving the analysis of very large numbers of biological samples, replication is not always practical. Therefore, there is a need for a method to select differentially expressed genes in a rational way from insufficiently replicated data. In this paper, we describe a simple method that uses bootstrapping to generate an error model from a replicated pilot study that can be used to identify differentially expressed genes in subsequent large-scale studies on the same platform, but in which there may be no replicated arrays. The method builds a stratified error model that includes array-to-array variability, feature-to-feature variability and the dependence of error on signal intensity. We apply this model to the characterization of the host response in a model of bacterial infection of human intestinal epithelial cells. We demonstrate the effectiveness of error model based microarray experiments and propose this as a general strategy for a microarray-based screening of large collections of biological samples. PMID:15800204

Greenness and school-wide test scores are not always positively associated – A replication of "linking student performance in Massachusetts elementary schools with the 'greenness' of school surroundings using remote sensing"

Treesearch

Matthew H.E.M. Browning; Ming Kuo; Sonya Sachdeva; Kangjae Lee; Lynne Westphal

2018-01-01

Recent studies find vegetation around schools correlates positively with student test scores. To test this relationship in schools with less green cover and more disadvantaged students, we replicated a leading study, using six years of NDVI-derived greenness data to predict school-level math and reading achievement in 404 Chicago public schools. A direct replication...

Replication of damaged DNA in vitro is blocked by p53

PubMed Central

Zhou, Jianmin; Prives, Carol

2003-01-01

The tumor suppressor protein p53 may have other roles and functions in addition to its well-documented ability to serve as a sequence-specific transcriptional activator in response to DNA damage. We showed previously that p53 can block the replication of polyomavirus origin-containing DNA (Py ori-DNA) in vitro when p53 binding sites are present on the late side of the Py ori. Here we have both further extended these observations and have also examined whether p53 might be able to bind directly to and inhibit the replication of damaged DNA. We found that p53 strongly inhibits replication of γ-irradiated Py ori-DNA and such inhibition requires both the central DNA binding domain and the extreme C-terminus of the p53 protein. An endogenous p53 binding site lies within the Py origin and is required for the ability of p53 to block initiation of replication from γ-irradiated Py ori-DNA, suggesting the possibility of DNA looping caused by p53 binding both non-specifically to sites of DNA damage and specifically to the endogenous site in the polyomavirus origin. Our results thus suggest the possibility that under some circumstances p53 might serve as a direct regulator of DNA replication and suggest as well an additional function for cooperation between its two autonomous DNA binding domains. PMID:12853603

Meta-replication reveals nonstationarity in multi-scale habitat selection of Mexican Spotted Owl

Treesearch

Ho Yi Wan; Kevin McGarigal; Joseph L. Ganey; Valentin Lauret; Brad C. Timm; Samuel A. Cushman

2017-01-01

Anthropogenic environmental changes are leading to habitat loss and degradation, driving many species to extinction. In this context, habitat models become increasingly important for effective species management and conservation. However, most habitat studies lack replicated study areas and do not properly address the role of nonstationarity and spatial scales in...

Costs by Major Program Category--A Budgetary Guide to Program Replication.

ERIC Educational Resources Information Center

Smith, Gary E.; And Others

Intended as a budgetary guide for potential replicators, this document presents a detailed discussion of estimated costs involved in setting up and operating a program similar to the Mountin-Plains Career Education Model IV, a residential, family-based education program developed to improve the economic potential and lifestyle of selected student…

Break-induced telomere synthesis underlies alternative telomere maintenance

PubMed Central

Dilley, Robert L.; Verma, Priyanka; Cho, Nam Woo; Winters, Harrison D.; Wondisford, Anne R.; Greenberg, Roger A.

2017-01-01

Homology-directed DNA repair is essential for genome maintenance through templated DNA synthesis. Alternative lengthening of telomeres (ALT) necessitates homology-directed DNA repair to maintain telomeres in about 10–15% of human cancers. How DNA damage induces assembly and execution of a DNA replication complex (break-induced replisome) at telomeres or elsewhere in the mammalian genome is poorly understood. Here we define break-induced telomere synthesis and demonstrate that it utilizes a specialized replisome, which underlies ALT telomere maintenance. DNA double-strand breaks enact nascent telomere synthesis by long-tract unidirectional replication. Proliferating cell nuclear antigen (PCNA) loading by replication factor C (RFC) acts as the initial sensor of telomere damage to establish predominance of DNA polymerase δ (Pol δ) through its POLD3 subunit. Break-induced telomere synthesis requires the RFC–PCNA–Pol δ axis, but is independent of other canonical replisome components, ATM and ATR, or the homologous recombination protein Rad51. Thus, the inception of telomere damage recognition by the break-induced replisome orchestrates homology-directed telomere maintenance. PMID:27760120

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.

In search of genetic constraints limiting the evolution of egg size: direct and correlated responses to artificial selection on a prenatal maternal effector.

PubMed

Pick, J L; Hutter, P; Tschirren, B

2016-06-01

Maternal effects are an important force in nature, but the evolutionary dynamics of the traits that cause them are not well understood. Egg size is known to be a key mediator of prenatal maternal effects with an established genetic basis. In contrast to theoretical expectations for fitness-related traits, there is a large amount of additive genetic variation in egg size observed in natural populations. One possible mechanism for the maintenance of this variation is through genetic constraints caused by a shared genetic basis among traits. Here we created replicated, divergent selection lines for maternal egg investment in Japanese quail (Coturnix japonica) to quantify the role of genetic constraints in the evolution of egg size. We found that egg size responds rapidly to selection, accompanied by a strong response in all egg components. Initially, we observed a correlated response in body size, but this response declined over time, showing that egg size and body size can evolve independently. Furthermore, no correlated response in fecundity (measured as the proportion of days on which a female laid an egg) was observed. However, the response to selection was asymmetrical, with egg size plateauing after one generation of selection in the high but not the low investment lines. We attribute this pattern to the presence of genetic asymmetries, caused by directional dominance or unequal allele frequencies. Such asymmetries may contribute to the evolutionary stasis in egg size observed in natural populations, despite a positive association between egg size and fitness.

In search of genetic constraints limiting the evolution of egg size: direct and correlated responses to artificial selection on a prenatal maternal effector

PubMed Central

Pick, J L; Hutter, P; Tschirren, B

2016-01-01

Maternal effects are an important force in nature, but the evolutionary dynamics of the traits that cause them are not well understood. Egg size is known to be a key mediator of prenatal maternal effects with an established genetic basis. In contrast to theoretical expectations for fitness-related traits, there is a large amount of additive genetic variation in egg size observed in natural populations. One possible mechanism for the maintenance of this variation is through genetic constraints caused by a shared genetic basis among traits. Here we created replicated, divergent selection lines for maternal egg investment in Japanese quail (Coturnix japonica) to quantify the role of genetic constraints in the evolution of egg size. We found that egg size responds rapidly to selection, accompanied by a strong response in all egg components. Initially, we observed a correlated response in body size, but this response declined over time, showing that egg size and body size can evolve independently. Furthermore, no correlated response in fecundity (measured as the proportion of days on which a female laid an egg) was observed. However, the response to selection was asymmetrical, with egg size plateauing after one generation of selection in the high but not the low investment lines. We attribute this pattern to the presence of genetic asymmetries, caused by directional dominance or unequal allele frequencies. Such asymmetries may contribute to the evolutionary stasis in egg size observed in natural populations, despite a positive association between egg size and fitness. PMID:26956564

Role of host protein Ebp1 in influenza virus growth: intracellular localization of Ebp1 in virus-infected and uninfected cells.

PubMed

Honda, Ayae

2008-01-20

The cellular protein Ebp1 was identified to interact with PB1 protein of influenza virus RNA polymerase, and inhibit both RNA synthesis in vitro and influenza virus replication in vivo [Honda, A., Okamoto, T., Ishihama, A., 2007. Host factor Ebp1: selective inhibitor of influenza virus transcriptase. Genes Cells 12, 133-142]. The intracellular localization of Ebp1 that is involved in cell proliferation control was analyzed by direct immunostaining of cells before and after influenza virus infection. Ebp1 was found to localize in the nuclear membrane of uninfected cells, and to form nuclear aggregates with viral P proteins in virus-infected cells.

Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha.

PubMed

Tikhomirova, L P; Ikonomova, R N; Kuznetsova, E N

1986-01-01

For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H. polymorpha or chromosomal DNA fragments of H. polymorpha as replicator sequences. The replication properties of chimeric plasmids in the yeast H. polymorpha were investigated. We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event. Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C. utilis. Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype. Cosmid pL2 was able to shuttle between Escherichia coli, S. cerevisiae and H. polymorpha, whereas plasmids with DNA fragments from H. polymorpha did not transform S. cerevisiae effectively.

Prebiotic chemistry and nucleic acid replication

NASA Technical Reports Server (NTRS)

Orgel, L. E.; Lohrmann, R.

1974-01-01

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

Initiation and Reinitiation of DNA Synthesis during Replication of Bacteriophage T7*

PubMed Central

Dressler, David; Wolfson, John; Magazin, Marilyn

1972-01-01

In its first round of replication, the T7 chromosome follows a simple pattern, as viewed in the electron microscope. The iniation of DNA synthesis occurs about 17% from the genetic left end of the viral DNA rod. Bidirectional DNA synthesis from this origin then generates a replicating intermediate that we call an “eye form.” In the eye form, when synthesis in the leftward direction reaches the left end of the viral chromosome, the molecule is converted into a Y-shaped replicating rod. The remaining growing point continues synthesis rightward, until presumably it runs off the right end of the DNA rod, thus terminating replication. Numerous T7 chromosomes were found in which a second round of replication had begun before the first round had finished. Analysis of these reinitiated DNA molecules showed that the second round of replication, like the first, began 17% from the end of the chromosome and involved bidirectional DNA synthesis. Images PMID:4554539

Nucleosomes influence multiple steps during replication initiation

PubMed Central

Azmi, Ishara F; Watanabe, Shinya; Maloney, Michael F; Kang, Sukhyun; Belsky, Jason A; MacAlpine, David M; Peterson, Craig L; Bell, Stephen P

2017-01-01

Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin. DOI: http://dx.doi.org/10.7554/eLife.22512.001 PMID:28322723

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.

An investigation into the use of recorded music as a surgical intervention: A systematic, critical review of methodologies used in recent adult controlled trials.

PubMed

Williams, Courtney; Hine, Trevor

2018-04-01

While music is being increasingly used as a surgical intervention, the types of music used and the reasons underlying their selection remain inconsistent. Empirical research into the efficacy of such musical interventions is therefore problematic. To provide clear guidelines for musical selection and employment in surgical interventions, created through a synthesis of the literature. The aim is to examine how music is implemented in surgical situations, and to provide guidance for the selection and composition of music for future interventions. English language quantitative surgical intervention studies from Science Direct, ProQuest, and Sage Journals Online, all published within the last 10 years and featuring recorded music, were systematically reviewed. Variables investigated included: the time the intervention was performed, the intervention length, the outcomes targeted, music description (general and specific), theoretical frameworks underlying the selection of the music, whether or not a musical expert was involved, participant music history, and the participants' feedback on the chosen music. Several aspects contribute to the lack of scientific rigour regarding music selection in this field, including the lack of a theoretical framework or frameworks, no involvement of musical experts, failure to list the music tracks used, and the use of vague and subjective terms in general music descriptions. Patients are frequently allowed to select music (risking both choosing music that has an adverse effect and making study replication difficult), and patient music history and listening habits are rarely considered. Crucially, five primary theoretical frameworks underlying the effectiveness of music arose in the literature (distraction, relaxation, emotional shift, entrainment, and endogenous analgesia), however music was rarely selected to enhance any of these mechanisms. Further research needs to be conducted to ensure that music is selected according to a theoretical framework and more rigorous and replicable methodology. Music interventions can be made more effective at improving psychological states and reducing physiological arousal by selecting music conducive to specific mechanisms, and also by considering at what point during the surgical experience the music would be most effective. Greater involvement of music experts in interventions would help to ensure that the most appropriate music was chosen, and that it is clearly and precisely described. Copyright © 2018. Published by Elsevier Ltd.

The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria.

PubMed

Muñoz-Espín, Daniel; Daniel, Richard; Kawai, Yoshikazu; Carballido-López, Rut; Castilla-Llorente, Virginia; Errington, Jeff; Meijer, Wilfried J J; Salas, Margarita

2009-08-11

Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage ϕ29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the ϕ29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the ϕ29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.

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.

Use of tissue-specific microRNA to control pathology of wild-type adenovirus without attenuation of its ability to kill cancer cells.

PubMed

Cawood, Ryan; Chen, Hannah H; Carroll, Fionnadh; Bazan-Peregrino, Miriam; van Rooijen, Nico; Seymour, Leonard W

2009-05-01

Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines; however, uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild-type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus adenovirus, we have engineered a hepatocyte-safe wild-type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this, we have included binding sites for hepatocyte-selective microRNA mir-122 within the 3' UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of mir-122 binding sites caused up to 80-fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild-type Ad5 (5x10(10) viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild-type virus retained full activity within cancer cells and provided a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral diseases.

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

PubMed Central

Schwab, Rebekka A.V.; Niedzwiedz, Wojciech

2011-01-01

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development. Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope. PMID:22064662

Directed evolution of DNA polymerase, RNA polymerase and reverse transcriptase activity in a single polypeptide.

PubMed

Ong, Jennifer L; Loakes, David; Jaroslawski, Szymon; Too, Kathleen; Holliger, Philipp

2006-08-18

DNA polymerases enable key technologies in modern biology but for many applications, native polymerases are limited by their stringent substrate recognition. Here we describe short-patch compartmentalized self-replication (spCSR), a novel strategy to expand the substrate spectrum of polymerases in a targeted way. spCSR is based on the previously described CSR, but unlike CSR only a short region (a "patch") of the gene under investigation is diversified and replicated. This allows the selection of polymerases under conditions where catalytic activity and processivity are compromised to the extent that full self-replication is inefficient. We targeted two specific motifs involved in substrate recognition in the active site of DNA polymerase I from Thermus aquaticus (Taq) and selected for incorporation of both ribonucleotide- (NTP) and deoxyribonucleotide-triphosphates (dNTPs) using spCSR. This allowed the isolation of multiple variants of Taq with apparent dual substrate specificity. They were able to synthesize RNA, while still retaining essentially wild-type (wt) DNA polymerase activity as judged by PCR. One such mutant (AA40: E602V, A608V, I614M, E615G) was able to incorporate both NTPs and dNTPs with the same catalytic efficiency as the wt enzyme incorporates dNTPs. AA40 allowed the generation of mixed RNA-DNA amplification products in PCR demonstrating DNA polymerase, RNA polymerase as well as reverse transcriptase activity within the same polypeptide. Furthermore, AA40 displayed an expanded substrate spectrum towards other 2'-substituted nucleotides and was able to synthesize nucleic acid polymers in which each base bore a different 2'-substituent. Our results suggest that spCSR will be a powerful strategy for the generation of polymerases with altered substrate specificity for applications in nano- and biotechnology and in the enzymatic synthesis of antisense and RNAi probes.

Biochemical Evaluation of the Inhibition Properties of Favipiravir and 2′-C-Methyl-Cytidine Triphosphates against Human and Mouse Norovirus RNA Polymerases

PubMed Central

Tucker, Kathryn; Lin, Xiaoyan; Kao, C. Cheng; Shaw, Ken; Tan, Hua; Symons, Julian; Behera, Ishani; Rajwanshi, Vivek K.; Dyatkina, Natalia; Wang, Guangyi; Beigelman, Leo

2015-01-01

Norovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2′-C-methyl-cytidine (2CM-C) inhibit NoV replication in vitro and in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC50s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2′-C-methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities. PMID:26392512

Genome-wide scan of 29,141 African Americans finds no evidence of directional selection since admixture.

PubMed

Bhatia, Gaurav; Tandon, Arti; Patterson, Nick; Aldrich, Melinda C; Ambrosone, Christine B; Amos, Christopher; Bandera, Elisa V; Berndt, Sonja I; Bernstein, Leslie; Blot, William J; Bock, Cathryn H; Caporaso, Neil; Casey, Graham; Deming, Sandra L; Diver, W Ryan; Gapstur, Susan M; Gillanders, Elizabeth M; Harris, Curtis C; Henderson, Brian E; Ingles, Sue A; Isaacs, William; De Jager, Phillip L; John, Esther M; Kittles, Rick A; Larkin, Emma; McNeill, Lorna H; Millikan, Robert C; Murphy, Adam; Neslund-Dudas, Christine; Nyante, Sarah; Press, Michael F; Rodriguez-Gil, Jorge L; Rybicki, Benjamin A; Schwartz, Ann G; Signorello, Lisa B; Spitz, Margaret; Strom, Sara S; Tucker, Margaret A; Wiencke, John K; Witte, John S; Wu, Xifeng; Yamamura, Yuko; Zanetti, Krista A; Zheng, Wei; Ziegler, Regina G; Chanock, Stephen J; Haiman, Christopher A; Reich, David; Price, Alkes L

2014-10-02

The extent of recent selection in admixed populations is currently an unresolved question. We scanned the genomes of 29,141 African Americans and failed to find any genome-wide-significant deviations in local ancestry, indicating no evidence of selection influencing ancestry after admixture. A recent analysis of data from 1,890 African Americans reported that there was evidence of selection in African Americans after their ancestors left Africa, both before and after admixture. Selection after admixture was reported on the basis of deviations in local ancestry, and selection before admixture was reported on the basis of allele-frequency differences between African Americans and African populations. The local-ancestry deviations reported by the previous study did not replicate in our very large sample, and we show that such deviations were expected purely by chance, given the number of hypotheses tested. We further show that the previous study's conclusion of selection in African Americans before admixture is also subject to doubt. This is because the FST statistics they used were inflated and because true signals of unusual allele-frequency differences between African Americans and African populations would be best explained by selection that occurred in Africa prior to migration to the Americas. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

HLA-B27 Selects for Rare Escape Mutations that Significantly Impair Hepatitis C Virus Replication and Require Compensatory Mutations

PubMed Central

Neumann-Haefelin, Christoph; Oniangue-Ndza, Cesar; Kuntzen, Thomas; Schmidt, Julia; Nitschke, Katja; Sidney, John; Caillet-Saguy, Célia; Binder, Marco; Kersting, Nadine; Kemper, Michael W.; Power, Karen A.; Ingber, Susan; Reyor, Laura L.; Hills-Evans, Kelsey; Kim, Arthur Y.; Lauer, Georg M.; Lohmann, Volker; Sette, Alessandro; Henn, Matthew R.; Bressanelli, Stéphane; Thimme, Robert; Allen, Todd M.

2011-01-01

HLA-B27 is associated with spontaneous viral clearance in hepatitis C virus (HCV) infection. Viral escape within the immunodominant HLA-B27 restricted HCV-specific CD8+ T cell epitope NS5B2841-2849 (ARMILMTHF) has been shown to be limited by viral fitness costs as well as broad T cell cross-recognition, suggesting a potential mechanism of protection by HLA-B27. Here, we studied the subdominant HLA-B27 restricted epitope NS5B2936-2944 (GRAAICGKY) in order to further define the mechanisms of protection by HLA-B27. We identified a unique pattern of escape mutations within this epitope in a large cohort of HCV genotype 1a infected patients. The predominant escape mutations represented conservative substitutions at the main HLA-B27 anchor residue or a T cell receptor contact site, neither of which impaired viral replication capacity as assessed in a subgenomic HCV replicon system. In contrast, however, in a subset of HLA-B27+ subjects rare escape mutations arose at the HLA-B27 anchor residue R2937, which nearly abolished viral replication. Notably, these rare mutations only occurred in conjunction with the selection of two equally rare, and structurally proximal, upstream mutations. Co-expression of these upstream mutations with the rare escape mutations dramatically restored viral replication capacity from <5% to ≥70% of wild-type levels. Conclusion The selection of rare CTL escape mutations in this HLA-B27 restricted epitope dramatically impairs viral replicative fitness unless properly compensated. These data support a role for the targeting of highly-constrained regions by HLA-B27 in its ability to assert immune control of HCV and other highly variable pathogens. PMID:22006856

An evolutionary framework for cultural change: Selectionism versus communal exchange

NASA Astrophysics Data System (ADS)

Gabora, Liane

2013-06-01

Dawkins' replicator-based conception of evolution has led to widespread mis-application of selectionism across the social sciences because it does not address the paradox that necessitated the theory of natural selection in the first place: how do organisms accumulate change when traits acquired over their lifetime are obliterated? This is addressed by von Neumann's concept of a self-replicating automaton (SRA). A SRA consists of a self-assembly code that is used in two distinct ways: (1) actively deciphered during development to construct a self-similar replicant, and (2) passively copied to the replicant to ensure that it can reproduce. Information that is acquired over a lifetime is not transmitted to offspring, whereas information that is inherited during copying is transmitted. In cultural evolution there is no mechanism for discarding acquired change. Acquired change can accumulate orders of magnitude faster than, and quickly overwhelm, inherited change due to differential replication of variants in response to selection. This prohibits a selectionist but not an evolutionary framework for culture and the creative processes that fuel it. The importance non-Darwinian processes in biological evolution is increasingly recognized. Recent work on the origin of life suggests that early life evolved through a non-Darwinian process referred to as communal exchange that does not involve a self-assembly code, and that natural selection emerged from this more haphazard, ancestral evolutionary process. It is proposed that communal exchange provides an evolutionary framework for culture that enables specification of cognitive features necessary for a (real or artificial) societies to evolve culture. This is supported by a computational model of cultural evolution and a conceptual network based program for documenting material cultural history, and it is consistent with high levels of human cooperation.

Chromosomal context and replication properties of ARS plasmids in Schizosaccharomyces pombe.

PubMed

Pratihar, Aditya S; Tripathi, Vishnu P; Yadav, Mukesh P; Dubey, Dharani D

2015-12-01

Short, specific DNA sequences called as Autonomously Replicating Sequence (ARS) elements function as plasmid as well as chromosomal replication origins in yeasts. As compared to ARSs, different chromosomal origins vary greatly in their efficiency and timing of replication probably due to their wider chromosomal context. The two Schizosaccharomyces pombe ARS elements, ars727 and ars2004, represent two extremities in their chromosomal origin activity - ars727 is inactive and late replicating, while ars2004 is a highly active, early-firing origin. To determine the effect of chromosomal context on the activity of these ARS elements, we have cloned them with their extended chromosomal context as well as in the context of each other in both orientations and analysed their replication efficiency by ARS and plasmid stability assays. We found that these ARS elements retain their origin activity in their extended/altered context. However, deletion of a 133-bp region of the previously reported ars727- associated late replication enforcing element (LRE) caused advancement in replication timing of the resulting plasmid. These results confirm the role of LRE in directing plasmid replication timing and suggest that the plasmid origin efficiency of ars2004 or ars727 remains unaltered by the extended chromosomal context.

Cooperation of catalysts and templates

NASA Technical Reports Server (NTRS)

White, D. H.; Kanavarioti, A.; Nibley, C. W.; Macklin, J. W.

1986-01-01

In order to understand how self-reproducing molecules could have originated on the primitive Earth or extraterrestrial bodies, it would be useful to find laboratory models of simple molecules which are able to carry out processes of catalysis and templating. Furthermore, it may be anticipated that systems in which several components are acting cooperatively to catalyze each other's synthesis will have different behavior with respect to natural selection than those of purely replicating systems. As the major focus of this work, laboratory models are devised to study the influence of short peptide catalysts on template reactions which produce oligonucleotides or additional peptides. Such catalysts could have been the earliest protoenzymes of selective advantage produced by replicating oligonucleotides. Since this is a complex problem, simpler systems are also studied which embody only one aspect at a time, such as peptide formation with and without a template, peptide catalysis of nontemplated peptide synthesis, and model reactions for replication of the type pioneered by Orgel.

Active Ebola Virus Replication and Heterogeneous Evolutionary Rates in EVD Survivors.

PubMed

Whitmer, Shannon L M; Ladner, Jason T; Wiley, Michael R; Patel, Ketan; Dudas, Gytis; Rambaut, Andrew; Sahr, Foday; Prieto, Karla; Shepard, Samuel S; Carmody, Ellie; Knust, Barbara; Naidoo, Dhamari; Deen, Gibrilla; Formenty, Pierre; Nichol, Stuart T; Palacios, Gustavo; Ströher, Ute

2018-01-30

Following cessation of continuous Ebola virus (EBOV) transmission within Western Africa, sporadic EBOV disease (EVD) cases continued to re-emerge beyond the viral incubation period. Epidemiological and genomic evidence strongly suggests that this represented transmission from EVD survivors. To investigate whether persistent infections are characterized by ongoing viral replication, we sequenced EBOV from the semen of nine EVD survivors and a subset of corresponding acute specimens. EBOV evolutionary rates during persistence were either similar to or reduced relative to acute infection rates. Active EBOV replication/transcription continued during convalescence, but decreased over time, consistent with viral persistence rather than viral latency. Patterns of genetic divergence suggest a moderate relaxation of selective constraints within the sGP carboxy-terminal tail during persistent infections, but do not support widespread diversifying selection. Altogether, our data illustrate that EBOV persistence in semen, urine, and aqueous humor is not a quiescent or latent infection. Published by Elsevier Inc.

Women's Stereotypic Roles: A Replication and Standardization of the AWS and PAQ for Selected Ethnic Groups.

ERIC Educational Resources Information Center

Wheeler, Edwin E.; And Others

A replication of two previous studies, this study examined the effect of both sex and ethnicity on attitudes toward women, self-reported masculinity-femininity, and masculine-feminine stereotypic attitudes. The Attitudes Toward Women Scale (AWS) and the Personal Attributes Questionnaire (PAQ) were administered to 367 college students (112 Anglos,…

PCM-SABRE: a platform for benchmarking and comparing outcome prediction methods in precision cancer medicine.

PubMed

Eyal-Altman, Noah; Last, Mark; Rubin, Eitan

2017-01-17

Numerous publications attempt to predict cancer survival outcome from gene expression data using machine-learning methods. A direct comparison of these works is challenging for the following reasons: (1) inconsistent measures used to evaluate the performance of different models, and (2) incomplete specification of critical stages in the process of knowledge discovery. There is a need for a platform that would allow researchers to replicate previous works and to test the impact of changes in the knowledge discovery process on the accuracy of the induced models. We developed the PCM-SABRE platform, which supports the entire knowledge discovery process for cancer outcome analysis. PCM-SABRE was developed using KNIME. By using PCM-SABRE to reproduce the results of previously published works on breast cancer survival, we define a baseline for evaluating future attempts to predict cancer outcome with machine learning. We used PCM-SABRE to replicate previous work that describe predictive models of breast cancer recurrence, and tested the performance of all possible combinations of feature selection methods and data mining algorithms that was used in either of the works. We reconstructed the work of Chou et al. observing similar trends - superior performance of Probabilistic Neural Network (PNN) and logistic regression (LR) algorithms and inconclusive impact of feature pre-selection with the decision tree algorithm on subsequent analysis. PCM-SABRE is a software tool that provides an intuitive environment for rapid development of predictive models in cancer precision medicine.

Bypassing bacterial infection in phage display by sequencing DNA released from phage particles.

PubMed

Villequey, Camille; Kong, Xu-Dong; Heinis, Christian

2017-11-01

Phage display relies on a bacterial infection step in which the phage particles are replicated to perform multiple affinity selection rounds and to enable the identification of isolated clones by DNA sequencing. While this process is efficient for wild-type phage, the bacterial infection rate of phage with mutant or chemically modified coat proteins can be low. For example, a phage mutant with a disulfide-free p3 coat protein, used for the selection of bicyclic peptides, has a more than 100-fold reduced infection rate compared to the wild-type. A potential strategy for bypassing the bacterial infection step is to directly sequence DNA extracted from phage particles after a single round of phage panning using high-throughput sequencing. In this work, we have quantified the fraction of phage clones that can be identified by directly sequencing DNA from phage particles. The results show that the DNA of essentially all of the phage particles can be 'decoded', and that the sequence coverage for mutants equals that of amplified DNA extracted from cells infected with wild-type phage. This procedure is particularly attractive for selections with phage that have a compromised infection capacity, and it may allow phage display to be performed with particles that are not infective at all. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Direct replication of Gervais & Norenzayan (2012): No evidence that analytic thinking decreases religious belief

PubMed Central

Sanchez, Clinton; Sundermeier, Brian; Gray, Kenneth

2017-01-01

Gervais & Norenzayan (2012) reported in Science a series of 4 experiments in which manipulations intended to foster analytic thinking decreased religious belief. We conducted a precise, large, multi-site pre-registered replication of one of these experiments. We observed little to no effect of the experimental manipulation on religious belief (d = 0.07 in the wrong direction, 95% CI[-0.12, 0.25], N = 941). The original finding does not seem to provide reliable or valid evidence that analytic thinking causes a decrease in religious belief. PMID:28234942

Direct replication of Gervais & Norenzayan (2012): No evidence that analytic thinking decreases religious belief.

PubMed

Sanchez, Clinton; Sundermeier, Brian; Gray, Kenneth; Calin-Jageman, Robert J

2017-01-01

Gervais & Norenzayan (2012) reported in Science a series of 4 experiments in which manipulations intended to foster analytic thinking decreased religious belief. We conducted a precise, large, multi-site pre-registered replication of one of these experiments. We observed little to no effect of the experimental manipulation on religious belief (d = 0.07 in the wrong direction, 95% CI[-0.12, 0.25], N = 941). The original finding does not seem to provide reliable or valid evidence that analytic thinking causes a decrease in religious belief.

Dengue and Zika viruses subvert reticulophagy by NS2B3-mediated cleavage of FAM134B.

PubMed

Lennemann, Nicholas J; Coyne, Carolyn B

2017-02-01

The endoplasmic reticulum (ER) is exploited by several diverse viruses during their infectious life cycles. Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), utilize the ER as a source of membranes to establish their replication organelles and to facilitate their assembly and eventual maturation along the secretory pathway. To maintain normal homeostasis, host cells have evolved highly efficient processes to dynamically regulate the ER, such as through reticulophagy, a selective form of autophagy that leads to ER degradation. Here, we identify the ER-localized reticulophagy receptor FAM134B as a host cell restriction factor for both DENV and ZIKV. We show that RNAi-mediated depletion of FAM134B significantly enhances both DENV and ZIKV replication at an early stage of the viral life cycle. Consistent with its role as an antiviral host factor, we found that several flaviviruses including DENV, ZIKV, and West Nile virus (WNV), utilize their NS3 virally-encoded proteases to directly cleave FAM134B at a single site within its reticulon homology domain (RHD). Mechanistically, we show that NS3-mediated cleavage of FAM134B blocks the formation of ER and viral protein-enriched autophagosomes, suggesting that the cleavage of FAM134B serves to specifically suppress the reticulophagy pathway. These findings thus point to an important role for FAM134B and reticulophagy in the regulation of flavivirus infection and suggest that these viruses specifically target these pathways to promote viral replication.

Co-existence of Distinct Prion Types Enables Conformational Evolution of Human PrPSc by Competitive Selection*

PubMed Central

Haldiman, Tracy; Kim, Chae; Cohen, Yvonne; Chen, Wei; Blevins, Janis; Qing, Liuting; Cohen, Mark L.; Langeveld, Jan; Telling, Glenn C.; Kong, Qingzhong; Safar, Jiri G.

2013-01-01

The unique phenotypic characteristics of mammalian prions are thought to be encoded in the conformation of pathogenic prion proteins (PrPSc). The molecular mechanism responsible for the adaptation, mutation, and evolution of prions observed in cloned cells and upon crossing the species barrier remains unsolved. Using biophysical techniques and conformation-dependent immunoassays in tandem, we isolated two distinct populations of PrPSc particles with different conformational stabilities and aggregate sizes, which frequently co-exist in the most common human prion disease, sporadic Creutzfeldt-Jakob disease. The protein misfolding cyclic amplification replicates each of the PrPSc particle types independently and leads to the competitive selection of those with lower initial conformational stability. In serial propagation with a nonglycosylated mutant PrPC substrate, the dominant PrPSc conformers are subject to further evolution by natural selection of the subpopulation with the highest replication rate due to its lowest stability. Cumulatively, the data show that sporadic Creutzfeldt-Jakob disease PrPSc is not a single conformational entity but a dynamic collection of two distinct populations of particles. This implies the co-existence of different prions, whose adaptation and evolution are governed by the selection of progressively less stable, faster replicating PrPSc conformers. PMID:23974118

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

PubMed

Dembowski, Jill A; DeLuca, Neal A

2015-05-01

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

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.

A Re-Examination of Global Suppression of RNA Interference by HIV-1

PubMed Central

Sanghvi, Viraj R.; Steel, Laura F.

2011-01-01

The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing. PMID:21386885

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.

Families Who Care: Assisting African American and Rural Caregivers Dealing with Dementia. A Replication Plan.

ERIC Educational Resources Information Center

Coogle, Constance L., Ed.; Finley, Ruth B., Ed.

This Replication Plan describes an Administration on Aging funded Project, "Assisting Caregivers of Black and Rural Elders with Dementia: Progressive Training through Trusted Resources." The project directly developed the capacities of trusted community leaders (n=69) who served as trainers of African American and rural family caregivers…

Rhythmic Engagement with Music in Early Childhood: A Replication and Extension

ERIC Educational Resources Information Center

Ilari, Beatriz

2015-01-01

The purpose of this study was to replicate and extend previous findings on spontaneous movement and rhythmic engagement with music in infancy. Using the identical stimuli and procedures from the original study, I investigated spontaneous rhythmic movements in response to music, infant-directed speech, and contrasting rhythmic patterns in 30…

Manipulating 3D-Printed and Paper Models Enhances Student Understanding of Viral Replication

ERIC Educational Resources Information Center

Couper, Lisa; Johannes, Kristen; Powers, Jackie; Silberglitt, Matt; Davenport, Jodi

2016-01-01

Understanding key concepts in molecular biology requires reasoning about molecular processes that are not directly observable and, as such, presents a challenge to students and teachers. We ask whether novel interactive physical models and activities can help students understand key processes in viral replication. Our 3D tangible models are…

Direct Binding to Replication Protein A (RPA)-coated Single-stranded DNA Allows Recruitment of the ATR Activator TopBP1 to Sites of DNA Damage*

PubMed Central

Acevedo, Julyana; Yan, Shan; Michael, W. Matthew

2016-01-01

A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated single-stranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes. PMID:27129245

Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major.

PubMed

Lombraña, Rodrigo; Álvarez, Alba; Fernández-Justel, José Miguel; Almeida, Ricardo; Poza-Carrión, César; Gomes, Fábia; Calzada, Arturo; Requena, José María; Gómez, María

2016-08-09

Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs). Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

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

Environmental change drives accelerated adaptation through stimulated copy number variation

PubMed Central

Hull, Ryan M.; Cruz, Cristina; Jack, Carmen V.

2017-01-01

Copy number variation (CNV) is rife in eukaryotic genomes and has been implicated in many human disorders, particularly cancer, in which CNV promotes both tumorigenesis and chemotherapy resistance. CNVs are considered random mutations but often arise through replication defects; transcription can interfere with replication fork progression and stability, leading to increased mutation rates at highly transcribed loci. Here we investigate whether inducible promoters can stimulate CNV to yield reproducible, environment-specific genetic changes. We propose a general mechanism for environmentally-stimulated CNV and validate this mechanism for the emergence of copper resistance in budding yeast. By analysing a large cohort of individual cells, we directly demonstrate that CNV of the copper-resistance gene CUP1 is stimulated by environmental copper. CNV stimulation accelerates the formation of novel alleles conferring enhanced copper resistance, such that copper exposure actively drives adaptation to copper-rich environments. Furthermore, quantification of CNV in individual cells reveals remarkable allele selectivity in the rate at which specific environments stimulate CNV. We define the key mechanistic elements underlying this selectivity, demonstrating that CNV is regulated by both promoter activity and acetylation of histone H3 lysine 56 (H3K56ac) and that H3K56ac is required for CUP1 CNV and efficient copper adaptation. Stimulated CNV is not limited to high-copy CUP1 repeat arrays, as we find that H3K56ac also regulates CNV in 3 copy arrays of CUP1 or SFA1 genes. The impact of transcription on DNA damage is well understood, but our research reveals that this apparently problematic association forms a pathway by which mutations can be directed to particular loci in particular environments and furthermore that this mutagenic process can be regulated through histone acetylation. Stimulated CNV therefore represents an unanticipated and remarkably controllable pathway facilitating organismal adaptation to new environments. PMID:28654659

Predators select against high growth rates and risk-taking behaviour in domestic trout populations.

PubMed

Biro, Peter A; Abrahams, Mark V; Post, John R; Parkinson, Eric A

2004-11-07

Domesticated (farm) salmonid fishes display an increased willingness to accept risk while foraging, and achieve high growth rates not observed in nature. Theory predicts that elevated growth rates in domestic salmonids will result in greater risk-taking to access abundant food, but low survival in the presence of predators. In replicated whole-lake experiments, we observed that domestic trout (selected for high growth rates) took greater risks while foraging and grew faster than a wild strain. However, survival consequences for greater growth rates depended upon the predation environment. Domestic trout experienced greater survival when risk was low, but lower survival when risk was high. This suggests that animals with high intrinsic growth rates are selected against in populations with abundant predators, explaining the absence of such phenotypes in nature. This is, to our knowledge, the first large-scale field experiment to directly test this theory and simultaneously quantify the initial invasibility of domestic salmonid strains that escape into the wild from aquaculture operations, and the ecological conditions affecting their survival.

Replicating DNA by cell factories: roles of central carbon metabolism and transcription in the control of DNA replication in microbes, and implications for understanding this process in human cells

PubMed Central

2013-01-01

Precise regulation of DNA replication is necessary to ensure the inheritance of genetic features by daughter cells after each cell division. Therefore, determining how the regulatory processes operate to control DNA replication is crucial to our understanding and application to biotechnological processes. Contrary to early concepts of DNA replication, it appears that this process is operated by large, stationary nucleoprotein complexes, called replication factories, rather than by single enzymes trafficking along template molecules. Recent discoveries indicated that in bacterial cells two processes, central carbon metabolism (CCM) and transcription, significantly and specifically influence the control of DNA replication of various replicons. The impact of these discoveries on our understanding of the regulation of DNA synthesis is discussed in this review. It appears that CCM may influence DNA replication by either action of specific metabolites or moonlighting activities of some enzymes involved in this metabolic pathway. The role of transcription in the control of DNA replication may arise from either topological changes in nucleic acids which accompany RNA synthesis or direct interactions between replication and transcription machineries. Due to intriguing similarities between some prokaryotic and eukaryotic regulatory systems, possible implications of studies on regulation of microbial DNA replication on understanding such a process occurring in human cells are discussed. PMID:23714207

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

Thresher: an improved algorithm for peak height thresholding of microbial community profiles.

PubMed

Starke, Verena; Steele, Andrew

2014-11-15

This article presents Thresher, an improved technique for finding peak height thresholds for automated rRNA intergenic spacer analysis (ARISA) profiles. We argue that thresholds must be sample dependent, taking community richness into account. In most previous fragment analyses, a common threshold is applied to all samples simultaneously, ignoring richness variations among samples and thereby compromising cross-sample comparison. Our technique solves this problem, and at the same time provides a robust method for outlier rejection, selecting for removal any replicate pairs that are not valid replicates. Thresholds are calculated individually for each replicate in a pair, and separately for each sample. The thresholds are selected to be the ones that minimize the dissimilarity between the replicates after thresholding. If a choice of threshold results in the two replicates in a pair failing a quantitative test of similarity, either that threshold or that sample must be rejected. We compare thresholded ARISA results with sequencing results, and demonstrate that the Thresher algorithm outperforms conventional thresholding techniques. The software is implemented in R, and the code is available at http://verenastarke.wordpress.com or by contacting the author. vstarke@ciw.edu or http://verenastarke.wordpress.com Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach.

PubMed

Frimodt-Møller, Jakob; Charbon, Godefroid; Krogfelt, Karen A; Løbner-Olesen, Anders

2017-09-11

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Diffusion of Energy Efficient Technology in Commercial Buildings: An Analysis of the Commercial Building Partnerships Program

NASA Astrophysics Data System (ADS)

Antonopoulos, Chrissi Argyro

This study presents findings from survey and interview data investigating replication of green building measures by Commercial Building Partnership (CBP) partners that worked directly with the Pacific Northwest National Laboratory (PNNL). PNNL partnered directly with 12 organizations on new and retrofit construction projects, which represented approximately 28 percent of the entire U.S. Department of Energy (DOE) CBP program. Through a feedback survey mechanism, along with personal interviews, quantitative and qualitative data were gathered relating to replication efforts by each organization. These data were analyzed to provide insight into two primary research areas: 1) CBP partners' replication efforts of green building approaches used in the CBP project to the rest of the organization's building portfolio, and, 2) the market potential for technology diffusion into the total U.S. commercial building stock, as a direct result of the CBP program. The first area of this research focused specifically on replication efforts underway or planned by each CBP program participant. The second area of this research develops a diffusion of innovations model to analyze potential broad market impacts of the CBP program on the commercial building industry in the United States. Findings from this study provided insight into motivations and objectives CBP partners had for program participation. Factors that impact replication include motivation, organizational structure and objectives firms have for implementation of energy efficient technologies. Comparing these factors between different CBP partners revealed patterns in motivation for constructing energy efficient buildings, along with better insight into market trends for green building practices. The optimized approach to the CBP program allows partners to develop green building parameters that fit the specific uses of their building, resulting in greater motivation for replication. In addition, the diffusion model developed for this analysis indicates that this method of market prediction may be used to adequately capture cumulative construction metrics for a whole-building analysis as opposed to individual energy efficiency measures used in green building.

Towards scalable Byzantine fault-tolerant replication

NASA Astrophysics Data System (ADS)

Zbierski, Maciej

2017-08-01

Byzantine fault-tolerant (BFT) replication is a powerful technique, enabling distributed systems to remain available and correct even in the presence of arbitrary faults. Unfortunately, existing BFT replication protocols are mostly load-unscalable, i.e. they fail to respond with adequate performance increase whenever new computational resources are introduced into the system. This article proposes a universal architecture facilitating the creation of load-scalable distributed services based on BFT replication. The suggested approach exploits parallel request processing to fully utilize the available resources, and uses a load balancer module to dynamically adapt to the properties of the observed client workload. The article additionally provides a discussion on selected deployment scenarios, and explains how the proposed architecture could be used to increase the dependability of contemporary large-scale distributed systems.

Direct and Conceptual Replications of Burgmer & Englich (2012): Power May Have Little to No Effect on Motor Performance

PubMed Central

Gottschalk, Christopher; Calin-Jageman, Robert J.

2015-01-01

Burgmer and Englich (2012) have reported that manipulating feelings of power can substantially improve performance on two motor tasks: golf and darts. We conducted two high-powered direct replications of the effects of power on golf, two online conceptual replications using mirror-tracing as a performance measure, and an additional conceptual replication using a cognitive performance measure (word-search). Overall, we found little to no effect of power on motor skill (d = 0.09, 95% CI[-0.07, 0.22], n = 603). We varied task difficulty, re-analyzed data without participants showing weak responses on manipulation checks, and tried adjusting performance scores for age, gender, and initial task skill. None of these secondary analyses revealed a strong effect of power on performance. A meta-analysis integrating our data with Burgmer & Englich leaves open the possibility that manipulating power could provide a modest boost in motor skill (d = 0.19, 95% CI [0.001, 0.38], n = 685). Unfortunately, the pattern of performance changes we observed was unrelated to group differences in perceived and rated power, suggesting that what motor effects do occur with this protocol may not be directly related to the construct of power. [Burgmer, P., &Englich, B. (2012). Bullseye!: How Power Improves Motor Performance. Social Psychological and Personality Science, 4(2), 224–232.] PMID:26536592

Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase.

PubMed

Liew, Li Phing; Lim, Zun Yi; Cohen, Matan; Kong, Ziqing; Marjavaara, Lisette; Chabes, Andrei; Bell, Stephen D

2016-11-01

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

On meme--gene coevolution.

PubMed

Bull, L; Holland, O; Blackmore, S

2000-01-01

In this article we examine the effects of the emergence of a new replicator, memes, on the evolution of a pre-existing replicator, genes. Using a version of the NKCS model we examine the effects of increasing the rate of meme evolution in relation to the rate of gene evolution, for various degrees of interdependence between the two replicators. That is, the effects of memes' (suggested) more rapid rate of evolution in comparison to that of genes is investigated using a tunable model of coevolution. It is found that, for almost any degree of interdependence between the two replicators, as the rate of meme evolution increases, a phase transition-like dynamic occurs under which memes have a significantly detrimental effect on the evolution of genes, quickly resulting in the cessation of effective gene evolution. Conversely, the memes experience a sharp increase in benefit from increasing their rate of evolution. We then examine the effects of enabling genes to reduce the percentage of gene-detrimental evolutionary steps taken by memes. Here a critical region emerges as the comparative rate of meme evolution increases, such that if genes cannot effectively select memes a high percentage of the time, they suffer from meme evolution as if they had almost no selective capability.

Hard X-Ray and Wide Focusing Telescopes

NASA Technical Reports Server (NTRS)

Gorenstein, Paul

1998-01-01

Studies are being carried out to compare the performance of several different separation materials used in the replication process. This report presents the results obtained during the second year of a program which consists of replicating smooth, thin substrates, depositing multilayer coatings upon them, and evaluating their performance. Replication and multilayer coatings are both critically important to the development of focussing hard X-ray telescopes that function up to 100 keV. The activities of the current year include extending the comparison between sputtered amorphous carbon and evaporated gold to include sputtered as well as evaporated gold. The figure of merit being the smoothness of the replica which has a direct effect on the specular reflectivity. These results were obtained with epoxy replication, but they should be applicable to electroformed nickel, the process we expect to use for the ultimate replicated optics.

Selective replication of oncolytic virus M1 results in a bystander killing effect that is potentiated by Smac mimetics.

PubMed

Cai, Jing; Lin, Yuan; Zhang, Haipeng; Liang, Jiankai; Tan, Yaqian; Cavenee, Webster K; Yan, Guangmei

2017-06-27

Oncolytic virotherapy is a treatment modality that uses native or genetically modified viruses that selectively replicate in and kill tumor cells. Viruses represent a type of pathogen-associated molecular pattern and thereby induce the up-regulation of dozens of cytokines via activating the host innate immune system. Second mitochondria-derived activator of caspases (Smac) mimetic compounds (SMCs), which antagonize the function of inhibitor of apoptosis proteins (IAPs) and induce apoptosis, sensitize tumor cells to multiple cytokines. Therefore, we sought to determine whether SMCs sensitize tumor cells to cytokines induced by the oncolytic M1 virus, thus enhancing a bystander killing effect. Here, we report that SMCs potentiate the oncolytic effect of M1 in vitro, in vivo, and ex vivo. This strengthened oncolytic efficacy resulted from the enhanced bystander killing effect caused by the M1 virus via cytokine induction. Through a microarray analysis and subsequent validation using recombinant cytokines, we identified IL-8, IL-1A, and TRAIL as the key cytokines in the bystander killing effect. Furthermore, SMCs increased the replication of M1, and the accumulation of virus protein induced irreversible endoplasmic reticulum stress- and c-Jun N-terminal kinase-mediated apoptosis. Nevertheless, the combined treatment with M1 and SMCs had little effect on normal and human primary cells. Because SMCs selectively and significantly enhance the bystander killing effect and the replication of oncolytic virus M1 specifically in cancer cells, this combined treatment may represent a promising therapeutic strategy.

Codon usage in Chlamydia trachomatis is the result of strand-specific mutational biases and a complex pattern of selective forces

PubMed Central

Romero, Héctor; Zavala, Alejandro; Musto, Héctor

2000-01-01

The patterns of synonymous codon choices of the completely sequenced genome of the bacterium Chlamydia trachomatis were analysed. We found that the most important source of variation among the genes results from whether the sequence is located on the leading or lagging strand of replication, resulting in an over representation of G or C, respectively. This can be explained by different mutational biases associated to the different enzymes that replicate each strand. Next we found that most highly expressed sequences are located on the leading strand of replication. From this result, replicational-transcriptional selection can be invoked. Then, when the genes located on the leading strand are studied separately, the correspondence analysis detects a principal trend which discriminates between lowly and highly expressed sequences, the latter displaying a different codon usage pattern than the former, suggesting selection for translation, which is reinforced by the fact that Ks values between orthologous sequences from C.trachomatis and Chlamydia pneumoniae are much smaller in highly expressed genes. Finally, synonymous codon choices appear to be influenced by the hydropathy of each encoded protein and by the degree of amino acid conservation. Therefore, synonymous codon usage in C.trachomatis seems to be the result of a very complex balance among different factors, which rises the problem of whether the forces driving codon usage patterns among microorganisms are rather more complex than generally accepted. PMID:10773076

Codon usage in Chlamydia trachomatis is the result of strand-specific mutational biases and a complex pattern of selective forces.

PubMed

Romero, H; Zavala, A; Musto, H

2000-05-15

The patterns of synonymous codon choices of the completely sequenced genome of the bacterium Chlamydia trachomatis were analysed. We found that the most important source of variation among the genes results from whether the sequence is located on the leading or lagging strand of replication, resulting in an over representation of G or C, respectively. This can be explained by different mutational biases associated to the different enzymes that replicate each strand. Next we found that most highly expressed sequences are located on the leading strand of replication. From this result, replicational-transcriptional selection can be invoked. Then, when the genes located on the leading strand are studied separately, the correspondence analysis detects a principal trend which discriminates between lowly and highly expressed sequences, the latter displaying a different codon usage pattern than the former, suggesting selection for translation, which is reinforced by the fact that Ks values between orthologous sequences from C. trachomatis and Chlamydia pneumoniae are much smaller in highly expressed genes. Finally, synonymous codon choices appear to be influenced by the hydropathy of each encoded protein and by the degree of amino acid conservation. Therefore, synonymous codon usage in C.trachomatis seems to be the result of a very complex balance among different factors, which rises the problem of whether the forces driving codon usage patterns among microorganisms are rather more complex than generally accepted.

Life history trade-offs and relaxed selection can decrease bacterial virulence in environmental reservoirs.

PubMed

Mikonranta, Lauri; Friman, Ville-Petri; Laakso, Jouni

2012-01-01

Pathogen virulence is usually thought to evolve in reciprocal selection with the host. While this might be true for obligate pathogens, the life histories of opportunistic pathogens typically alternate between within-host and outside-host environments during the infection-transmission cycle. As a result, opportunistic pathogens are likely to experience conflicting selection pressures across different environments, and this could affect their virulence through life-history trait correlations. We studied these correlations experimentally by exposing an opportunistic bacterial pathogen Serratia marcescens to its natural protist predator Tetrahymena thermophila for 13 weeks, after which we measured changes in bacterial traits related to both anti-predator defence and virulence. We found that anti-predator adaptation (producing predator-resistant biofilm) caused a correlative attenuation in virulence. Even though the direct mechanism was not found, reduction in virulence was most clearly connected to a predator-driven loss of a red bacterial pigment, prodigiosin. Moreover, life-history trait evolution was more divergent among replicate populations in the absence of predation, leading also to lowered virulence in some of the 'predator absent' selection lines. Together these findings suggest that the virulence of non-obligatory, opportunistic bacterial pathogens can decrease in environmental reservoirs through life history trade-offs, or random accumulation of mutations that impair virulence traits under relaxed selection.

Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle.

PubMed

Serre, Stéphanie B N; Jensen, Sanne B; Ghanem, Lubna; Humes, Daryl G; Ramirez, Santseharay; Li, Yi-Ping; Krarup, Henrik; Bukh, Jens; Gottwein, Judith M

2016-06-01

Hepatitis C virus (HCV) NS3 protease inhibitors (PIs) are important components of novel HCV therapy regimens. Studies of PI resistance initially focused on genotype 1. Therefore, knowledge about the determinants of PI resistance for the highly prevalent genotypes 2 to 6 remains limited. Using Huh7.5 cell culture-infectious HCV recombinants with genotype 1 to 6 NS3 protease, we identified protease positions 54, 155, and 156 as hot spots for the selection of resistance substitutions under treatment with the first licensed PIs, telaprevir and boceprevir. Treatment of a genotype 2 isolate with the newer PIs vaniprevir, faldaprevir, simeprevir, grazoprevir, paritaprevir, and deldeprevir identified positions 156 and 168 as hot spots for resistance; the Y56H substitution emerged for three newer PIs. Substitution selection also depended on the specific recombinant. The substitutions identified conferred cross-resistance to several PIs; however, most substitutions selected under telaprevir or boceprevir treatment conferred less resistance to certain newer PIs. In a single-cycle production assay, across genotypes, PI treatment primarily decreased viral replication, which was rescued by PI resistance substitutions. The substitutions identified resulted in differential effects on viral fitness, depending on the original recombinant and the substitution. Across genotypes, fitness impairment induced by resistance substitutions was due primarily to decreased replication. Most combinations of substitutions that were identified increased resistance or fitness. Combinations of resistance substitutions with fitness-compensating substitutions either rescued replication or compensated for decreased replication by increasing assembly. This comprehensive study provides insight into the selection patterns and effects of PI resistance substitutions for HCV genotypes 1 to 6 in the context of the infectious viral life cycle, which is of interest for clinical and virological HCV research. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Selection of homeotic proteins for binding to a human DNA replication origin.

PubMed

de Stanchina, E; Gabellini, D; Norio, P; Giacca, M; Peverali, F A; Riva, S; Falaschi, A; Biamonti, G

2000-06-09

We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene. Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence. All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators. We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene. The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays. In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells. We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation. Copyright 2000 Academic Press.

Targeting Nucleotide Biosynthesis: A Strategy for Improving the Oncolytic Potential of DNA Viruses

PubMed Central

Irwin, Chad R.; Hitt, Mary M.; Evans, David H.

2017-01-01

The rapid growth of tumors depends upon elevated levels of dNTPs, and while dNTP concentrations are tightly regulated in normal cells, this control is often lost in transformed cells. This feature of cancer cells has been used to advantage to develop oncolytic DNA viruses. DNA viruses employ many different mechanisms to increase dNTP levels in infected cells, because the low concentration of dNTPs found in non-cycling cells can inhibit virus replication. By disrupting the virus-encoded gene(s) that normally promote dNTP biosynthesis, one can assemble oncolytic versions of these agents that replicate selectively in cancer cells. This review covers the pathways involved in dNTP production, how they are dysregulated in cancer cells, and the various approaches that have been used to exploit this biology to improve the tumor specificity of oncolytic viruses. In particular, we compare and contrast the ways that the different types of oncolytic virus candidates can directly modulate these processes. We limit our review to the large DNA viruses that naturally encode homologs of the cellular enzymes that catalyze dNTP biogenesis. Lastly, we consider how this knowledge might guide future development of oncolytic viruses. PMID:29018771

Biological Characteristics of H9N2 Avian Influenza Viruses from Healthy Chickens in Shanghai, China.

PubMed

Shi, Qingfeng; Wang, Qianli; Ju, Liwen; Xiong, Haiyan; Chen, Yue; Jiang, Lufang; Jiang, Qingwu

2016-12-10

BACKGROUND H9N2 avian influenza viruses that circulate in domestic poultry in eastern China pose challenges to human health. However, few studies have compared the biological characteristics of H9N2 viruses isolated from healthy chickens in Shanghai. MATERIAL AND METHODS Three H9N2 viruses - CK/SH/Y1/07, CK/SH/Y1/02, and CK/SH/23/13 - isolated from healthy chickens in Shanghai between 2002 and 2013, were selected and their biological characteristics were determined. RESULTS All 3 H9N2 viruses showed a preference for both the avian- and human-like receptors, and they replicated well in MDCK and A549 cells. All H9N2 viruses were non-pathogenic to mini-pigs and were detected in the trachea and lung tissues. The CK/SH/Y1/07 and CK/SH/Y1/02 viruses were transmitted to mini-pigs through direct-contact or respiratory droplet exposure, but CK/SH/23/13 virus was not. CONCLUSIONS These results suggest that H9N2 viruses isolated from healthy chickens in Shanghai efficiently replicate and transmit among pigs and other mammals.

Kin Selection in the RNA World.

PubMed

Levin, Samuel R; West, Stuart A

2017-12-05

Various steps in the RNA world required cooperation. Why did life's first inhabitants, from polymerases to synthetases, cooperate? We develop kin selection models of the RNA world to answer these questions. We develop a very simple model of RNA cooperation and then elaborate it to model three relevant issues in RNA biology: (1) whether cooperative RNAs receive the benefits of cooperation; (2) the scale of competition in RNA populations; and (3) explicit replicator diffusion and survival. We show: (1) that RNAs are likely to express partial cooperation; (2) that RNAs will need mechanisms for overcoming local competition; and (3) in a specific example of RNA cooperation, persistence after replication and offspring diffusion allow for cooperation to overcome competition. More generally, we show how kin selection can unify previously disparate answers to the question of RNA world cooperation.

A novel 3,4-dihydropyrimidin-2(1H)-one: HIV-1 replication inhibitors with improved metabolic stability.

PubMed

Kim, Junwon; Ok, Taedong; Park, Changmin; So, Wonyoung; Jo, Mina; Kim, Youngmi; Seo, Minjung; Lee, Doohyun; Jo, Suyeon; Ko, Yoonae; Choi, Inhee; Park, Youngsam; Yoon, Jaewan; Ju, Moon Kyeong; Ahn, JiYe; Kim, Junghwan; Han, Sung-Jun; Kim, Tae-Hee; Cechetto, Jonathan; Nam, Jiyoun; Liuzzi, Michel; Sommer, Peter; No, Zaesung

2012-04-01

Following the previous SAR of a novel dihydropyrimidinone scaffold as HIV-1 replication inhibitors a detailed study directed towards optimizing the metabolic stability of the ester functional group in the dihydropyrimidinone (DHPM) scaffold is described. Replacement of the ester moiety by thiazole ring significantly improved the metabolic stability while retaining antiviral activity against HIV-1 replication. These novel and potent DHPMs with bioisosteres could serve as advanced leads for further optimization. Copyright © 2012 Elsevier Ltd. All rights reserved.

Initiation preference at a yeast origin of replication.

PubMed

Brewer, B J; Fangman, W L

1994-04-12

Replication origins in the yeast Saccharomyces cerevisiae are identified as autonomous replication sequence (ARS) elements. To examine the effect of origin density on replication initiation, we have analyzed the replication of a plasmid that contains two copies of the same origin, ARS1. The activation of origins and the direction that replication forks move through flanking sequences can be physically determined by analyzing replication intermediates on two-dimensional agarose gels. We find that only one of the two identical ARSs on the plasmid initiates replication on any given plasmid molecule; that is, this close spacing of ARSs results in an apparent interference between the potential origins. Moreover, in the particular plasmid that we constructed, one of the two identical copies of ARS1 is used four times more frequently than the other one. These results show that the plasmid context is critical for determining the preferred origin. This origin preference is also exhibited when the tandem copies of ARS1 are introduced into a yeast chromosome. The sequences responsible for establishing the origin preference have been identified by deletion analysis and are found to reside in a portion of the yeast URA3 gene.

Asynchronous Replication and Autosome-Pair Non-Equivalence in Human Embryonic Stem Cells

PubMed Central

Dutta, Devkanya; Ensminger, Alexander W.; Zucker, Jacob P.; Chess, Andrew

2009-01-01

A number of mammalian genes exhibit the unusual properties of random monoallelic expression and random asynchronous replication. Such exceptional genes include genes subject to X inactivation and autosomal genes including odorant receptors, immunoglobulins, interleukins, pheromone receptors, and p120 catenin. In differentiated cells, random asynchronous replication of interspersed autosomal genes is coordinated at the whole chromosome level, indicative of chromosome-pair non-equivalence. Here we have investigated the replication pattern of the random asynchronously replicating genes in undifferentiated human embryonic stem cells, using fluorescence in situ hybridization based assay. We show that allele-specific replication of X-linked genes and random monoallelic autosomal genes occur in human embryonic stem cells. The direction of replication is coordinated at the whole chromosome level and can cross the centromere, indicating the existence of autosome-pair non-equivalence in human embryonic stem cells. These results suggest that epigenetic mechanism(s) that randomly distinguish between two parental alleles are emerging in the cells of the inner cell mass, the source of human embryonic stem cells. PMID:19325893

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.

PriC-mediated DNA replication restart requires PriC complex formation with the single-stranded DNA-binding protein.

PubMed

Wessel, Sarah R; Marceau, Aimee H; Massoni, Shawn C; Zhou, Ruobo; Ha, Taekjip; Sandler, Steven J; Keck, James L

2013-06-14

Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated with DNA replication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step of DNA replication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Single-molecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading.

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.

Discovery of 3,4-dihydropyrimidin-2(1H)-ones with inhibitory activity against HIV-1 replication.

PubMed

Kim, Junwon; Park, Changmin; Ok, Taedong; So, Wonyoung; Jo, Mina; Seo, Minjung; Kim, Youngmi; Sohn, Jeong-Hun; Park, Youngsam; Ju, Moon Kyeong; Kim, Junghwan; Han, Sung-Jun; Kim, Tae-Hee; Cechetto, Jonathan; Nam, Jiyoun; Sommer, Peter; No, Zaesung

2012-03-01

3,4-Dihydropyrimidin-2(1H)-ones (DHPMs) were selected and derivatized through a HIV-1 replication assay based on GFP reporter cells. Compounds 14, 25, 31, and 36 exhibited significant inhibition of HIV-1 replication with a good safety profile. Chiral separation of each enantiomer by fractional crystallization showed that only the S enantiomer retained anti-HIV activity. Compound (S)-40, a novel and potent DHPM analog, could serve as an advanced lead for further development and the determination of the mechanism of action. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

A 1-1/2-level on-chip-decoding bubble memory chip design

NASA Technical Reports Server (NTRS)

Chen, T. T.

1975-01-01

Design includes multi-channel replicator which can reduce chip-writing requirement, selective annihilating switch which can effectively annihilate bubbles with minimum delay, and modified transfer switch which can be used as selective steering-type decoder.

Spacetime Replication of Quantum Information Using (2 , 3) Quantum Secret Sharing and Teleportation

NASA Astrophysics Data System (ADS)

Wu, Yadong; Khalid, Abdullah; Davijani, Masoud; Sanders, Barry

The aim of this work is to construct a protocol to replicate quantum information in any valid configuration of causal diamonds and assess resources required to physically realize spacetime replication. We present a set of codes to replicate quantum information along with a scheme to realize these codes using continuous-variable quantum optics. We use our proposed experimental realizations to determine upper bounds on the quantum and classical resources required to simulate spacetime replication. For four causal diamonds, our implementation scheme is more efficient than the one proposed previously. Our codes are designed using a decomposition algorithm for complete directed graphs, (2 , 3) quantum secret sharing, quantum teleportation and entanglement swapping. These results show the simulation of spacetime replication of quantum information is feasible with existing experimental methods. Alberta Innovates, NSERC, China's 1000 Talent Plan and the Institute for Quantum Information and Matter, which is an NSF Physics Frontiers Center (NSF Grant PHY-1125565) with support of the Gordon and Betty Moore Foundation (GBMF-2644).

Histone H4 acetylation required for chromatin decompaction during DNA replication.

PubMed

Ruan, Kun; Yamamoto, Takaharu G; Asakawa, Haruhiko; Chikashige, Yuji; Kimura, Hiroshi; Masukata, Hisao; Haraguchi, Tokuko; Hiraoka, Yasushi

2015-07-30

Faithful DNA replication is a prerequisite for cell proliferation. Several cytological studies have shown that chromosome structures alter in the S-phase of the cell cycle. However, the molecular mechanisms behind the alteration of chromosome structures associated with DNA replication have not been elucidated. Here, we investigated chromatin structures and acetylation of specific histone residues during DNA replication using the meiotic nucleus of the fission yeast Schizosaccharomyces pombe. The S. pombe meiotic nucleus provides a unique opportunity for measuring the levels of compaction of chromatin along the chromosome in a defined orientation. By direct measurement of chromatin compaction in living cells, we demonstrated that decompaction of chromatin occurs during meiotic DNA replication. This chromatin decompaction was suppressed by depletion of histone acetyltransferase Mst1 or by arginine substitution of specific lysine residues (K8 and K12) of histone H4. These results suggest that acetylation of histone H4 residues K8 and K12 plays a critical role in loosening chromatin structures during DNA replication.

COPI Is Required for Enterovirus 71 Replication

PubMed Central

Wang, Jianmin; Wu, Zhiqiang; Jin, Qi

2012-01-01

Enterovirus 71 (EV71), a member of the Picornaviridae family, is found in Asian countries where it causes a wide range of human diseases. No effective therapy is available for the treatment of these infections. Picornaviruses undergo RNA replication in association with membranes of infected cells. COPI and COPII have been shown to be involved in the formation of picornavirus-induced vesicles. Replication of several picornaviruses, including poliovirus and Echovirus 11 (EV11), is dependent on COPI or COPII. Here, we report that COPI, but not COPII, is required for EV71 replication. Replication of EV71 was inhibited by brefeldin A and golgicide A, inhibitors of COPI activity. Furthermore, we found EV71 2C protein interacted with COPI subunits by co-immunoprecipitation and GST pull-down assay, indicating that COPI coatomer might be directed to the viral replication complex through viral 2C protein. Additionally, because the pathway is conserved among different species of enteroviruses, it may represent a novel target for antiviral therapies. PMID:22662263

Animal Mitochondrial DNA Replication

PubMed Central

Ciesielski, Grzegorz L.; Oliveira, Marcos T.; Kaguni, Laurie S.

2016-01-01

Recent advances in the field of mitochondrial DNA (mtDNA) replication highlight the diversity of both the mechanisms utilized and the structural and functional organization of the proteins at mtDNA replication fork, despite the simplicity of the animal mtDNA genome. DNA polymerase γ, mtDNA helicase and mitochondrial single-stranded DNA-binding protein- the key replisome proteins, have evolved distinct structural features and biochemical properties. These appear to be correlated with mtDNA genomic features in different metazoan taxa and with their modes of DNA replication, although a substantial integrative research is warranted to establish firmly these links. To date, several modes of mtDNA replication have been described for animals: rolling circle, theta, strand-displacement, and RITOLS/bootlace. Resolution of a continuing controversy relevant to mtDNA replication in mammals/vertebrates will have a direct impact on the mechanistic interpretation of mtDNA-related human diseases. Here we review these subjects, integrating earlier and recent data to provide a perspective on the major challenges for future research. PMID:27241933

Multiscale analysis of replication technique efficiency for 3D roughness characterization of manufactured surfaces

NASA Astrophysics Data System (ADS)

Jolivet, S.; Mezghani, S.; El Mansori, M.

2016-09-01

The replication of topography has been generally restricted to optimizing material processing technologies in terms of statistical and single-scale features such as roughness. By contrast, manufactured surface topography is highly complex, irregular, and multiscale. In this work, we have demonstrated the use of multiscale analysis on replicates of surface finish to assess the precise control of the finished replica. Five commercial resins used for surface replication were compared. The topography of five standard surfaces representative of common finishing processes were acquired both directly and by a replication technique. Then, they were characterized using the ISO 25178 standard and multiscale decomposition based on a continuous wavelet transform, to compare the roughness transfer quality at different scales. Additionally, atomic force microscope force modulation mode was used in order to compare the resins’ stiffness properties. The results showed that less stiff resins are able to replicate the surface finish along a larger wavelength band. The method was then tested for non-destructive quality control of automotive gear tooth surfaces.

Function of the Plant DNA Polymerase Epsilon in Replicative Stress Sensing, a Genetic Analysis.

PubMed

Pedroza-García, José-Antonio; Mazubert, Christelle; Del Olmo, Ivan; Bourge, Mickael; Domenichini, Séverine; Bounon, Rémi; Tariq, Zakia; Delannoy, Etienne; Piñeiro, Manuel; Jarillo, José A; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

2017-03-01

Faithful transmission of the genetic information is essential in all living organisms. DNA replication is therefore a critical step of cell proliferation, because of the potential occurrence of replication errors or DNA damage when progression of a replication fork is hampered causing replicative stress. Like other types of DNA damage, replicative stress activates the DNA damage response, a signaling cascade allowing cell cycle arrest and repair of lesions. The replicative DNA polymerase ε (Pol ε) was shown to activate the S-phase checkpoint in yeast in response to replicative stress, but whether this mechanism functions in multicellular eukaryotes remains unclear. Here, we explored the genetic interaction between Pol ε and the main elements of the DNA damage response in Arabidopsis ( Arabidopsis thaliana ). We found that mutations affecting the polymerase domain of Pol ε trigger ATR-dependent signaling leading to SOG1 activation, WEE1-dependent cell cycle inhibition, and tolerance to replicative stress induced by hydroxyurea, but result in enhanced sensitivity to a wide range of DNA damaging agents. Using knock-down lines, we also provide evidence for the direct role of Pol ε in replicative stress sensing. Together, our results demonstrate that the role of Pol ε in replicative stress sensing is conserved in plants, and provide, to our knowledge, the first genetic dissection of the downstream signaling events in a multicellular eukaryote. © 2017 American Society of Plant Biologists. All Rights Reserved.

New Therapeutic Strategies for Antibiotic-Resistant Select Agents

DTIC Science & Technology

2007-12-31

activity possibly due to inherent differences between mesophilic versus thermophilic replication enzymes (Figure 7A). E. coli helicase stimulated Y...primase activity possibly due to inherent differences between mesophilic versus thermophilic replication enzymes (Figure 7A). B E. coli...either lower the enzyme and template to 20 nM, or use higher compound concentrations that are the same as the enzyme and template. A series of

Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication, SirA, with domain I of DnaA

PubMed Central

Jameson, Katie H; Rostami, Nadia; Fogg, Mark J; Turkenburg, Johan P; Grahl, Anne; Murray, Heath; Wilkinson, Anthony J

2014-01-01

Chromosome copy number in cells is controlled so that the frequency of initiation of DNA replication matches that of cell division. In bacteria, this is achieved through regulation of the interaction between the initiator protein DnaA and specific DNA elements arrayed at the origin of replication. DnaA assembles at the origin and promotes DNA unwinding and the assembly of a replication initiation complex. SirA is a DnaA-interacting protein that inhibits initiation of replication in diploid Bacillus subtilis cells committed to the developmental pathway leading to formation of a dormant spore. Here we present the crystal structure of SirA in complex with the N-terminal domain of DnaA revealing a heterodimeric complex. The interacting surfaces of both proteins are α-helical with predominantly apolar side-chains packing in a hydrophobic interface. Site-directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo. Localization of GFP–SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA. The SirA interacting surface of DnaA corresponds closely to the HobA-interacting surface of DnaA from Helicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor. PMID:25041308

A Drop in the Bucket or a Pebble in a Pond: Commercial Building Partners’ Replication of EEMs Across Their Portfolios

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

Antonopoulos, Chrissi A.; Baechler, Michael C.; Dillon, Heather E.

This study presents findings from questionnaire and interview data investigating replication efforts of Commercial Building Partnership (CBP) partners that worked directly with the Pacific Northwest National Laboratory (PNNL). PNNL partnered with 12 organizations on new and retrofit construction projects as part of the U.S. Department of Energy (DOE) CBP program. PNNL and other national laboratories collaborate with industry leaders that own large portfolios of buildings to develop high performance projects for new construction and renovation. This project accelerates market adoption of commercially available energy saving technologies into the design process for new and upgraded commercial buildings. The labs provide assistancemore » to the partners’ design teams and make a business case for energy investments. From the owner’s perspective, a sound investment results in energy savings based on corporate objectives and design. Through a feedback questionnaire, along with personal interviews, PNNL gathered qualitative and quantitative information relating to replication efforts by each organization. Data through this process were analyzed to provide insight into two primary research areas: 1) CBP partners’ replication efforts of technologies and approaches used in the CBP project to the rest of the organization’s building portfolio (including replication verification), and, 2) the market potential for technology diffusion into the total U.S. commercial building stock, as a direct result of the CBP entire program.« less

Neural modulation of directed forgetting by valence and arousal: An event-related potential study.

PubMed

Gallant, Sara N; Dyson, Benjamin J

2016-10-01

Intentional forgetting benefits memory by removing no longer needed information and promoting processing of more relevant materials. This study sought to understand how the behavioural and neurophysiological representation of intentional forgetting would be impacted by emotion. We took a novel approach by examining the unique contribution of both valence and arousal on emotional directed forgetting. Participants completed an item directed forgetting task for positive, negative, and neutral words at high and lower levels of arousal while brain activity was recorded using electroencephalography (EEG). Behaviourally, recognition of to-be-remembered (TBR) and to-be-forgotten (TBF) items varied as a function of valence and arousal with reduced directed forgetting for high arousing negative and neutral words. In the brain, patterns of frontal and posterior activation in response to TBF and TBR cues respectively replicated prior EEG evidence to support involvement of inhibitory and selective rehearsal mechanisms in item directed forgetting. Interestingly, emotion only impacted cue-related posterior activity, which varied depending on specific interactions between valence and arousal. Together, results suggest that the brain handles valence and arousal differently and highlights the importance of considering in a collective manner the multidimensional nature of emotion in experimentation. Copyright © 2016 Elsevier B.V. All rights reserved.

Homologous recombination occurs in a distinct retroviral subpopulation and exhibits high negative interference.

PubMed Central

Hu, W S; Bowman, E H; Delviks, K A; Pathak, V K

1997-01-01

Homologous recombination and deletions occur during retroviral replication when reverse transcriptase switches templates. While recombination occurs solely by intermolecular template switching (between copackaged RNAs), deletions can occur by an intermolecular or an intramolecular template switch (within the same RNA). To directly compare the rates of intramolecular and intermolecular template switching, two spleen necrosis virus-based vectors were constructed. Each vector contained a 110-bp direct repeat that was previously shown to delete at a high rate. The 110-bp direct repeat was flanked by two different sets of restriction site markers. These vectors were used to form heterozygotic virions containing RNAs of each parental vector, from which recombinant viruses were generated. By analyses of the markers flanking the direct repeats in recombinant and nonrecombinant proviruses, the rates of intramolecular and intermolecular template switching were determined. The results of these analyses indicate that intramolecular template switching is much more efficient than intermolecular template switching and that direct repeat deletions occur primarily through intramolecular template switching events. These studies also indicate that retroviral recombination occurs within a distinct viral subpopulation and exhibits high negative interference, whereby the selection of one recombination event increases the probability that a second recombination event will be observed. PMID:9223494

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

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.

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.

Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.

PubMed

Burbank, Lindsey P; Stenger, Drake C

2016-08-01

The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

Non-adaptive plasticity potentiates rapid adaptive evolution of gene expression in nature.

PubMed

Ghalambor, Cameron K; Hoke, Kim L; Ruell, Emily W; Fischer, Eva K; Reznick, David N; Hughes, Kimberly A

2015-09-17

Phenotypic plasticity is the capacity for an individual genotype to produce different phenotypes in response to environmental variation. Most traits are plastic, but the degree to which plasticity is adaptive or non-adaptive depends on whether environmentally induced phenotypes are closer or further away from the local optimum. Existing theories make conflicting predictions about whether plasticity constrains or facilitates adaptive evolution. Debate persists because few empirical studies have tested the relationship between initial plasticity and subsequent adaptive evolution in natural populations. Here we show that the direction of plasticity in gene expression is generally opposite to the direction of adaptive evolution. We experimentally transplanted Trinidadian guppies (Poecilia reticulata) adapted to living with cichlid predators to cichlid-free streams, and tested for evolutionary divergence in brain gene expression patterns after three to four generations. We find 135 transcripts that evolved parallel changes in expression within the replicated introduction populations. These changes are in the same direction exhibited in a native cichlid-free population, suggesting rapid adaptive evolution. We find 89% of these transcripts exhibited non-adaptive plastic changes in expression when the source population was reared in the absence of predators, as they are in the opposite direction to the evolved changes. By contrast, the remaining transcripts exhibiting adaptive plasticity show reduced population divergence. Furthermore, the most plastic transcripts in the source population evolved reduced plasticity in the introduction populations, suggesting strong selection against non-adaptive plasticity. These results support models predicting that adaptive plasticity constrains evolution, whereas non-adaptive plasticity potentiates evolution by increasing the strength of directional selection. The role of non-adaptive plasticity in evolution has received relatively little attention; however, our results suggest that it may be an important mechanism that predicts evolutionary responses to new environments.

Measurements of experimental precision for trials with cowpea (Vigna unguiculata L. Walp.) genotypes.

PubMed

Teodoro, P E; Torres, F E; Santos, A D; Corrêa, A M; Nascimento, M; Barroso, L M A; Ceccon, G

2016-05-09

The aim of this study was to evaluate the suitability of statistics as experimental precision degree measures for trials with cowpea (Vigna unguiculata L. Walp.) genotypes. Cowpea genotype yields were evaluated in 29 trials conducted in Brazil between 2005 and 2012. The genotypes were evaluated with a randomized block design with four replications. Ten statistics that were estimated for each trial were compared using descriptive statistics, Pearson correlations, and path analysis. According to the class limits established, selective accuracy and F-test values for genotype, heritability, and the coefficient of determination adequately estimated the degree of experimental precision. Using these statistics, 86.21% of the trials had adequate experimental precision. Selective accuracy and the F-test values for genotype, heritability, and the coefficient of determination were directly related to each other, and were more suitable than the coefficient of variation and the least significant difference (by the Tukey test) to evaluate experimental precision in trials with cowpea genotypes.

Low-level sensory plasticity during task-irrelevant perceptual learning: Evidence from conventional and double training procedures

PubMed Central

Pilly, Praveen K.; Grossberg, Stephen; Seitz, Aaron R.

2009-01-01

Studies of perceptual learning have focused on aspects of learning that are related to early stages of sensory processing. However, conclusions that perceptual learning results in low-level sensory plasticity are controversial, since such learning may also be attributed to plasticity in later stages of sensory processing or in readout from sensory to decision stages, or to changes in high-level central processing. To address this controversy, we developed a novel random dot motion (RDM) stimulus to target motion cells selective to contrast polarity by ensuring the motion direction information arises only from signal dot onsets and not their offsets, and used these stimuli in the paradigm of task-irrelevant perceptual learning (TIPL). In TIPL, learning is achieved in response to a stimulus by subliminally pairing that stimulus with the targets of an unrelated training task. In this manner, we are able to probe learning for an aspect of motion processing thought to be a function of directional V1 simple cells with a learning procedure that dissociates the learned stimulus from the decision processes relevant to the training task. Our results show direction-selective learning for the designated contrast polarity that does not transfer to the opposite contrast polarity. This polarity specificity was replicated in a double training procedure in which subjects were additionally exposed to the opposite polarity. Taken together, these results suggest that TIPL for motion stimuli may occur at the stage of directional V1 simple cells. Finally, a theoretical explanation is provided to understand the data. PMID:19800358

Genetic parameters and path analysis in cowpea genotypes grown in the Cerrado/Pantanal ecotone.

PubMed

Lopes, K V; Teodoro, P E; Silva, F A; Silva, M T; Fernandes, R L; Rodrigues, T C; Faria, T C; Corrêa, A M

2017-05-18

Estimating genetic parameters in plant breeding allows us to know the population potential for selecting and designing strategies that can maximize the achievement of superior genotypes. The objective of this study was to evaluate the genetic potential of a population of 20 cowpea genotypes by estimating genetic parameters and path analysis among the traits to guide the selection strategies. The trial was conducted in randomized block design with four replications. Its morphophysiological components, components of green grain production and dry grain yield were estimated from genetic use and correlations between the traits. Phenotypic correlations were deployed through path analysis into direct and indirect effects of morphophysiological traits and yield components on dry grain yield. There were significant differences (P < 0.01) between the genotypes for most the traits, indicating the presence of genetic variability in the population and the possibility of practicing selection. The population presents the potential for future genetic breeding studies and is highly promising for the selection of traits dry grain yield, the number of grains per pod, and hundred grains mass. A number of grains per green pod is the main determinant trait of dry grain yield that is also influenced by the cultivar cycle and that the selection for the dry grain yield can be made indirectly by selecting the green pod mass and green pod length.

Correlation, path analysis and heritability estimation for agronomic traits contribute to yield on soybean

NASA Astrophysics Data System (ADS)

Sulistyo, A.; Purwantoro; Sari, K. P.

2018-01-01

Selection is a routine activity in plant breeding programs that must be done by plant breeders in obtaining superior plant genotypes. The use of appropriate selection criteria will determine the effectiveness of selection activities. The purpose of this study was to analysis the inheritable agronomic traits that contribute to soybean yield. A total of 91 soybean lines were planted in Muneng Experimental Station, Probolinggo District, East Java Province, Indonesia in 2016. All soybean lines were arranged in randomized complete block design with two replicates. Correlation analysis, path analysis and heritability estimation were performed on days to flowering, days to maturing, plant height, number of branches, number of fertile nodes, number of filled pods, weight of 100 seeds, and yield to determine selection criteria on soybean breeding program. The results showed that the heritability value of almost all agronomic traits observed is high except for the number of fertile nodes with low heritability. The result of correlation analysis shows that days to flowering, plant height and number of fertile nodes have positive correlation with seed yield per plot (0.056, 0.444, and 0.100, respectively). In addition, path analysis showed that plant height and number of fertile nodes have highest positive direct effect on soybean yield. Based on this result, plant height can be selected as one of selection criteria in soybean breeding program to obtain high yielding soybean variety.

The two faces of selective memory retrieval: recall specificity of the detrimental but not the beneficial effect.

PubMed

Bäuml, Karl-Heinz T; Dobler, Ina M

2015-01-01

Depending on the degree to which the original study context is accessible, selective memory retrieval can be detrimental or beneficial for the recall of other memories (Bäuml & Samenieh, 2012). Prior work has shown that the detrimental effect of memory retrieval is typically recall specific and does not arise after restudy trials, whereas recall specificity of the beneficial effect has not been examined to date. Addressing the issue, we compared in 2 experiments the effects of retrieval and restudy on recall of other items, when access to the study context was (largely) maintained and when access to the study context was impaired (in Experiment 1 by using the listwise directed-forgetting task, in Experiment 2 by using a prolonged retention interval). In both experiments, selective retrieval but not restudy induced forgetting of other items when context access was maintained, which replicates prior work. In contrast, when context access was impaired, both selective retrieval and restudy induced beneficial effects on other memories. These findings suggest that the detrimental but not the beneficial effect of selective memory retrieval is recall specific. The results are consistent with a recent 2-factor account of selective memory retrieval that attributes the detrimental effect to inhibition or blocking but the beneficial effect to context reactivation processes. PsycINFO Database Record (c) 2015 APA, 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

Replication Origins and Timing of Temporal Replication in Budding Yeast: How to Solve the Conundrum?

PubMed Central

Barberis, Matteo; Spiesser, Thomas W.; Klipp, Edda

2010-01-01

Similarly to metazoans, the budding yeast Saccharomyces cereviasiae replicates its genome with a defined timing. In this organism, well-defined, site-specific origins, are efficient and fire in almost every round of DNA replication. However, this strategy is neither conserved in the fission yeast Saccharomyces pombe, nor in Xenopus or Drosophila embryos, nor in higher eukaryotes, in which DNA replication initiates asynchronously throughout S phase at random sites. Temporal and spatial controls can contribute to the timing of replication such as Cdk activity, origin localization, epigenetic status or gene expression. However, a debate is going on to answer the question how individual origins are selected to fire in budding yeast. Two opposing theories were proposed: the “replicon paradigm” or “temporal program” vs. the “stochastic firing”. Recent data support the temporal regulation of origin activation, clustering origins into temporal blocks of early and late replication. Contrarily, strong evidences suggest that stochastic processes acting on origins can generate the observed kinetics of replication without requiring a temporal order. In mammalian cells, a spatiotemporal model that accounts for a partially deterministic and partially stochastic order of DNA replication has been proposed. Is this strategy the solution to reconcile the conundrum of having both organized replication timing and stochastic origin firing also for budding yeast? In this review we discuss this possibility in the light of our recent study on the origin activation, suggesting that there might be a stochastic component in the temporal activation of the replication origins, especially under perturbed conditions. PMID:21037857

Fabrication and testing of Wolter type-I mirrors for soft x-ray microscopes

NASA Astrophysics Data System (ADS)

Hoshino, Masato; Aoki, Sadao; Watanabe, Norio; Hirai, Shinichiro

2004-10-01

Development of a small Wolter type-I mirror that is mainly used as an objective for the X-ray microscope is described. Small Wolter mirrors for X-ray microscopes are fabricated by the vacuum replication method because of their long aspherical shape. Master mandrel is ground and polished by an ultra-precision NC lathe. Tungsten carbide was selected as a material because its thermal expansion coefficient is a little larger than the replica glass. It was ground by ELID (Electrolytic In-process Dressing) grinding technique that is appropriate for the efficient mirror surface grinding. After ultra-precision grinding, the figure error of master mandrel was better than 0.5μm except the boundary between the hyperboloid and the ellipsoid. Before vacuum replication, the mandrel was coated with Au (thickness 50nm) as the parting layer. Pyrex glass was empirically selected as mirror material. The master mandrel was inserted into the Pyrex glass tube and heated up to 675°C in the electric furnace. Although vacuum replication is a proper technique in terms of its high replication accuracy, the surface roughness characterized by the high spatial frequency of the mandrel was replicated less accurate than the figure error characterized by the low spatial frequency. This indicates that the surface roughness and the figure error depend on the glass surface and the figure error of the master mandrel, respectively. A fabricated mirror was evaluated by the imaging performance with a laser plasma X-ray source (λ=3.2nm).

Selective and uncoupled role of substrate elasticity in the regulation of replication and transcription in epithelial cells.

PubMed

Kocgozlu, Leyla; Lavalle, Philippe; Koenig, Géraldine; Senger, Bernard; Haikel, Youssef; Schaaf, Pierre; Voegel, Jean-Claude; Tenenbaum, Henri; Vautier, Dominique

2010-01-01

Actin cytoskeleton forms a physical connection between the extracellular matrix, adhesion complexes and nuclear architecture. Because tissue stiffness plays key roles in adhesion and cytoskeletal organization, an important open question concerns the influence of substrate elasticity on replication and transcription. To answer this major question, polyelectrolyte multilayer films were used as substrate models with apparent elastic moduli ranging from 0 to 500 kPa. The sequential relationship between Rac1, vinculin adhesion assembly, and replication becomes efficient at above 200 kPa because activation of Rac1 leads to vinculin assembly, actin fiber formation and, subsequently, to initiation of replication. An optimal window of elasticity (200 kPa) is required for activation of focal adhesion kinase through auto-phosphorylation of tyrosine 397. Transcription, including nuclear recruitment of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), occurred above 50 kPa. Actin fiber and focal adhesion signaling are not required for transcription. Above 50 kPa, transcription was correlated with alphav-integrin engagement together with histone H3 hyperacetylation and chromatin decondensation, allowing little cell spreading. By contrast, soft substrate (below 50 kPa) promoted morphological changes characteristic of apoptosis, including cell rounding, nucleus condensation, loss of focal adhesions and exposure of phosphatidylserine at the outer cell surface. On the basis of our data, we propose a selective and uncoupled contribution from the substrate elasticity to the regulation of replication and transcription activities for an epithelial cell model.

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

PubMed Central

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

2012-01-01

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

Cellular replication limits in the Luria-Delbrück mutation model

NASA Astrophysics Data System (ADS)

Rodriguez-Brenes, Ignacio A.; Wodarz, Dominik; Komarova, Natalia L.

2016-08-01

Originally developed to elucidate the mechanisms of natural selection in bacteria, the Luria-Delbrück model assumed that cells are intrinsically capable of dividing an unlimited number of times. This assumption however, is not true for human somatic cells which undergo replicative senescence. Replicative senescence is thought to act as a mechanism to protect against cancer and the escape from it is a rate-limiting step in cancer progression. Here we introduce a Luria-Delbrück model that explicitly takes into account cellular replication limits in the wild type cell population and models the emergence of mutants that escape replicative senescence. We present results on the mean, variance, distribution, and asymptotic behavior of the mutant population in terms of three classical formulations of the problem. More broadly the paper introduces the concept of incorporating replicative limits as part of the Luria-Delbrück mutational framework. Guidelines to extend the theory to include other types of mutations and possible applications to the modeling of telomere crisis and fluctuation analysis are also discussed.

Maximal gene number maintainable by stochastic correction - The second error threshold.

PubMed

Hubai, András G; Kun, Ádám

2016-09-21

There is still no general solution to Eigen׳s Paradox, the chicken-or-egg problem of the origin of life: neither accurate copying, nor long genomes could have evolved without one another being established beforehand. But an array of small, individually replicating genes might offer a workaround, provided that multilevel selection assists the survival of the ensemble. There are two key difficulties that such a system has to overcome: the non-synchronous replication of genes, and their random assortment into daughter cells (the units of higher-level selection) upon fission. Here we find, using the Stochastic Corrector Model framework, that a large number (τ≥90) of genes can coexist. Furthermore, the system can tolerate about 10% replication rate asymmetry (competition) among the genes. On this basis, we put forward a plausible (and testable!) scenario for how novel genes could have been incorporated into early living systems: a route to complex metabolism. Copyright © 2016 Elsevier Ltd. All rights reserved.

A dynamic replication management strategy in distributed GIS

NASA Astrophysics Data System (ADS)

Pan, Shaoming; Xiong, Lian; Xu, Zhengquan; Chong, Yanwen; Meng, Qingxiang

2018-03-01

Replication strategy is one of effective solutions to meet the requirement of service response time by preparing data in advance to avoid the delay of reading data from disks. This paper presents a brand-new method to create copies considering the selection of replicas set, the number of copies for each replica and the placement strategy of all copies. First, the popularities of all data are computed considering both the historical access records and the timeliness of the records. Then, replica set can be selected based on their recent popularities. Also, an enhanced Q-value scheme is proposed to assign the number of copies for each replica. Finally, a reasonable copies placement strategy is designed to meet the requirement of load balance. In addition, we present several experiments that compare the proposed method with techniques that use other replication management strategies. The results show that the proposed model has better performance than other algorithms in all respects. Moreover, the experiments based on different parameters also demonstrated the effectiveness and adaptability of the proposed algorithm.

Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields

PubMed Central

Carnes, Aaron E.; Luke, Jeremy M.; Vincent, Justin M.; Anderson, Sheryl; Schukar, Angela; Hodgson, Clague P.; Williams, James A.

2010-01-01

Background For safety considerations, regulatory agencies recommend elimination of antibiotic resistance markers and nonessential sequences from plasmid DNA-based gene medicines. In the present study we analyzed antibiotic-free (AF) vector design criteria impacting bacterial production and mammalian transgene expression. Methods Both CMV-HTLV-I R RNA Pol II promoter (protein transgene) and murine U6 RNA Pol III promoter (RNA transgene) vector designs were studied. Plasmid production yield was assessed through inducible fed-batch fermentation. RNA Pol II-directed EGFP and RNA Pol III-directed RNA expression were quantified by fluorometry and quantitative real-time polymerase chain reaction (RT-PCR), respectively, after transfection of human HEK293 cells. Results Sucrose-selectable minimalized protein and therapeutic RNA expression vector designs that combined an RNA-based AF selection with highly productive fermentation manufacturing (>1,000 mg/L plasmid DNA) and high level in vivo expression of encoded products were identified. The AF selectable marker was also successfully applied to convert existing kanamycin-resistant DNA vaccine plasmids gWIZ and pVAX1 into AF vectors, demonstrating a general utility for retrofitting existing vectors. A minimum vector size for high yield plasmid fermentation was identified. A strategy for stable fermentation of plasmid dimers with improved vector potency and fermentation yields up to 1,740 mg/L was developed. Conclusions We report the development of potent high yield AF gene medicine expression vectors for protein or RNA (e.g. short hairpin RNA or microRNA) products. These AF expression vectors were optimized to exceed a newly identified size threshold for high copy plasmid replication and direct higher transgene expression levels than alternative vectors. PMID:20806425

Validation of reliable and selective methods for direct determination of glyphosate and aminomethylphosphonic acid in milk and urine using LC-MS/MS.

PubMed

Jensen, Pamela K; Wujcik, Chad E; McGuire, Michelle K; McGuire, Mark A

2016-01-01

Simple high-throughput procedures were developed for the direct analysis of glyphosate [N-(phosphonomethyl)glycine] and aminomethylphosphonic acid (AMPA) in human and bovine milk and human urine matrices. Samples were extracted with an acidified aqueous solution on a high-speed shaker. Stable isotope labeled internal standards were added with the extraction solvent to ensure accurate tracking and quantitation. An additional cleanup procedure using partitioning with methylene chloride was required for milk matrices to minimize the presence of matrix components that can impact the longevity of the analytical column. Both analytes were analyzed directly, without derivatization, by liquid chromatography tandem mass spectrometry using two separate precursor-to-product transitions that ensure and confirm the accuracy of the measured results. Method performance was evaluated during validation through a series of assessments that included linearity, accuracy, precision, selectivity, ionization effects and carryover. Limits of quantitation (LOQ) were determined to be 0.1 and 10 µg/L (ppb) for urine and milk, respectively, for both glyphosate and AMPA. Mean recoveries for all matrices were within 89-107% at three separate fortification levels including the LOQ. Precision for replicates was ≤ 7.4% relative standard deviation (RSD) for milk and ≤ 11.4% RSD for urine across all fortification levels. All human and bovine milk samples used for selectivity and ionization effects assessments were free of any detectable levels of glyphosate and AMPA. Some of the human urine samples contained trace levels of glyphosate and AMPA, which were background subtracted for accuracy assessments. Ionization effects testing showed no significant biases from the matrix. A successful independent external validation was conducted using the more complicated milk matrices to demonstrate method transferability.

Registered Replication Report: Strack, Martin, & Stepper (1988).

PubMed

Acosta, Alberto; Adams, Reginald B; Albohn, Daniel N; Allard, Eric S; Beek, Titia; Benning, Stephen D; Blouin- Hudon, Eve-Marie; Bulnes, Luis Carlo; Caldwell, Tracy L; Calin-Jageman, Robert J; Capaldi, Colin A; Carfagno, Nicholas S; Chasten, Kelsie T; Cleeremans, Axel; Connell, Louise; DeCicco, Jennifer M.; Dijkhoff, Laura; Dijkstra, Katinka; Fischer, Agneta H; Foroni, Francesco; Gronau, Quentin F; Hess, Ursula; Holmes, Kevin J; Jones, Jacob L H; Klein, Olivier; Koch, Christopher; Korb, Sebastian; Lewinski, Peter; Liao, Julia D; Lund, Sophie; Lupiáñez, Juan; Lynott, Dermot; Nance, Christin N; Oosterwijk, Suzanne; Özdog˘ru, Asil Ali; Pacheco-Unguetti, Antonia Pilar; Pearson, Bethany; Powis, Christina; Riding, Sarah; Roberts, Tomi-Ann; Rumiati, Raffaella I; Senden, Morgane; Shea-Shumsky, Noah B; Sobocko, Karin; Soto, Jose A; Steiner, Troy G; Talarico, Jennifer M; vanAllen, Zack M; Wagenmakers, E-J; Vandekerckhove, Marie; Wainwright, Bethany; Wayand, Joseph F; Zeelenberg, Rene; Zetzer, Emily E; Zwaan, Rolf A

2016-11-01

According to the facial feedback hypothesis, people's affective responses can be influenced by their own facial expression (e.g., smiling, pouting), even when their expression did not result from their emotional experiences. For example, Strack, Martin, and Stepper (1988) instructed participants to rate the funniness of cartoons using a pen that they held in their mouth. In line with the facial feedback hypothesis, when participants held the pen with their teeth (inducing a "smile"), they rated the cartoons as funnier than when they held the pen with their lips (inducing a "pout"). This seminal study of the facial feedback hypothesis has not been replicated directly. This Registered Replication Report describes the results of 17 independent direct replications of Study 1 from Strack et al. (1988), all of which followed the same vetted protocol. A meta-analysis of these studies examined the difference in funniness ratings between the "smile" and "pout" conditions. The original Strack et al. (1988) study reported a rating difference of 0.82 units on a 10-point Likert scale. Our meta-analysis revealed a rating difference of 0.03 units with a 95% confidence interval ranging from -0.11 to 0.16. © The Author(s) 2016.

Unreliable Yet Still Replicable: A Comment on LeBel and Paunonen (2011)

PubMed Central

De Schryver, Maarten; Hughes, Sean; Rosseel, Yves; De Houwer, Jan

2016-01-01

Lebel and Paunonen (2011) highlight that despite their importance and popularity in both theoretical and applied research, many implicit measures continue to be plagued by a persistent and troublesome issue—low reliability. In their paper, they offer a conceptual analysis of the relationship between reliability, power and replicability, and then provide a series of recommendations for researchers interested in using implicit measures in an experimental setting. At the core of their account is the idea that reliability can be equated with statistical power, such that “lower levels of reliability are associated with decreasing probabilities of detecting a statistically significant effect, given one exists in the population” (p. 573). They also take the additional step of equating reliability and replicability. In our commentary, we draw attention to the fact that there is no direct, fixed or one-to-one relation between reliability and power or replicability. More specifically, we argue that when adopting an experimental (rather than a correlational) approach, researchers strive to minimize inter-individual variation, which has a direct impact on sample based reliability estimates. We evaluate the strengths and weaknesses of the LeBel and Paunonen's recommendations and refine them where appropriate. PMID:26793150

The fundamental units, processes and patterns of evolution, and the Tree of Life conundrum

PubMed Central

Koonin, Eugene V; Wolf, Yuri I

2009-01-01

Background The elucidation of the dominant role of horizontal gene transfer (HGT) in the evolution of prokaryotes led to a severe crisis of the Tree of Life (TOL) concept and intense debates on this subject. Concept Prompted by the crisis of the TOL, we attempt to define the primary units and the fundamental patterns and processes of evolution. We posit that replication of the genetic material is the singular fundamental biological process and that replication with an error rate below a certain threshold both enables and necessitates evolution by drift and selection. Starting from this proposition, we outline a general concept of evolution that consists of three major precepts. 1. The primary agency of evolution consists of Fundamental Units of Evolution (FUEs), that is, units of genetic material that possess a substantial degree of evolutionary independence. The FUEs include both bona fide selfish elements such as viruses, viroids, transposons, and plasmids, which encode some of the information required for their own replication, and regular genes that possess quasi-independence owing to their distinct selective value that provides for their transfer between ensembles of FUEs (genomes) and preferential replication along with the rest of the recipient genome. 2. The history of replication of a genetic element without recombination is isomorphously represented by a directed tree graph (an arborescence, in the graph theory language). Recombination within a FUE is common between very closely related sequences where homologous recombination is feasible but becomes negligible for longer evolutionary distances. In contrast, shuffling of FUEs occurs at all evolutionary distances. Thus, a tree is a natural representation of the evolution of an individual FUE on the macro scale, but not of an ensemble of FUEs such as a genome. 3. The history of life is properly represented by the "forest" of evolutionary trees for individual FUEs (Forest of Life, or FOL). Search for trends and patterns in the FOL is a productive direction of study that leads to the delineation of ensembles of FUEs that evolve coherently for a certain time span owing to a shared history of vertical inheritance or horizontal gene transfer; these ensembles are commonly known as genomes, taxa, or clades, depending on the level of analysis. A small set of genes (the universal genetic core of life) might show a (mostly) coherent evolutionary trend that transcends the entire history of cellular life forms. However, it might not be useful to denote this trend "the tree of life", or organismal, or species tree because neither organisms nor species are fundamental units of life. Conclusion A logical analysis of the units and processes of biological evolution suggests that the natural fundamental unit of evolution is a FUE, that is, a genetic element with an independent evolutionary history. Evolution of a FUE on the macro scale is naturally represented by a tree. Only the full compendium of trees for individual FUEs (the FOL) is an adequate depiction of the evolution of life. Coherent evolution of FUEs over extended evolutionary intervals is a crucial aspect of the history of life but a "species" or "organismal" tree is not a fundamental concept. Reviewers This articles was reviewed by Valerian Dolja, W. Ford Doolittle, Nicholas Galtier, and William Martin PMID:19788730

A new processing scheme for ultra-high resolution direct infusion mass spectrometry data

NASA Astrophysics Data System (ADS)

Zielinski, Arthur T.; Kourtchev, Ivan; Bortolini, Claudio; Fuller, Stephen J.; Giorio, Chiara; Popoola, Olalekan A. M.; Bogialli, Sara; Tapparo, Andrea; Jones, Roderic L.; Kalberer, Markus

2018-04-01

High resolution, high accuracy mass spectrometry is widely used to characterise environmental or biological samples with highly complex composition enabling the identification of chemical composition of often unknown compounds. Despite instrumental advancements, the accurate molecular assignment of compounds acquired in high resolution mass spectra remains time consuming and requires automated algorithms, especially for samples covering a wide mass range and large numbers of compounds. A new processing scheme is introduced implementing filtering methods based on element assignment, instrumental error, and blank subtraction. Optional post-processing incorporates common ion selection across replicate measurements and shoulder ion removal. The scheme allows both positive and negative direct infusion electrospray ionisation (ESI) and atmospheric pressure photoionisation (APPI) acquisition with the same programs. An example application to atmospheric organic aerosol samples using an Orbitrap mass spectrometer is reported for both ionisation techniques resulting in final spectra with 0.8% and 8.4% of the peaks retained from the raw spectra for APPI positive and ESI negative acquisition, respectively.

Developing approaches for linear mixed modeling in landscape genetics through landscape-directed dispersal simulations

USGS Publications Warehouse

Row, Jeffrey R.; Knick, Steven T.; Oyler-McCance, Sara J.; Lougheed, Stephen C.; Fedy, Bradley C.

2017-01-01

Dispersal can impact population dynamics and geographic variation, and thus, genetic approaches that can establish which landscape factors influence population connectivity have ecological and evolutionary importance. Mixed models that account for the error structure of pairwise datasets are increasingly used to compare models relating genetic differentiation to pairwise measures of landscape resistance. A model selection framework based on information criteria metrics or explained variance may help disentangle the ecological and landscape factors influencing genetic structure, yet there are currently no consensus for the best protocols. Here, we develop landscape-directed simulations and test a series of replicates that emulate independent empirical datasets of two species with different life history characteristics (greater sage-grouse; eastern foxsnake). We determined that in our simulated scenarios, AIC and BIC were the best model selection indices and that marginal R2 values were biased toward more complex models. The model coefficients for landscape variables generally reflected the underlying dispersal model with confidence intervals that did not overlap with zero across the entire model set. When we controlled for geographic distance, variables not in the underlying dispersal models (i.e., nontrue) typically overlapped zero. Our study helps establish methods for using linear mixed models to identify the features underlying patterns of dispersal across a variety of landscapes.

Preschool children adapt grasping movements to upcoming object manipulations: Evidence from a dial rotation task.

PubMed

Herbort, Oliver; Büschelberger, Juliane; Janczyk, Markus

2018-03-01

In adults, the motor plans for object-directed grasping movements reflects the anticipated requirements of intended future object manipulations. This prospective mode of planning has been termed second-order planning. Surprisingly, second-order planning is thought to be fully developed only by 10 years of age, when children master seemingly more complex motor skills. In this study, we tested the hypothesis that already 5- and 6-year-old children consistently use second-order planning but that this ability does not become apparent in tasks that are traditionally used to probe it. We asked 5- and 6-year-olds and adults to grasp and rotate a circular dial in a clockwise or counterclockwise direction. Although children's grasp selections were less consistent on an intra- and inter-individual level than adults' grasp selections, all children adjusted their grasps to the upcoming dial rotations. By contrast, in an also administered bar rotation task, only a subset of children adjusted their grasps to different bar rotations, thereby replicating previous results. The results indicate that 5- and 6-year-olds consistently use second-order planning in a dial rotation task, although this ability does not become apparent in bar rotation tasks. Copyright © 2017 Elsevier Inc. All rights reserved.

Kin Selection in the RNA World

PubMed Central

West, Stuart A.

2017-01-01

Various steps in the RNA world required cooperation. Why did life’s first inhabitants, from polymerases to synthetases, cooperate? We develop kin selection models of the RNA world to answer these questions. We develop a very simple model of RNA cooperation and then elaborate it to model three relevant issues in RNA biology: (1) whether cooperative RNAs receive the benefits of cooperation; (2) the scale of competition in RNA populations; and (3) explicit replicator diffusion and survival. We show: (1) that RNAs are likely to express partial cooperation; (2) that RNAs will need mechanisms for overcoming local competition; and (3) in a specific example of RNA cooperation, persistence after replication and offspring diffusion allow for cooperation to overcome competition. More generally, we show how kin selection can unify previously disparate answers to the question of RNA world cooperation. PMID:29206171

Hormones and the Evolution of Complex Traits: Insights from Artificial Selection on Behavior.

PubMed

Garland, Theodore; Zhao, Meng; Saltzman, Wendy

2016-08-01

Although behavior may often be a fairly direct target of natural or sexual selection, it cannot evolve without changes in subordinate traits that cause or permit its expression. In principle, changes in endocrine function could be a common mechanism underlying behavioral evolution because they are well positioned to mediate integrated responses to behavioral selection. More specifically, hormones can influence both motivational (e.g., brain) and performance (e.g., muscles) components of behavior simultaneously and in a coordinated fashion. If the endocrine system is often "used" as a general mechanism to effect responses to selection, then correlated responses in other aspects of behavior, life history, and organismal performance (e.g., locomotor abilities) should commonly occur because any cell with appropriate receptors could be affected. Ways in which behavior coadapts with other aspects of the phenotype can be studied directly through artificial selection and experimental evolution. Several studies have targeted rodent behavior for selective breeding and reported changes in other aspects of behavior, life history, and lower-level effectors of these organismal traits, including endocrine function. One example involves selection for high levels of voluntary wheel running, one aspect of physical activity, in four replicate High Runner (HR) lines of mice. Circulating levels of several hormones (including insulin, testosterone, thyroxine, triiodothyronine) have been characterized, three of which-corticosterone, leptin, and adiponectin-differ between HR and control lines, depending on sex, age, and generation. Potential changes in circulating levels of other behaviorally and metabolically relevant hormones, as well as in other components of the endocrine system (e.g., receptors), have yet to be examined. Overall, results to date identify promising avenues for further studies on the endocrine basis of activity levels. © The Author 2016. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Comparative metagenomic, phylogenetic and physiological analyses of soil microbial communities across nitrogen gradients.

PubMed

Fierer, Noah; Lauber, Christian L; Ramirez, Kelly S; Zaneveld, Jesse; Bradford, Mark A; Knight, Rob

2012-05-01

Terrestrial ecosystems are receiving elevated inputs of nitrogen (N) from anthropogenic sources and understanding how these increases in N availability affect soil microbial communities is critical for predicting the associated effects on belowground ecosystems. We used a suite of approaches to analyze the structure and functional characteristics of soil microbial communities from replicated plots in two long-term N fertilization experiments located in contrasting systems. Pyrosequencing-based analyses of 16S rRNA genes revealed no significant effects of N fertilization on bacterial diversity, but significant effects on community composition at both sites; copiotrophic taxa (including members of the Proteobacteria and Bacteroidetes phyla) typically increased in relative abundance in the high N plots, with oligotrophic taxa (mainly Acidobacteria) exhibiting the opposite pattern. Consistent with the phylogenetic shifts under N fertilization, shotgun metagenomic sequencing revealed increases in the relative abundances of genes associated with DNA/RNA replication, electron transport and protein metabolism, increases that could be resolved even with the shallow shotgun metagenomic sequencing conducted here (average of 75 000 reads per sample). We also observed shifts in the catabolic capabilities of the communities across the N gradients that were significantly correlated with the phylogenetic and metagenomic responses, indicating possible linkages between the structure and functioning of soil microbial communities. Overall, our results suggest that N fertilization may, directly or indirectly, induce a shift in the predominant microbial life-history strategies, favoring a more active, copiotrophic microbial community, a pattern that parallels the often observed replacement of K-selected with r-selected plant species with elevated N.

Visual statistical learning is not reliably modulated by selective attention to isolated events

PubMed Central

Musz, Elizabeth; Weber, Matthew J.; Thompson-Schill, Sharon L.

2014-01-01

Recent studies of visual statistical learning (VSL) indicate that the visual system can automatically extract temporal and spatial relationships between objects. We report several attempts to replicate and extend earlier work (Turk-Browne et al., 2005) in which observers performed a cover task on one of two interleaved stimulus sets, resulting in learning of temporal relationships that occur in the attended stream, but not those present in the unattended stream. Across four experiments, we exposed observers to a similar or identical familiarization protocol, directing attention to one of two interleaved stimulus sets; afterward, we assessed VSL efficacy for both sets using either implicit response-time measures or explicit familiarity judgments. In line with prior work, we observe learning for the attended stimulus set. However, unlike previous reports, we also observe learning for the unattended stimulus set. When instructed to selectively attend to only one of the stimulus sets and ignore the other set, observers could extract temporal regularities for both sets. Our efforts to experimentally decrease this effect by changing the cover task (Experiment 1) or the complexity of the statistical regularities (Experiment 3) were unsuccessful. A fourth experiment using a different assessment of learning likewise failed to show an attentional effect. Simulations drawing random samples our first three experiments (n=64) confirm that the distribution of attentional effects in our sample closely approximates the null. We offer several potential explanations for our failure to replicate earlier findings, and discuss how our results suggest limiting conditions on the relevance of attention to VSL. PMID:25172196

Concise Review: Fabrication, Customization, and Application of Cell Mimicking Microparticles in Stem Cell Science

PubMed Central

Labriola, Nicholas R.; Azagury, Aharon; Gutierrez, Robert; Mathiowitz, Edith

2018-01-01

Abstract Stem and non‐stem cell behavior is heavily influenced by the surrounding microenvironment, which includes other cells, matrix, and potentially biomaterials. Researchers have been successful in developing scaffolds and encapsulation techniques to provide stem cells with mechanical, topographical, and chemical cues to selectively direct them toward a desired differentiation pathway. However, most of these systems fail to present truly physiological replications of the in vivo microenvironments that stem cells are typically exposed to in tissues. Thus, cell mimicking microparticles (CMMPs) have been developed to more accurately recapitulate the properties of surrounding cells while still offering ways to tailor what stimuli are presented. This nascent field holds the promise of reducing, or even eliminating, the need for live cells in select, regenerative medicine therapies, and diagnostic applications. Recent, CMMP‐based studies show great promise for the technology, yet only reproduce a small subset of cellular characteristics from among those possible: size, morphology, topography, mechanical properties, surface molecules, and tailored chemical release to name the most prominent. This Review summarizes the strengths, weaknesses, and ideal applications of micro/nanoparticle fabrication and customization methods relevant to cell mimicking and provides an outlook on the future of this technology. Moving forward, researchers should seek to combine multiple techniques to yield CMMPs that replicate as many cellular characteristics as possible, with an emphasis on those that most strongly influence the desired therapeutic effects. The level of flexibility in customizing CMMP properties allows them to substitute for cells in a variety of regenerative medicine, drug delivery, and diagnostic systems. Stem Cells Translational Medicine 2018;7:232–240 PMID:29316362

Adaptive population divergence and directional gene flow across steep elevational gradients in a climate‐sensitive mammal

USGS Publications Warehouse

Waterhouse, Matthew D.; Erb, Liesl P.; Beever, Erik; Russello, Michael A.

2018-01-01

The American pika is a thermally sensitive, alpine lagomorph species. Recent climate-associated population extirpations and genetic signatures of reduced population sizes range-wide indicate the viability of this species is sensitive to climate change. To test for potential adaptive responses to climate stress, we sampled pikas along two elevational gradients (each ~470 to 1640 m) and employed three outlier detection methods, BAYESCAN, LFMM, and BAYPASS, to scan for genotype-environment associations in samples genotyped at 30,763 SNP loci. We resolved 173 loci with robust evidence of natural selection detected by either two independent analyses or replicated in both transects. A BLASTN search of these outlier loci revealed several genes associated with metabolic function and oxygen transport, indicating natural selection from thermal stress and hypoxia. We also found evidence of directional gene flow primarily downslope from large high-elevation populations and reduced gene flow at outlier loci, a pattern suggesting potential impediments to the upward elevational movement of adaptive alleles in response to contemporary climate change. Finally, we documented evidence of reduced genetic diversity associated the south-facing transect and an increase in corticosterone stress levels associated with inbreeding. This study suggests the American pika is already undergoing climate-associated natural selection at multiple genomic regions. Further analysis is needed to determine if the rate of climate adaptation in the American pika and other thermally sensitive species will be able to keep pace with rapidly changing climate conditions.

High Resolution Genomic Scans Reveal Genetic Architecture Controlling Alcohol Preference in Bidirectionally Selected Rat Model.

PubMed

Lo, Chiao-Ling; Lossie, Amy C; Liang, Tiebing; Liu, Yunlong; Xuei, Xiaoling; Lumeng, Lawrence; Zhou, Feng C; Muir, William M

2016-08-01

Investigations on the influence of nature vs. nurture on Alcoholism (Alcohol Use Disorder) in human have yet to provide a clear view on potential genomic etiologies. To address this issue, we sequenced a replicated animal model system bidirectionally-selected for alcohol preference (AP). This model is uniquely suited to map genetic effects with high reproducibility, and resolution. The origin of the rat lines (an 8-way cross) resulted in small haplotype blocks (HB) with a corresponding high level of resolution. We sequenced DNAs from 40 samples (10 per line of each replicate) to determine allele frequencies and HB. We achieved ~46X coverage per line and replicate. Excessive differentiation in the genomic architecture between lines, across replicates, termed signatures of selection (SS), were classified according to gene and region. We identified SS in 930 genes associated with AP. The majority (50%) of the SS were confined to single gene regions, the greatest numbers of which were in promoters (284) and intronic regions (169) with the least in exon's (4), suggesting that differences in AP were primarily due to alterations in regulatory regions. We confirmed previously identified genes and found many new genes associated with AP. Of those newly identified genes, several demonstrated neuronal function involved in synaptic memory and reward behavior, e.g. ion channels (Kcnf1, Kcnn3, Scn5a), excitatory receptors (Grin2a, Gria3, Grip1), neurotransmitters (Pomc), and synapses (Snap29). This study not only reveals the polygenic architecture of AP, but also emphasizes the importance of regulatory elements, consistent with other complex traits.

Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae

PubMed Central

Szyjka, Shawn J.; Aparicio, Jennifer G.; Viggiani, Christopher J.; Knott, Simon; Xu, Weihong; Tavaré, Simon; Aparicio, Oscar M.

2008-01-01

Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase, which plays a vital role in survival by stabilizing stalled replication forks. However, evidence that Rad53 directly modulates the activity of replication forks has been lacking, and the nature of fork stabilization has remained unclear. Recently, cells lacking the Psy2–Pph3 phosphatase were shown to be defective in dephosphorylation of Rad53 as well as replication fork restart after DNA damage, suggesting a mechanistic link between Rad53 deactivation and fork restart. To test this possibility we examined the progression of replication forks in methyl-methanesulfonate (MMS)-damaged cells, under different conditions of Rad53 activity. Hyperactivity of Rad53 in pph3Δ cells slows fork progression in MMS, whereas deactivation of Rad53, through expression of dominant-negative Rad53-KD, is sufficient to allow fork restart during recovery. Furthermore, combined deletion of PPH3 and PTC2, a second, unrelated Rad53 phosphatase, results in complete replication fork arrest and lethality in MMS, demonstrating that Rad53 deactivation is a key mechanism controlling fork restart. We propose a model for regulation of replication fork progression through damaged DNA involving a cycle of Rad53 activation and deactivation that coordinates replication restart with DNA repair. PMID:18628397

Universal Temporal Profile of Replication Origin Activation in Eukaryotes

NASA Astrophysics Data System (ADS)

Goldar, Arach

2011-03-01

The complete and faithful transmission of eukaryotic genome to daughter cells involves the timely duplication of mother cell's DNA. DNA replication starts at multiple chromosomal positions called replication origin. From each activated replication origin two replication forks progress in opposite direction and duplicate the mother cell's DNA. While it is widely accepted that in eukaryotic organisms replication origins are activated in a stochastic manner, little is known on the sources of the observed stochasticity. It is often associated to the population variability to enter S phase. We extract from a growing Saccharomyces cerevisiae population the average rate of origin activation in a single cell by combining single molecule measurements and a numerical deconvolution technique. We show that the temporal profile of the rate of origin activation in a single cell is similar to the one extracted from a replicating cell population. Taking into account this observation we exclude the population variability as the origin of observed stochasticity in origin activation. We confirm that the rate of origin activation increases in the early stage of S phase and decreases at the latter stage. The population average activation rate extracted from single molecule analysis is in prefect accordance with the activation rate extracted from published micro-array data, confirming therefore the homogeneity and genome scale invariance of dynamic of replication process. All these observations point toward a possible role of replication fork to control the rate of origin activation.

The Location of the Bacterial Origin of Replication is Critical for Initial Ciproflaxcin Antibiotic Resistance

NASA Astrophysics Data System (ADS)

Bos, Julia; Nehring, Ralph; Cruz, Diane; Austin, Doug; Rosenberg, Susan; Austin, Robert

By using E. coli cells in which the unique origin of replication has been moved to a ectopic chromosome location distant from the native one, we probe how perturbation of gene order near the origin of replication impacts genome stability and survival under genomic attack. We find that when challenged with sub-inhibitory doses of ciprofloxacin, an antibiotic that generates replication fork stalling, cells with the ectopic origin show significant fitness loss. We show that genes functionally relevant to the cipro-induced stress response are largely located near the native origin, even in distantly related species. We show that while cipro induces increased copy number of genes proximal to the origin of replication as a direct consequence of replication fork stalling, gene copy number variation was reduced near the ectopic origin. Altered gene dosage in cells with an ectopic origin resulted in impaired replication fork repair and chromosome instability. We propose that gene distribution in the origin region acts as a fundamental first line of defense when the integrity of the genome is threatened and that genes proximal to the origin of replication serve as a mechanism of genetic innovation and a driving force of genome evolution in the presence of genotoxic antibiotics. Lewis Sigler Institute for Integrative Genomics and the Physics Department at Princeton University.

G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV

PubMed Central

Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P.; Robertson, Erle S.; Schildkraut, Carl L.; Verma, Subhash C.

2016-01-01

Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases. PMID:26837574

Template Directed Replication Supports the Maintenance of the Metabolically Coupled Replicator System

NASA Astrophysics Data System (ADS)

Könnyű, Balázs; Czárán, Tamás

2015-06-01

The RNA World scenario of prebiotic chemical evolution is among the most plausible conceptual framework available today for modelling the origin of life. RNA offers genetic and catalytic (metabolic) functionality embodied in a single chemical entity, and a metabolically cooperating community of RNA molecules would constitute a viable infrabiological subsystem with a potential to evolve into proto-cellular life. Our Metabolically Coupled Replicator System (MCRS) model is a spatially explicit computer simulation implementation of the RNA-World scenario, in which replicable ribozymes cooperate in supplying each other with monomers for their own replication. MCRS has been repeatedly demonstrated to be viable and evolvable, with different versions of the model improved in depth (chemical detail of metabolism) or in extension (additional functions of RNA molecules). One of the dynamically relevant extensions of the MCRS approach to prebiotic RNA evolution is the explicit inclusion of template replication into its assumptions, which we have studied in the present version. We found that this modification has not changed the behaviour of the system in the qualitative sense, just the range of the parameter space which is optimal for the coexistence of metabolically cooperating replicators has shifted in terms of metabolite mobility. The system also remains resistant and tolerant to parasitic replicators.

Effects of replicative fitness on competing HIV strains.

PubMed

Chirove, Faraimunashe; Lungu, Edward M

2013-07-01

We develop an n-strain model to show the effects of replicative fitness of competing viral strains exerting selective density-dependant infective pressure on each other. A two strain model is used to illustrate the results. A perturbation technique and numerical simulations were used to establish the existence and stability of steady states. More than one infected steady states governed by the replicative fitness resulted from the model exhibiting either strain replacement or co-infection. We found that the presence of two or more HIV strains could result in a disease-free state that, in general, is not globally stable. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Elementary Science-Magnet School Student Attitudes toward Science as Measured by Selected National Assessment of Educational Progress Items and Achievement in Science: A Replication and Extension.

ERIC Educational Resources Information Center

Solomon, Alan; Rachild, Bruce

Attitudes toward science of magnet school students were compared with those of their counterparts in two regular schools. This study attempted to replicate the findings of a 1988 study by A. Solomon and J. Wroblewski involving the same magnet school, the John Moffett Neighborhood Elementary Science Magnet School located in North Philadelphia…

Stability, chromatin association and functional activity of mammalian pre-replication complex proteins during the cell cycle

PubMed Central

Okuno, Yukiko; McNairn, Adrian J.; den Elzen, Nicole; Pines, Jonathon; Gilbert, David M.

2001-01-01

We have examined the behavior of pre-replication complex (pre-RC) proteins in relation to key cell cycle transitions in Chinese Hamster Ovary (CHO) cells. ORC1, ORC4 and Cdc6 were stable (T1/2 >2 h) and associated with a chromatin-containing fraction throughout the cell cycle. Green fluorescent protein-tagged ORC1 associated with chromatin throughout mitosis in living cells and co-localized with ORC4 in metaphase spreads. Association of Mcm proteins with chromatin took place during telophase, ∼30 min after the destruction of geminin and cyclins A and B, and was coincident with the licensing of chromatin to replicate in geminin-supplemented Xenopus egg extracts. Neither Mcm recruitment nor licensing required protein synthesis throughout mitosis. Moreover, licensing could be uncoupled from origin specification in geminin-supplemented extracts; site-specific initiation within the dihydrofolate reductase locus required nuclei from cells that had passed through the origin decision point (ODP). These results demonstrate that mammalian pre-RC assembly takes place during telophase, mediated by post-translational modifications of pre-existing proteins, and is not sufficient to select specific origin sites. A subsequent, as yet undefined, step selects which pre-RCs will function as replication origins. PMID:11483529

Genotypic character relationship and phenotypic path coefficient analysis in chili pepper genotypes grown under tropical condition.

PubMed

Usman, Magaji G; Rafii, Mohd Y; Martini, Mohammad Y; Oladosu, Yusuff; Kashiani, Pedram

2017-03-01

Studies on genotypic and phenotypic correlations among characters of crop plants are useful in planning, evaluating and setting selection criteria for the desired characters in a breeding program. The present study aimed to estimate the phenotypic correlation coefficients among yield and yield attributed characters and to work out the direct and indirect effects of yield-related characters on yield per plant using path coefficient analysis. Twenty-six genotypes of chili pepper were laid out in a randomized complete block design with three replications. Yield per plant showed positive and highly significant (P ≤ 0.01) correlations with most of the characters studied at both the phenotypic and genotypic levels. By contrast, disease incidence and days to flowering showed a significant negative association with yield. Fruit weight and number of fruits exerted positive direct effect on yield and also had a positive and significant (P ≤ 0.01) correlation with yield per plant. However, fruit length showed a low negative direct effect with a strong and positive indirect effect through fruit weight on yield and had a positive and significant association with yield. Longer fruits, heavy fruits and a high number of fruits are variables that are related to higher yields of chili pepper under tropical conditions and hence could be used as a reliable indicator in indirect selection for yield. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Oncolytic herpes simplex virus-based strategies: toward a breakthrough in glioblastoma therapy

PubMed Central

Ning, Jianfang; Wakimoto, Hiroaki

2014-01-01

Oncolytic viruses (OV) are a class of antitumor agents that selectively kill tumor cells while sparing normal cells. Oncolytic herpes simplex virus (oHSV) has been investigated in clinical trials for patients with the malignant brain tumor glioblastoma for more than a decade. These clinical studies have shown the safety of oHSV administration to the human brain, however, therapeutic efficacy of oHSV as a single treatment remains unsatisfactory. Factors that could hamper the anti-glioblastoma efficacy of oHSV include: attenuated potency of oHSV due to deletion or mutation of viral genes involved in virulence, restricting viral replication and spread within the tumor; suboptimal oHSV delivery associated with intratumoral injection; virus infection-induced inflammatory and cellular immune responses which could inhibit oHSV replication and promote its clearance; lack of effective incorporation of oHSV into standard-of-care, and poor knowledge about the ability of oHSV to target glioblastoma stem cells (GSCs). In an attempt to address these issues, recent research efforts have been directed at: (1) design of new engineered viruses to enhance potency, (2) better understanding of the role of the cellular immunity elicited by oHSV infection of tumors, (3) combinatorial strategies with different antitumor agents with a mechanistic rationale, (4) “armed” viruses expressing therapeutic transgenes, (5) use of GSC-derived models in oHSV evaluation, and (6) combinations of these. In this review, we will describe the current status of oHSV clinical trials for glioblastoma, and discuss recent research advances and future directions toward successful oHSV-based therapy of glioblastoma. PMID:24999342

Evidence for Sequential and Increasing Activation of Replication Origins along Replication Timing Gradients in the Human Genome

PubMed Central

Guilbaud, Guillaume; Rappailles, Aurélien; Baker, Antoine; Chen, Chun-Long; Arneodo, Alain; Goldar, Arach; d'Aubenton-Carafa, Yves; Thermes, Claude; Audit, Benjamin; Hyrien, Olivier

2011-01-01

Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics. PMID:22219720

Friendly fire: redirecting herpes simplex virus-1 for therapeutic applications.

PubMed

Advani, S J; Weichselbaum, R R; Whitley, R J; Roizman, B

2002-09-01

Herpes simplex virus-1 (HSV-1) is a relatively large double-stranded DNA virus encoding at least 89 proteins with well characterized disease pathology. An understanding of the functions of viral proteins together with the ability to genetically engineer specific viral mutants has led to the development of attenuated HSV-1 for gene therapy. This review highlights the progress in creating attenuated genetically engineered HSV-1 mutants that are either replication competent (viral non-essential gene deleted) or replication defective (viral essential gene deleted). The choice between a replication-competent or -defective virus is based on the end-goal of the therapeutic intervention. Replication-competent HSV-1 mutants have primarily been employed as antitumor oncolytic viruses, with the lytic nature of the virus harnessed to destroy tumor cells selectively. In replacement gene therapy, replication-defective viruses have been utilized as delivery vectors. The advantages of HSV-1 vectors are that they infect quiescent and dividing cells efficiently and can encode for relatively large transgenes.

Cohesin Can Remain Associated with Chromosomes during DNA Replication.

PubMed

Rhodes, James D P; Haarhuis, Judith H I; Grimm, Jonathan B; Rowland, Benjamin D; Lavis, Luke D; Nasmyth, Kim A

2017-09-19

To ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in situ after replication. We developed a non-fluorescent HaloTag ligand to discriminate the fluorescence recovery signal from labeling of newly synthesized Halo-tagged Scc1 protein (pulse-chase or pcFRAP). In cells where cohesin turnover is inactivated by deletion of WAPL, Scc1 can remain associated with chromatin throughout S phase. These findings suggest that cohesion might be generated by cohesin that is already bound to un-replicated DNA. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Replication and contradiction of highly cited research papers in psychiatry: 10-year follow-up.

PubMed

Tajika, Aran; Ogawa, Yusuke; Takeshima, Nozomi; Hayasaka, Yu; Furukawa, Toshi A

2015-10-01

Contradictions and initial overestimates are not unusual among highly cited studies. However, this issue has not been researched in psychiatry. Aims: To assess how highly cited studies in psychiatry are replicated by subsequent studies. We selected highly cited studies claiming effective psychiatric treatments in the years 2000 through 2002. For each of these studies we searched for subsequent studies with a better-controlled design, or with a similar design but a larger sample. Among 83 articles recommending effective interventions, 40 had not been subject to any attempt at replication, 16 were contradicted, 11 were found to have substantially smaller effects and only 16 were replicated. The standardised mean differences of the initial studies were overestimated by 132%. Studies with a total sample size of 100 or more tended to produce replicable results. Caution is needed when a study with a small sample size reports a large effect. © The Royal College of Psychiatrists 2015.

Human replication protein Cdc6 is selectively cleaved by caspase 3 during apoptosis

PubMed Central

Pelizon, Cristina; d’Adda di Fagagna, Fabrizio; Farrace, Lorena; Laskey, Ronald A.

2002-01-01

In eukaryotes, the initiation of DNA replication involves the ordered assembly on chromatin of pre-replicative complexes (pre-RCs), including the origin recognition complex (ORC), Cdc6, Cdt1 and the minichromosome maintenance proteins (MCMs). In light of its indispensable role in the formation of pre-RCs, Cdc6 binding to chromatin represents a key step in the regulation of DNA replication and cell proliferation. Here, we study the human Cdc6 (HuCdc6) protein during programmed cell death (apoptosis). We find that HuCdc6, but not HuOrc2 (a member of the ORC) or HuMcm5 (one of the MCMs), is specifically cleaved in several human cell lines induced to undergo apoptosis by a variety of stimuli. Expression of caspase-uncleavable mutant HuCdc6 attenuates apoptosis, delaying cell death. Therefore, an important function for cleavage of HuCdc6 is to prevent a wounded cell from replicating and to facilitate death. PMID:12151338

Hair whorl direction and sexual orientation in human males.

PubMed

Rahman, Qazi; Clarke, Kenneth; Morera, Tirma

2009-04-01

Hair whorl direction is a somatic feature that is organized early during neurodevelopment and unlikely to be influenced by social factors. This study aimed to replicate a widely reported association by A. J. S. Klar (2003) between counterclockwise hair whorl direction and homosexuality in men, using more objective methodology. The authors took digital photographs of parietal surface hair whorls from 100 heterosexual men and 100 homosexual men who were predominantly right-handed. These images were rated for clockwise and counterclockwise direction (for which no more than 1 hair whorl was present) by 2 raters unaware of sexual orientation. The authors found no significant difference between heterosexual and homosexual men in hair whorl direction, but the authors did replicate the fraternal birth order effect (more older brothers for homosexual men). Number of older sisters was positively correlated with counterclockwise hair whorls in heterosexual men. These data were discussed in relation to prenatal factors assumed to play a role in the neurodevelopment of male homosexuality. (c) 2009 APA, all rights reserved.

Absence of MutSbeta leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks

PubMed Central

Slean, Meghan M.; Panigrahi, Gagan B.; Castel, Arturo López; Pearson, August B.; Tomkinson, Alan E.; Pearson, Christopher E.

2016-01-01

Typically disease-causing CAG/CTG repeats expand, but rare affected families can display high levels of contraction of the expanded repeat amongst offspring. Understanding instability is important since arresting expansions or enhancing contractions could be clinically beneficial. The MutSβ mismatch repair complex is required for CAG/CTG expansions in mice and patients. Oddly, by unknown mechanisms MutSβ-deficient mice incur contractions instead of expansions. Replication using CTG or CAG as the lagging strand template is known to cause contractions or expansions respectively; however, the interplay between replication and repair leading to this instability remains unclear. Towards understanding how repeat contractions may arise, we performed in vitro SV40-mediated replication of repeat-containing plasmids in the presence or absence of mismatch repair. Specifically, we separated repair from replication: Replication mediated by MutSβ- and MutSα-deficient human cells or cell extracts produced slipped-DNA heteroduplexes in the contraction- but not expansion-biased replication direction. Replication in the presence of MutSβ disfavoured the retention of replication products harbouring slipped-DNA heteroduplexes. Post-replication repair of slipped-DNAs by MutSβ-proficient extracts eliminated slipped-DNAs. Thus, a MutSβ-deficiency likely enhances repeat contractions because MutSβ protects against contractions by repairing template strand slip-outs. Replication deficient in LigaseI or PCNA-interaction mutant LigaseI revealed slipped-DNA formation at lagging strands. Our results reveal that distinct mechanisms lead to expansions or contractions and support inhibition of MutSβ as a therapeutic strategy to enhance the contraction of expanded repeats. PMID:27155933

Nuclear Lamins

PubMed Central

Dechat, Thomas; Adam, Stephen A.; Taimen, Pekka; Shimi, Takeshi; Goldman, Robert D.

2010-01-01

The nuclear lamins are type V intermediate filament proteins that are critically important for the structural properties of the nucleus. In addition, they are involved in the regulation of numerous nuclear processes, including DNA replication, transcription and chromatin organization. The developmentally regulated expression of lamins suggests that they are involved in cellular differentiation. Their assembly dynamic properties throughout the cell cycle, particularly in mitosis, are influenced by posttranslational modifications. Lamins may regulate nuclear functions by direct interactions with chromatin and determining the spatial organization of chromosomes within the nuclear space. They may also regulate chromatin functions by interacting with factors that epigenetically modify the chromatin or directly regulate replication or transcription. PMID:20826548

Evidence that compatibility of closely related replicons in Clostridium perfringens depends on linkage to parMRC-like partitioning systems of different subfamilies.

PubMed

Watts, Thomas D; Johanesen, Priscilla A; Lyras, Dena; Rood, Julian I; Adams, Vicki

2017-05-01

Clostridium perfringens produces an extensive repertoire of toxins and extracellular enzymes, many of which are intimately involved in the progression of disease and are encoded by genes on conjugative plasmids. In addition, many C. perfringens strains can carry up to five of these conjugative toxin or antimicrobial resistance plasmids, each of which has a similar 35kb backbone. This conserved backbone includes the tcp conjugation locus and the central control region (CCR), which encodes genes involved in plasmid regulation, replication and partitioning, including a parMRC partitioning locus. Most conjugative plasmids in C. perfringens have a conserved replication protein, raising questions as to how multiple, closely related plasmids are maintained within a single strain. Bioinformatics analysis has highlighted the presence of at least 10 different parMRC partitioning system families (parMRC A-J ) in these plasmids, with differences in amino acid sequence identity between each ParM family ranging from 15% to 54%. No two plasmids that encode genes belonging to the same partitioning family have been observed in a single strain, suggesting that these families represent the basis for plasmid incompatibility. In an attempt to validate the proposed parMRC incompatibility groups, genetically marked C. perfringens plasmids encoding identical parMRC C or parMRC D homologues or different combinations of parMRC A , parMRC C and parMRC D family homologues were introduced into a single strain via conjugation. The stability of each plasmid was determined using an incompatibility assay in which the plasmid profile of each strain was monitored over the course of two days in the absence of direct selection. The results showed that plasmids with identical parMRC C or parMRC D homologues were incompatible and could not coexist in the absence of external selection. By contrast, plasmids that encoded different parMRC homologues were compatible and could coexist in the same cell in the absence of selection, with the exception of strains housing parMRC C and parMRC D combinations, which showed a minor incompatibility phenotype. In conclusion, we have provided the first direct evidence of plasmid incompatibility in Clostridium spp. and have shown experimentally that the compatibility of conjugative C. perfringens plasmids correlates with the presence of parMRC-like partitioning systems of different phylogenetic subfamilies. Copyright © 2017 Elsevier Inc. All rights reserved.

The function of Xenopus Bloom's syndrome protein homolog (xBLM) in DNA replication

PubMed Central

Liao, Shuren; Graham, Jeanine; Yan, Hong

2000-01-01

The Bloom's syndrome gene (BLM) plays a pivotal role in the maintenance of genomic stability in somatic cells. It encodes a DNA helicase (BLM) of the RecQ family, but the exact function of BLM remains elusive. To study this question, we have cloned the BLM homolog of the frog Xenopus laevis (xBLM) and have raised antibodies to it. Immunodepletion of xBLM from a Xenopus egg extract severely inhibits the replication of DNA in reconstituted nuclei. Moreover, the inhibition can be rescued by the addition of the recombinant xBLM protein. These results provide the first direct evidence that BLM plays an important role in DNA replication, suggesting that Bloom's syndrome may be the consequence of defective DNA replication. PMID:11040210

A study of an adaptive replication framework for orchestrated composite web services.

PubMed

Mohamed, Marwa F; Elyamany, Hany F; Nassar, Hamed M

2013-01-01

Replication is considered one of the most important techniques to improve the Quality of Services (QoS) of published Web Services. It has achieved impressive success in managing resource sharing and usage in order to moderate the energy consumed in IT environments. For a robust and successful replication process, attention should be paid to suitable time as well as the constraints and capabilities in which the process runs. The replication process is time-consuming since outsourcing some new replicas into other hosts is lengthy. Furthermore, nowadays, most of the business processes that might be implemented over the Web are composed of multiple Web services working together in two main styles: Orchestration and Choreography. Accomplishing a replication over such business processes is another challenge due to the complexity and flexibility involved. In this paper, we present an adaptive replication framework for regular and orchestrated composite Web services. The suggested framework includes a number of components for detecting unexpected and unhappy events that might occur when consuming the original published web services including failure or overloading. It also includes a specific replication controller to manage the replication process and select the best host that would encapsulate a new replica. In addition, it includes a component for predicting the incoming load in order to decrease the time needed for outsourcing new replicas, enhancing the performance greatly. A simulation environment has been created to measure the performance of the suggested framework. The results indicate that adaptive replication with prediction scenario is the best option for enhancing the performance of the replication process in an online business environment.

Crystallization and preliminary X-ray characterization of the eukaryotic replication terminator Reb1-Ter DNA complex.

PubMed

Jaiswal, Rahul; Singh, Samarendra K; Bastia, Deepak; Escalante, Carlos R

2015-04-01

The Reb1 protein from Schizosaccharomyces pombe is a member of a family of proteins that control programmed replication termination and/or transcription termination in eukaryotic cells. These events occur at naturally occurring replication fork barriers (RFBs), where Reb1 binds to termination (Ter) DNA sites and coordinates the polar arrest of replication forks and transcription approaching in opposite directions. The Reb1 DNA-binding and replication-termination domain was expressed in Escherichia coli, purified and crystallized in complex with a 26-mer DNA Ter site. Batch crystallization under oil was required to produce crystals of good quality for data collection. Crystals grew in space group P2₁, with unit-cell parameters a = 68.9, b = 162.9, c = 71.1 Å, β = 94.7°. The crystals diffracted to a resolution of 3.0 Å. The crystals were mosaic and required two or three cycles of annealing. This study is the first to yield structural information about this important family of proteins and will provide insights into the mechanism of replication and transcription termination.

Heterosexual Transmission of Subtype C HIV-1 Selects Consensus-Like Variants without Increased Replicative Capacity or Interferon-α Resistance

PubMed Central

Fenton-May, Angharad E.; Dilernia, Dario A.; Kilembe, William; Allen, Susan A.; Borrow, Persephone; Hunter, Eric

2015-01-01

Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential. PMID:26378795

Analysis of response to 20 generations of selection for body composition in mice: fit to infinitesimal model assumptions

PubMed Central

Martinez, Victor; Bünger, Lutz; Hill, William G

2000-01-01

Data were analysed from a divergent selection experiment for an indicator of body composition in the mouse, the ratio of gonadal fat pad to body weight (GFPR). Lines were selected for 20 generations for fat (F), lean (L) or were unselected (C), with three replicates of each. Selection was within full-sib families, 16 families per replicate for the first seven generations, eight subsequently. At generation 20, GFPR in the F lines was twice and in the L lines half that of C. A log transformation removed both asymmetry of response and heterogeneity of variance among lines, and so was used throughout. Estimates of genetic variance and heritability (approximately 50%) obtained using REML with an animal model were very similar, whether estimated from the first few generations of selection, or from all 20 generations, or from late generations having fitted pedigree. The estimates were also similar when estimated from selected or control lines. Estimates from REML also agreed with estimates of realised heritability. The results all accord with expectations under the infinitesimal model, despite the four-fold changes in mean. Relaxed selection lines, derived from generation 20, showed little regression in fatness after 40 generations without selection. PMID:14736404

The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

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

Ziehr, Ben; Lenarcic, Erik; Cecil, Chad

Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compoundmore » that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.« less

Genome-wide mapping reveals single-origin chromosome replication in Leishmania, a eukaryotic microbe.

PubMed

Marques, Catarina A; Dickens, Nicholas J; Paape, Daniel; Campbell, Samantha J; McCulloch, Richard

2015-10-19

DNA replication initiates on defined genome sites, termed origins. Origin usage appears to follow common rules in the eukaryotic organisms examined to date: all chromosomes are replicated from multiple origins, which display variations in firing efficiency and are selected from a larger pool of potential origins. To ask if these features of DNA replication are true of all eukaryotes, we describe genome-wide origin mapping in the parasite Leishmania. Origin mapping in Leishmania suggests a striking divergence in origin usage relative to characterized eukaryotes, since each chromosome appears to be replicated from a single origin. By comparing two species of Leishmania, we find evidence that such origin singularity is maintained in the face of chromosome fusion or fission events during evolution. Mapping Leishmania origins suggests that all origins fire with equal efficiency, and that the genomic sites occupied by origins differ from related non-origins sites. Finally, we provide evidence that origin location in Leishmania displays striking conservation with Trypanosoma brucei, despite the latter parasite replicating its chromosomes from multiple, variable strength origins. The demonstration of chromosome replication for a single origin in Leishmania, a microbial eukaryote, has implications for the evolution of origin multiplicity and associated controls, and may explain the pervasive aneuploidy that characterizes Leishmania chromosome architecture.

The cytoprotective enzyme heme oxygenase-1 suppresses Ebola virus replication.

PubMed

Hill-Batorski, Lindsay; Halfmann, Peter; Neumann, Gabriele; Kawaoka, Yoshihiro

2013-12-01

Ebola virus (EBOV) is the causative agent of a severe hemorrhagic fever in humans with reported case fatality rates as high as 90%. There are currently no licensed vaccines or antiviral therapeutics to combat EBOV infections. Heme oxygenase-1 (HO-1), an enzyme that catalyzes the rate-limiting step in heme degradation, has antioxidative properties and protects cells from various stresses. Activated HO-1 was recently shown to have antiviral activity, potently inhibiting the replication of viruses such as hepatitis C virus and human immunodeficiency virus. However, the effect of HO-1 activation on EBOV replication remains unknown. To determine whether the upregulation of HO-1 attenuates EBOV replication, we treated cells with cobalt protoporphyrin (CoPP), a selective HO-1 inducer, and assessed its effects on EBOV replication. We found that CoPP treatment, pre- and postinfection, significantly suppressed EBOV replication in a manner dependent upon HO-1 upregulation and activity. In addition, stable overexpression of HO-1 significantly attenuated EBOV growth. Although the exact mechanism behind the antiviral properties of HO-1 remains to be elucidated, our data show that HO-1 upregulation does not attenuate EBOV entry or budding but specifically targets EBOV transcription/replication. Therefore, modulation of the cellular enzyme HO-1 may represent a novel therapeutic strategy against EBOV infection.

Relaxation of herbivore-mediated selection drives the evolution of genetic covariances between plant competitive and defense traits.

PubMed

Uesugi, Akane; Connallon, Tim; Kessler, André; Monro, Keyne

2017-06-01

Insect herbivores are important mediators of selection on traits that impact plant defense against herbivory and competitive ability. Although recent experiments demonstrate a central role for herbivory in driving rapid evolution of defense and competition-mediating traits, whether and how herbivory shapes heritable variation in these traits remains poorly understood. Here, we evaluate the structure and evolutionary stability of the G matrix for plant metabolites that are involved in defense and allelopathy in the tall goldenrod, Solidago altissima. We show that G has evolutionarily diverged between experimentally replicated populations that evolved in the presence versus the absence of ambient herbivory, providing direct evidence for the evolution of G by natural selection. Specifically, evolution in an herbivore-free habitat altered the orientation of G, revealing a negative genetic covariation between defense- and competition-related metabolites that is typically masked in herbivore-exposed populations. Our results may be explained by predictions of classical quantitative genetic theory, as well as the theory of acquisition-allocation trade-offs. The study provides compelling evidence that herbivory drives the evolution of plant genetic architecture. © 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.

Replication domains are self-interacting structural chromatin units of human chromosomes

NASA Astrophysics Data System (ADS)

Arneodo, Alain

2011-03-01

In higher eukaryotes, the absence of specific sequence motifs marking the origins of replication has been a serious hindrance to the understanding of the mechanisms that regulate the initiation and the maintenance of the replication program in different cell types. In silico analysis of nucleotide compositional skew has predicted the existence, in the germline, of replication N-domains bordered by putative replication origins and where the skew decreases rather linearly as the signature of a progressive inversion of the average fork polarity. Here, from the demonstration that the average fork polarity can be directly extracted from the derivative of replication timing profiles, we develop a wavelet-based pattern recognition methodology to delineate replication U-domains where the replication timing profile is shaped as a U and its derivative as a N. Replication U-domains are robustly found in seven cell lines as covering a significant portion (40-50%) of the human genome where the replication timing data actually displays some plasticity between cell lines. The early replication initiation zones at U-domains borders are found to be hypersensitive to DNase I cleavage, to be associated with transcriptional activity and to present a significant enrichment in insular-binding proteins CTCF, the hallmark of an open chromatin structure. A comparative analysis of genome-wide chromatin interaction (HiC) data shows that replication-U domains correspond to self-interacting structural high order chromatin units of megabase characteristic size. Taken together, these findings provide evidence that the epigenetic compartmentalization of the human genome into autonomous replication U-domains comes along with an extensive remodelling of the threedimensional chromosome architecture during development or in specific diseases. The observed cell specific conservation of the replication timing between the human and mouse genomes strongly suggests that this chromosome organization into self-interacting structural and functional units is a general feature of mammalian organisms.

Insect herbivores drive real-time ecological and evolutionary change in plant populations.

PubMed

Agrawal, Anurag A; Hastings, Amy P; Johnson, Marc T J; Maron, John L; Salminen, Juha-Pekka

2012-10-05

Insect herbivores are hypothesized to be major factors affecting the ecology and evolution of plants. We tested this prediction by suppressing insects in replicated field populations of a native plant, Oenothera biennis, which reduced seed predation, altered interspecific competitive dynamics, and resulted in rapid evolutionary divergence. Comparative genotyping and phenotyping of nearly 12,000 O. biennis individuals revealed that in plots protected from insects, resistance to herbivores declined through time owing to changes in flowering time and lower defensive ellagitannins in fruits, whereas plant competitive ability increased. This independent real-time evolution of plant resistance and competitive ability in the field resulted from the relaxation of direct selective effects of insects on plant defense and through indirect effects due to reduced herbivory on plant competitors.

Cytonuclear genetics of experimental fish hybrid zones inside Biosphere 2

USGS Publications Warehouse

Scribner, K.T.; Avise, John C.

1994-01-01

Two species of mosquitofish (family Poeciliidae) known to hybridize in nature were introduced into freshwater habitats inside Biosphere 2, and their population genetics were monitored after 2 years. Within four to six generations, nuclear and cytoplasmic markers characteristic of Gambusia holbrooki had risen greatly in frequency, although some Gambusia affinis alleles and haplotypes were retained primarily in recombinant genotypes, indicative of introgressive hybridization. The temporal cytonuclear dynamics proved similar to population genetic changes observed in replicated experimental hybrid populations outside of Biosphere 2, thus indicating strong directional selection favoring G. holbrooki genotypes across the range of environments monitored. When interpreted in the context of species-specific population demographies observed previously, results suggest that the extremely rapid evolution in these zones of secondary contact is attributable primarily to species differences in life-history traits.

Groundwater-quality data from the eastern Snake River Plain Aquifer, Jerome and Gooding Counties, south-central Idaho, 2017

USGS Publications Warehouse

Skinner, Kenneth D.

2018-05-11

Groundwater-quality samples and water-level data were collected from 36 wells in the Jerome/Gooding County area of the eastern Snake River Plain aquifer during June 2017. The wells included 30 wells sampled for the U.S. Geological Survey’s National Water-Quality Assessment project, plus an additional 6 wells were selected to increase spatial distribution. The data provide water managers with the ability for an improved understanding of groundwater quality and flow directions in the area. Groundwater-quality samples were analyzed for nutrients, major ions, trace elements, and stable isotopes of water. Quality-assurance and quality-control measures consisted of multiple blank samples and a sequential replicate sample. All data are available online at the USGS National Water Information System.

The role of calcium and predation on plate morph evolution in the three-spined stickleback (Gasterosteus aculeatus).

PubMed

Smith, Carl; Spence, Rowena; Barber, Iain; Przybylski, Mirosław; Wootton, Robert J

2014-09-01

While the genetic basis to plate morph evolution of the three-spined stickleback (Gasterosteus aculeatus) is well described, the environmental variables that select for different plate and spine morphs are incompletely understood. Using replicate populations of three-spined sticklebacks on North Uist, Scotland, we previously investigated the role of predation pressure and calcium limitation on the adaptive evolution of stickleback morphology and behavior. While dissolved calcium proved a significant predictor of plate and spine morph, predator abundance did not. Ecol. Evol., xxx, 2014 and xxx performed a comparable analysis to our own to address the same question. They failed to detect a significant effect of dissolved calcium on morphological evolution, but did establish a significant effect of predation; albeit in the opposite direction to their prediction.

A New Age-Structured Multiscale Model of the Hepatitis C Virus Life-Cycle During Infection and Therapy With Direct-Acting Antiviral Agents

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

Quintela, Barbara de M.; Conway, Jessica M.; Hyman, James M.

Here, the dynamics of hepatitis C virus (HCV) RNA during translation and replication within infected cells were added to a previous age-structured multiscale mathematical model of HCV infection and treatment. The model allows the study of the dynamics of HCV RNA inside infected cells as well as the release of virus from infected cells and the dynamics of subsequent new cell infections. The model was used to fit in vitro data and estimate parameters characterizing HCV replication. This is the first model to our knowledge to consider both positive and negative strands of HCV RNA with an age-structured multiscale modelingmore » approach. Using this model we also studied the effects of direct-acting antiviral agents (DAAs) in blocking HCV RNA intracellular replication and the release of new virions and fit the model to in vivo data obtained from HCV-infected subjects under therapy.« less

Direct-acting Antivirals and Host-targeting Agents against the Hepatitis A Virus

PubMed Central

Kanda, Tatsuo; Nakamoto, Shingo; Wu, Shuang; Nakamura, Masato; Jiang, Xia; Haga, Yuki; Sasaki, Reina; Yokosuka, Osamu

2015-01-01

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis and occasionally leads to acute liver failure in both developing and developed countries. Although effective vaccines for HAV are available, the development of new antivirals against HAV may be important for the control of HAV infection in developed countries where no universal vaccination program against HAV exists, such as Japan. There are two forms of antiviral agents against HAV: direct-acting antivirals (DAAs) and host-targeting agents (HTAs). Studies using small interfering ribonucleic acid (siRNA) have suggested that the HAV internal ribosomal entry site (IRES) is an attractive target for the control of HAV replication and infection. Among the HTAs, amantadine and interferon-lambda 1 (IL-29) inhibit HAV IRES-mediated translation and HAV replication. Janus kinase (JAK) inhibitors inhibit La protein expression, HAV IRES activity, and HAV replication. Based on this review, both DAAs and HTAs may be needed to control effectively HAV infection, and their use should continue to be explored. PMID:26623267

A New Age-Structured Multiscale Model of the Hepatitis C Virus Life-Cycle During Infection and Therapy With Direct-Acting Antiviral Agents

DOE PAGES

Quintela, Barbara de M.; Conway, Jessica M.; Hyman, James M.; ...

2018-04-04

Here, the dynamics of hepatitis C virus (HCV) RNA during translation and replication within infected cells were added to a previous age-structured multiscale mathematical model of HCV infection and treatment. The model allows the study of the dynamics of HCV RNA inside infected cells as well as the release of virus from infected cells and the dynamics of subsequent new cell infections. The model was used to fit in vitro data and estimate parameters characterizing HCV replication. This is the first model to our knowledge to consider both positive and negative strands of HCV RNA with an age-structured multiscale modelingmore » approach. Using this model we also studied the effects of direct-acting antiviral agents (DAAs) in blocking HCV RNA intracellular replication and the release of new virions and fit the model to in vivo data obtained from HCV-infected subjects under therapy.« less

Adaptive scaling of reward in episodic memory: a replication study.

PubMed

Mason, Alice; Ludwig, Casimir; Farrell, Simon

2017-11-01

Reward is thought to enhance episodic memory formation via dopaminergic consolidation. Bunzeck, Dayan, Dolan, and Duzel [(2010). A common mechanism for adaptive scaling of reward and novelty. Human Brain Mapping, 31, 1380-1394] provided functional magnetic resonance imaging (fMRI) and behavioural evidence that reward and episodic memory systems are sensitive to the contextual value of a reward-whether it is relatively higher or lower-as opposed to absolute value or prediction error. We carried out a direct replication of their behavioural study and did not replicate their finding that memory performance associated with reward follows this pattern of adaptive scaling. An effect of reward outcome was in the opposite direction to that in the original study, with lower reward outcomes leading to better memory than higher outcomes. There was a marginal effect of reward context, suggesting that expected value affected memory performance. We discuss the robustness of the reward memory relationship to variations in reward context, and whether other reward-related factors have a more reliable influence on episodic memory.

Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. II. Attributes of virus cloned at different dog kidney passage levels.

PubMed

Halstead, S B; Marchette, N J; Diwan, A R; Palumbo, N E; Putvatana, R

1984-07-01

Uncloned dengue (DEN) 4 (H-241) which had been passaged 15, 30 and 50 times in primary dog kidney (PDK) cells were subjected to two successive terminal dilution procedures. In the first (3Cl), virus was diluted in 10-fold steps in 10 replicate tubes. An infected tube from a dilution row with three or fewer virus-infected tubes was selected for two further passages. In the second (TD3), virus was triple terminal diluted using 2-fold dilution steps and selecting one positive tube out of 10. Both procedures selected virus population which differed from antecedents. Plaque size of PDK 15 was medium, PDK 30, small and PDK 50, pin-point. PDK 19-3Cl were medium and 56-3Cl, 24-TD3, 35-TD3 and 61-TD3 were all small. All cloned virus replication was completely shut-off at 38.5 degrees C; PDK 15 and 30 continued to replicate at this temperature. Uncloned viruses showed a graduated decrease in monkey virulence with PDK passage; cloned viruses were either avirulent for monkeys (19-3Cl, 56-31Cl, 24-TD3 and 35-TD3) or produced revertant large plaque parental-type viremia (35-3Cl and 61-TD3). Those cloned viruses which exhibited temperature sensitivity, reduced monkey virulence and stability after monkey passage may be suitable as vaccine candidates for evaluation in human beings.

Chemotherapy, within-host ecology and the fitness of drug-resistant malaria parasites.

PubMed

Huijben, Silvie; Nelson, William A; Wargo, Andrew R; Sim, Derek G; Drew, Damien R; Read, Andrew F

2010-10-01

A major determinant of the rate at which drug-resistant malaria parasites spread through a population is the ecology of resistant and sensitive parasites sharing the same host. Drug treatment can significantly alter this ecology by removing the drug-sensitive parasites, leading to competitive release of resistant parasites. Here, we test the hypothesis that the spread of resistance can be slowed by reducing drug treatment and hence restricting competitive release. Using the rodent malaria model Plasmodium chabaudi, we found that low-dose chemotherapy did reduce competitive release. A higher drug dose regimen exerted stronger positive selection on resistant parasites for no detectable clinical gain. We estimated instantaneous selection coefficients throughout the course of replicate infections to analyze the temporal pattern of the strength and direction of within-host selection. The strength of selection on resistance varied through the course of infections, even in untreated infections, but increased immediately following drug treatment, particularly in the high-dose groups. Resistance remained under positive selection for much longer than expected from the half life of the drug. Although there are many differences between mice and people, our data do raise the question whether the aggressive treatment regimens aimed at complete parasite clearance are the best resistance-management strategies for humans. © 2010 The Author(s). Journal compilation © 2010 The Society for the Study of Evolution.

Viral replication rate regulates clinical outcome and CD8 T cell responses during highly pathogenic H5N1 influenza virus infection in mice.

PubMed

Hatta, Yasuko; Hershberger, Karen; Shinya, Kyoko; Proll, Sean C; Dubielzig, Richard R; Hatta, Masato; Katze, Michael G; Kawaoka, Yoshihiro; Suresh, M

2010-10-07

Since the first recorded infection of humans with H5N1 viruses of avian origin in 1997, sporadic human infections continue to occur with a staggering mortality rate of >60%. Although sustained human-to-human transmission has not occurred yet, there is a growing concern that these H5N1 viruses might acquire this trait and raise the specter of a pandemic. Despite progress in deciphering viral determinants of pathogenicity, we still lack crucial information on virus/immune system interactions pertaining to severe disease and high mortality associated with human H5N1 influenza virus infections. Using two human isolates of H5N1 viruses that differ in their pathogenicity in mice, we have defined mechanistic links among the rate of viral replication, mortality, CD8 T cell responses, and immunopathology. The extreme pathogenicity of H5N1 viruses was directly linked to the ability of the virus to replicate rapidly, and swiftly attain high steady-state titers in the lungs within 48 hours after infection. The remarkably high replication rate of the highly pathogenic H5N1 virus did not prevent the induction of IFN-β or activation of CD8 T cells, but the CD8 T cell response was ineffective in controlling viral replication in the lungs and CD8 T cell deficiency did not affect viral titers or mortality. Additionally, BIM deficiency ameliorated lung pathology and inhibited T cell apoptosis without affecting survival of mice. Therefore, rapidly replicating, highly lethal H5N1 viruses could simply outpace and overwhelm the adaptive immune responses, and kill the host by direct cytopathic effects. However, therapeutic suppression of early viral replication and the associated enhancement of CD8 T cell responses improved the survival of mice following a lethal H5N1 infection. These findings suggest that suppression of early H5N1 virus replication is key to the programming of an effective host response, which has implications in treatment of this infection in humans.

Open-field behavior of house mice selectively bred for high voluntary wheel-running.

PubMed

Bronikowski, A M; Carter, P A; Swallow, J G; Girard, I A; Rhodes, J S; Garland, T

2001-05-01

Open-field behavioral assays are commonly used to test both locomotor activity and emotionality in rodents. We performed open-field tests on house mice (Mus domesticus) from four replicate lines genetically selected for high voluntary wheel-running for 22 generations and from four replicate random-bred control lines. Individual mice were recorded by video camera for 3 min in a 1-m2 open-field arena on 2 consecutive days. Mice from selected lines showed no statistical differences from control mice with respect to distance traveled, defecation, time spent in the interior, or average distance from the center of the arena during the trial. Thus, we found little evidence that open-field behavior, as traditionally defined, is genetically correlated with wheel-running behavior. This result is a useful converse test of classical studies that report no increased wheel-running in mice selected for increased open-field activity. However, mice from selected lines turned less in their travel paths than did control-line mice, and females from selected lines had slower travel times (longer latencies) to reach the wall. We discuss these results in the context of the historical open-field test and newly defined measures of open-field activity.

Natural selection underlies apparent stress-induced mutagenesis in a bacteriophage infection model.

PubMed

Yosef, Ido; Edgar, Rotem; Levy, Asaf; Amitai, Gil; Sorek, Rotem; Munitz, Ariel; Qimron, Udi

2016-04-18

The emergence of mutations following growth-limiting conditions underlies bacterial drug resistance, viral escape from the immune system and fundamental evolution-driven events. Intriguingly, whether mutations are induced by growth limitation conditions or are randomly generated during growth and then selected by growth limitation conditions remains an open question(1). Here, we show that bacteriophage T7 undergoes apparent stress-induced mutagenesis when selected for improved recognition of its host's receptor. In our unique experimental set-up, the growth limitation condition is physically and temporally separated from mutagenesis: growth limitation occurs while phage DNA is outside the host, and spontaneous mutations occur during phage DNA replication inside the host. We show that the selected beneficial mutations are not pre-existing and that the initial slow phage growth is enabled by the phage particle's low-efficiency DNA injection into the host. Thus, the phage particle allows phage populations to initially extend their host range without mutagenesis by virtue of residual recognition of the host receptor. Mutations appear during non-selective intracellular replication, and the frequency of mutant phages increases by natural selection acting on free phages, which are not capable of mutagenesis.

Selection of UV Resins for Nanostructured Molds for Thermal-NIL.

PubMed

Jia, Zheng; Choi, Junseo; Park, Sunggook

2018-06-18

Nanoimprint molds made of soft polymeric materials have advantages of low demolding force and low fabrication cost over Si or metal-based hard molds. However, such advantages are often sacrificed by their reduced replication fidelity associated with the low mechanical strength. In this paper, we studied replication fidelity of different UV-resin molds copied from a Si master mold via UV nanoimprint lithography (NIL) and their thermal imprinting performance into a thermoplastic polymer. Four different UV resins were studied: two were high surface energy UV resins based on tripropyleneglycol diacrylate (TPGDA resin) and polypropyleneglycol diacrylate (PPGDA resin), and the other two were commercially available, low surface energy poly-urethane acrylate (PUA resin) and fluorine-containing (MD 700) UV resins. The replication fidelity among the four UV-resins during UV nanoimprint lithograph from a Si master with sharp nanostructures was in the increasing order of (poorest) PUA resin < MD 700 < PPGDA resin < TPGDA resin (best). The results show that the high surface energy and small monomer size are keys to achieving good UV resin filling into sharp nanostructures over the viscosity of the resin solution. When the four UV-resin molds were used for thermal-NIL into a thermoplastic polymer, the replication fidelity was in the increasing order of (poorest) MD 700 < TPGDA resin < PUA resin (best), which follows the same order of their Young's moduli. Our results indicate that the selection of an appropriate UV resin for NIL molds requires consideration of the replication fidelities in the mold fabrication and the subsequent thermal-NIL into thermoplastic polymers. © 2018 IOP Publishing Ltd.

Chromatin Landscapes of Retroviral and Transposon Integration Profiles

PubMed Central

Badhai, Jitendra; Rust, Alistair G.; Rad, Roland; Hilkens, John; Berns, Anton; van Lohuizen, Maarten; Wessels, Lodewyk F. A.; de Ridder, Jeroen

2014-01-01

The ability of retroviruses and transposons to insert their genetic material into host DNA makes them widely used tools in molecular biology, cancer research and gene therapy. However, these systems have biases that may strongly affect research outcomes. To address this issue, we generated very large datasets consisting of to unselected integrations in the mouse genome for the Sleeping Beauty (SB) and piggyBac (PB) transposons, and the Mouse Mammary Tumor Virus (MMTV). We analyzed (epi)genomic features to generate bias maps at both local and genome-wide scales. MMTV showed a remarkably uniform distribution of integrations across the genome. More distinct preferences were observed for the two transposons, with PB showing remarkable resemblance to bias profiles of the Murine Leukemia Virus. Furthermore, we present a model where target site selection is directed at multiple scales. At a large scale, target site selection is similar across systems, and defined by domain-oriented features, namely expression of proximal genes, proximity to CpG islands and to genic features, chromatin compaction and replication timing. Notable differences between the systems are mainly observed at smaller scales, and are directed by a diverse range of features. To study the effect of these biases on integration sites occupied under selective pressure, we turned to insertional mutagenesis (IM) screens. In IM screens, putative cancer genes are identified by finding frequently targeted genomic regions, or Common Integration Sites (CISs). Within three recently completed IM screens, we identified 7%–33% putative false positive CISs, which are likely not the result of the oncogenic selection process. Moreover, results indicate that PB, compared to SB, is more suited to tag oncogenes. PMID:24721906

Histone acetylation regulates the time of replication origin firing.

PubMed

Vogelauer, Maria; Rubbi, Liudmilla; Lucas, Isabelle; Brewer, Bonita J; Grunstein, Michael

2002-11-01

The temporal firing of replication origins throughout S phase in yeast depends on unknown determinants within the adjacent chromosomal environment. We demonstrate here that the state of histone acetylation of surrounding chromatin is an important regulator of temporal firing. Deletion of RPD3 histone deacetylase causes earlier origin firing and concurrent binding of the replication factor Cdc45p to origins. In addition, increased acetylation of histones in the vicinity of the late origin ARS1412 by recruitment of the histone acetyltransferase Gcn5p causes ARS1412 alone to fire earlier. These data indicate that histone acetylation is a direct determinant of the timing of origin firing.

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.

Measurement of replication structures at the nanometer scale using super-resolution light microscopy

PubMed Central

Baddeley, D.; Chagin, V. O.; Schermelleh, L.; Martin, S.; Pombo, A.; Carlton, P. M.; Gahl, A.; Domaing, P.; Birk, U.; Leonhardt, H.; Cremer, C.; Cardoso, M. C.

2010-01-01

DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses. PMID:19864256

Molecular basis for PrimPol recruitment to replication forks by RPA.

PubMed

Guilliam, Thomas A; Brissett, Nigel C; Ehlinger, Aaron; Keen, Benjamin A; Kolesar, Peter; Taylor, Elaine M; Bailey, Laura J; Lindsay, Howard D; Chazin, Walter J; Doherty, Aidan J

2017-05-23

DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.

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.

A maize root tip system to study DNA replication programmes in somatic and endocycling nuclei during plant development.

PubMed

Bass, Hank W; Wear, Emily E; Lee, Tae-Jin; Hoffman, Gregg G; Gumber, Hardeep K; Allen, George C; Thompson, William F; Hanley-Bowdoin, Linda

2014-06-01

The progress of nuclear DNA replication is complex in both time and space, and may reflect several levels of chromatin structure and 3-dimensional organization within the nucleus. To understand the relationship between DNA replication and developmental programmes, it is important to examine replication and nuclear substructure in different developmental contexts including natural cell-cycle progressions in situ. Plant meristems offer an ideal opportunity to analyse such processes in the context of normal growth of an organism. Our current understanding of large-scale chromosomal DNA replication has been limited by the lack of appropriate tools to visualize DNA replication with high resolution at defined points within S phase. In this perspective, we discuss a promising new system that can be used to visualize DNA replication in isolated maize (Zea mays L.) root tip nuclei after in planta pulse labelling with the thymidine analogue, 5-ethynyl-2'-deoxyuridine (EdU). Mixed populations of EdU-labelled nuclei are then separated by flow cytometry into sequential stages of S phase and examined directly using 3-dimensional deconvolution microscopy to characterize spatial patterns of plant DNA replication. Combining spatiotemporal analyses with studies of replication and epigenetic inheritance at the molecular level enables an integrated experimental approach to problems of mitotic inheritance and cellular differentiation. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

End-to-end crosstalk within the hepatitis C virus genome mediates the conformational switch of the 3′X-tail region

PubMed Central

Romero-López, Cristina; Barroso-delJesus, Alicia; García-Sacristán, Ana; Briones, Carlos; Berzal-Herranz, Alfredo

2014-01-01

The hepatitis C virus (HCV) RNA genome contains multiple structurally conserved domains that make long-distance RNA–RNA contacts important in the establishment of viral infection. Microarray antisense oligonucelotide assays, improved dimethyl sulfate probing methods and 2′ acylation chemistry (selective 2’-hydroxyl acylation and primer extension, SHAPE) showed the folding of the genomic RNA 3′ end to be regulated by the internal ribosome entry site (IRES) element via direct RNA–RNA interactions. The essential cis-acting replicating element (CRE) and the 3′X-tail region adopted different 3D conformations in the presence and absence of the genomic RNA 5′ terminus. Further, the structural transition in the 3′X-tail from the replication-competent conformer (consisting of three stem-loops) to the dimerizable form (with two stem-loops), was found to depend on the presence of both the IRES and the CRE elements. Complex interplay between the IRES, the CRE and the 3′X-tail region would therefore appear to occur. The preservation of this RNA–RNA interacting network, and the maintenance of the proper balance between different contacts, may play a crucial role in the switch between different steps of the HCV cycle. PMID:24049069

Primary Role for Toll-Like Receptor-Driven Tumor Necrosis Factor Rather than Cytosolic Immune Detection in Restricting Coxiella burnetii Phase II Replication within Mouse Macrophages

PubMed Central

Bradley, William P.; Boyer, Mark A.; Nguyen, Hieu T.; Birdwell, L. Dillon; Yu, Janet; Ribeiro, Juliana M.; Roy, Craig R.

2016-01-01

Coxiella burnetii replicates within permissive host cells by employing a Dot/Icm type IV secretion system (T4SS) to translocate effector proteins that direct the formation of a parasitophorous vacuole. C57BL/6 mouse macrophages restrict the intracellular replication of the C. burnetii Nine Mile phase II (NMII) strain. However, eliminating Toll-like receptor 2 (TLR2) permits bacterial replication, indicating that the restriction of bacterial replication is immune mediated. Here, we examined whether additional innate immune pathways are employed by C57BL/6 macrophages to sense and restrict NMII replication. In addition to the known role of TLR2 in detecting and restricting NMII infection, we found that TLR4 also contributes to cytokine responses but is not required to restrict bacterial replication. Furthermore, the TLR signaling adaptors MyD88 and Trif are required for cytokine responses and restricting bacterial replication. The C. burnetii NMII T4SS translocates bacterial products into C57BL/6 macrophages. However, there was little evidence of cytosolic immune sensing of NMII, as there was a lack of inflammasome activation, T4SS-dependent cytokine responses, and robust type I interferon (IFN) production, and these pathways were not required to restrict bacterial replication. Instead, endogenous tumor necrosis factor (TNF) produced upon TLR sensing of C. burnetii NMII was required to control bacterial replication. Therefore, our findings indicate a primary role for TNF produced upon immune detection of C. burnetii NMII by TLRs, rather than cytosolic PRRs, in enabling C57BL/6 macrophages to restrict bacterial replication. PMID:26787725

Relationship between DNA damage response, initiated by camptothecin or oxidative stress, and DNA replication, analyzed by quantitative 3D image analysis.

PubMed

Berniak, K; Rybak, P; Bernas, T; Zarębski, M; Biela, E; Zhao, H; Darzynkiewicz, Z; Dobrucki, J W

2013-10-01

A method of quantitative analysis of spatial (3D) relationship between discrete nuclear events detected by confocal microscopy is described and applied in analysis of a dependence between sites of DNA damage signaling (γH2AX foci) and DNA replication (EdU incorporation) in cells subjected to treatments with camptothecin (Cpt) or hydrogen peroxide (H2O2). Cpt induces γH2AX foci, likely reporting formation of DNA double-strand breaks (DSBs), almost exclusively at sites of DNA replication. This finding is consistent with the known mechanism of induction of DSBs by DNA topoisomerase I (topo1) inhibitors at the sites of collisions of the moving replication forks with topo1-DNA "cleavable complexes" stabilized by Cpt. Whereas an increased level of H2AX histone phosphorylation is seen in S-phase of cells subjected to H2O2, only a minor proportion of γH2AX foci coincide with DNA replication sites. Thus, the increased level of H2AX phosphorylation induced by H2O2 is not a direct consequence of formation of DNA lesions at the sites of moving DNA replication forks. These data suggest that oxidative stress induced by H2O2 and formation of the primary H2O2-induced lesions (8-oxo-7,8-dihydroguanosine) inhibits replication globally and triggers formation of γH2AX at various distances from replication forks. Quantitative analysis of a frequency of DNA replication sites and γH2AX foci suggests also that stalling of replicating forks by Cpt leads to activation of new DNA replication origins. © 2013 International Society for Advancement of Cytometry. Copyright © 2013 International Society for Advancement of Cytometry.

Bromovirus RNA Replication Compartment Formation Requires Concerted Action of 1a's Self-Interacting RNA Capping and Helicase Domains

PubMed Central

Diaz, Arturo; Gallei, Andreas

2012-01-01

All positive-strand RNA viruses replicate their genomes in association with rearranged intracellular membranes such as single- or double-membrane vesicles. Brome mosaic virus (BMV) RNA synthesis occurs in vesicular endoplasmic reticulum (ER) membrane invaginations, each induced by many copies of viral replication protein 1a, which has N-terminal RNA capping and C-terminal helicase domains. Although the capping domain is responsible for 1a membrane association and ER targeting, neither this domain nor the helicase domain was sufficient to induce replication vesicle formation. Moreover, despite their potential for mutual interaction, the capping and helicase domains showed no complementation when coexpressed in trans. Cross-linking showed that the capping and helicase domains each form trimers and larger multimers in vivo, and the capping domain formed extended, stacked, hexagonal lattices in vivo. Furthermore, coexpressing the capping domain blocked the ability of full-length 1a to form replication vesicles and replicate RNA and recruited full-length 1a into mixed hexagonal lattices with the capping domain. Thus, BMV replication vesicle formation and RNA replication depend on the direct linkage and concerted action of 1a's self-interacting capping and helicase domains. In particular, the capping domain's strong dominant-negative effects showed that the ability of full-length 1a to form replication vesicles was highly sensitive to disruption by non-productively titrating lattice-forming self-interactions of the capping domain. These and other findings shed light on the roles and interactions of 1a domains in replication compartment formation and support prior results suggesting that 1a induces replication vesicles by forming a capsid-like interior shell. PMID:22090102

Derivation of simple rules for complex flow vector fields on the lower part of the human face for robot face design.

PubMed

Ishihara, Hisashi; Ota, Nobuyuki; Asada, Minoru

2017-11-27

It is quite difficult for android robots to replicate the numerous and various types of human facial expressions owing to limitations in terms of space, mechanisms, and materials. This situation could be improved with greater knowledge regarding these expressions and their deformation rules, i.e. by using the biomimetic approach. In a previous study, we investigated 16 facial deformation patterns and found that each facial point moves almost only in its own principal direction and different deformation patterns are created with different combinations of moving lengths. However, the replication errors caused by moving each control point of a face in only their principal direction were not evaluated for each deformation pattern at that time. Therefore, we calculated the replication errors in this study using the second principal component scores of the 16 sets of flow vectors at each point on the face. More than 60% of the errors were within 1 mm, and approximately 90% of them were within 3 mm. The average error was 1.1 mm. These results indicate that robots can replicate the 16 investigated facial expressions with errors within 3 mm and 1 mm for about 90% and 60% of the vectors, respectively, even if each point on the robot face moves in only its own principal direction. This finding seems promising for the development of robots capable of showing various facial expressions because significantly fewer types of movements than previously predicted are necessary.

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

PubMed

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

2018-03-01

Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication. IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly associated with the replicating single-stranded DNA viral genome and played a critical role in viral DNA replication. In contrast, the DNA damage response-induced phosphorylated forms of RPA32 were dispensable for viral DNA replication. Copyright © 2018 American Society for Microbiology.

The Human RNA Polymerase I Transcription Terminator Complex Acts as a Replication Fork Barrier That Coordinates the Progress of Replication with rRNA Transcription Activity.

PubMed

Akamatsu, Yufuko; Kobayashi, Takehiko

2015-05-01

In S phase, the replication and transcription of genomic DNA need to accommodate each other, otherwise their machineries collide, with chromosomal instability as a possible consequence. Here, we characterized the human replication fork barrier (RFB) that is present downstream from the 47S pre-rRNA gene (ribosomal DNA [rDNA]). We found that the most proximal transcription terminator, Sal box T1, acts as a polar RFB, while the other, Sal box T4/T5, arrests replication forks bidirectionally. The fork-arresting activity at these sites depends on polymerase I (Pol I) transcription termination factor 1 (TTF-1) and a replisome component, TIMELESS (TIM). We also found that the RFB activity was linked to rDNA copies with hypomethylated CpG and coincided with the time that actively transcribed rRNA genes are replicated. Failed fork arrest at RFB sites led to a slowdown of fork progression moving in the opposite direction to rRNA transcription. Chemical inhibition of transcription counteracted this deceleration of forks, indicating that rRNA transcription impedes replication in the absence of RFB activity. Thus, our results reveal a role of RFB for coordinating the progression of replication and transcription activity in highly transcribed rRNA genes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV.

PubMed

Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P; Robertson, Erle S; Schildkraut, Carl L; Verma, Subhash C

2016-05-05

Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Replication of the Escherichia coli chromosome in RNase HI-deficient cells: multiple initiation regions and fork dynamics.

PubMed

Maduike, Nkabuije Z; Tehranchi, Ashley K; Wang, Jue D; Kreuzer, Kenneth N

2014-01-01

DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation protein DnaA. However, replication can be initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic evidence indicates that initiation from oriK depends on RNA-DNA hybrids (R-loops), which are normally removed by enzymes such as RNase HI to prevent oriK from misfiring during normal growth. Initiation from oriK sites occurs in RNase HI-deficient mutants, and possibly in wild-type cells under certain unusual conditions. Despite previous work, the locations of oriK and their impact on genome stability remain unclear. We combined 2D gel electrophoresis and whole genome approaches to map genome-wide oriK locations. The DNA copy number profiles of various RNase HI-deficient strains contained multiple peaks, often in consistent locations, identifying candidate oriK sites. Removal of RNase HI protein also leads to global alterations of replication fork migration patterns, often opposite to normal replication directions, and presumably eukaryote-like replication fork merging. Our results have implications for genome stability, offering a new understanding of how RNase HI deficiency results in R-loop-mediated transcription-replication conflict, as well as inappropriate replication stalling or blockage at Ter sites outside of the terminus trap region and at ribosomal operons. © 2013 John Wiley & Sons Ltd.

Endonuclease G promotes mitochondrial genome cleavage and replication

PubMed Central

Wiehe, Rahel Stefanie; Gole, Boris; Chatre, Laurent; Walther, Paul; Calzia, Enrico; Ricchetti, Miria; Wiesmüller, Lisa

2018-01-01

Endonuclease G (EndoG) is a nuclear-encoded endonuclease, mostly localised in mitochondria. In the nucleus EndoG participates in site-specific cleavage during replication stress and genome-wide DNA degradation during apoptosis. However, the impact of EndoG on mitochondrial DNA (mtDNA) metabolism is poorly understood. Here, we investigated whether EndoG is involved in the regulation of mtDNA replication and removal of aberrant copies. We applied the single-cell mitochondrial Transcription and Replication Imaging Protocol (mTRIP) and PCR-based strategies on human cells after knockdown/knockout and re-expression of EndoG. Our analysis revealed that EndoG stimulates both mtDNA replication initiation and mtDNA depletion, the two events being interlinked and dependent on EndoG's nuclease activity. Stimulation of mtDNA replication by EndoG was independent of 7S DNA processing at the replication origin. Importantly, both mtDNA-directed activities of EndoG were promoted by oxidative stress. Inhibition of base excision repair (BER) that repairs oxidative stress-induced DNA damage unveiled a pronounced effect of EndoG on mtDNA removal, reminiscent of recently discovered links between EndoG and BER in the nucleus. Altogether with the downstream effects on mitochondrial transcription, protein expression, redox status and morphology, this study demonstrates that removal of damaged mtDNA by EndoG and compensatory replication play a critical role in mitochondria homeostasis. PMID:29719607

Double silencing of relevant genes suggests the existence of the direct link between DNA replication/repair and central carbon metabolism in human fibroblasts.

PubMed

Wieczorek, Aneta; Fornalewicz, Karolina; Mocarski, Łukasz; Łyżeń, Robert; Węgrzyn, Grzegorz

2018-04-15

Genetic evidence for a link between DNA replication and glycolysis has been demonstrated a decade ago in Bacillus subtilis, where temperature-sensitive mutations in genes coding for replication proteins could be suppressed by mutations in genes of glycolytic enzymes. Then, a strong influence of dysfunctions of particular enzymes from the central carbon metabolism (CCM) on DNA replication and repair in Escherichia coli was reported. Therefore, we asked if such a link occurs only in bacteria or it is a more general phenomenon. Here, we demonstrate that effects of silencing (provoked by siRNA) of expression of genes coding for proteins involved in DNA replication and repair (primase, DNA polymerase ι, ligase IV, and topoisomerase IIIβ) on these processes (less efficient entry into the S phase of the cell cycle and decreased level of DNA synthesis) could be suppressed by silencing of specific genes of enzymes from CMM. Silencing of other pairs of replication/repair and CMM genes resulted in enhancement of the negative effects of lower expression levels of replication/repair genes. We suggest that these results may be proposed as a genetic evidence for the link between DNA replication/repair and CMM in human cells, indicating that it is a common biological phenomenon, occurring from bacteria to humans. Copyright © 2018 Elsevier B.V. All rights reserved.

Selection enhanced estimates of marker effects on means and variances of beef tenderness

USDA-ARS?s Scientific Manuscript database

Genetic marker associations from surveys of industry cattle populations have low frequencies of rare homozygous animals. Selection for calpain (CAPN1) and calpastatin (CAST) genetic markers was replicated in two cattle populations (Angus and MARC III) at the U.S. Meat Animal Research Center. These...

A Formal Language Selection Process for Introductory Programming Courses

ERIC Educational Resources Information Center

Parker, Kevin R.; Chao, Joseph T.; Ottaway, Thomas A.; Chang, Jane

2006-01-01

The selection of a programming language for introductory courses has long been an informal process involving faculty evaluation, discussion, and consensus. As the number of faculty, students, and language options grows, this process becomes increasingly unwieldy. As it stands, the process currently lacks structure and replicability. Establishing a…

Registration of "Essex" lentil

USDA-ARS?s Scientific Manuscript database

‘Essex’ lentil (Lens culinaris Medik.) was released by the USDA-ARS in August 2009. Essex, selection LC01602307E, originated as an F5 selection from the cross of Richlea/PI 297754. Essex was released based on its superior yield performance relative to the variety Eston. Essex was tested in replicate...

Neural network models for spatial data mining, map production, and cortical direction selectivity

NASA Astrophysics Data System (ADS)

Parsons, Olga

A family of ARTMAP neural networks for incremental supervised learning has been developed over the last decade. The Sensor Exploitation Group of MIT Lincoln Laboratory (LL) has incorporated an early version of this network as the recognition engine of a hierarchical system for fusion and data mining of multiple registered geospatial images. The LL system has been successfully fielded, but it is limited to target vs. non-target identifications and does not produce whole maps. This dissertation expands the capabilities of the LL system so that it learns to identify arbitrarily many target classes at once and can thus produce a whole map. This new spatial data mining system is designed particularly to cope with the highly skewed class distributions of typical mapping problems. Specification of a consistent procedure and a benchmark testbed has permitted the evaluation of candidate recognition networks as well as pre- and post-processing and feature extraction options. The resulting default ARTMAP network and mapping methodology set a standard for a variety of related mapping problems and application domains. The second part of the dissertation investigates the development of cortical direction selectivity. The possible role of visual experience and oculomotor behavior in the maturation of cells in the primary visual cortex is studied. The responses of neurons in the thalamus and cortex of the cat are modeled when natural scenes are scanned by several types of eye movements. Inspired by the Hebbian-like synaptic plasticity, which is based upon correlations between cell activations, the second-order statistical structure of thalamo-cortical activity is examined. In the simulations, patterns of neural activity that lead to a correct refinement of cell responses are observed during visual fixation, when small ocular movements occur, but are not observed in the presence of large saccades. Simulations also replicate experiments in which kittens are reared under stroboscopic illumination. The abnormal fixational eye movements of these cats may account for the puzzling finding of a specific loss of cortical direction selectivity but preservation of orientation selectivity. This work indicates that the oculomotor behavior of visual fixation may play an important role in the refinement of cell response selectivity.

Running on empty: does mitochondrial DNA mutation limit replicative lifespan in yeast?: Mutations that increase the division rate of cells lacking mitochondrial DNA also extend replicative lifespan in Saccharomyces cerevisiae.

PubMed

Dunn, Cory D

2011-10-01

Mitochondrial DNA (mtDNA) mutations escalate with increasing age in higher organisms. However, it has so far been difficult to experimentally determine whether mtDNA mutation merely correlates with age or directly limits lifespan. A recent study shows that budding yeast can also lose functional mtDNA late in life. Interestingly, independent studies of replicative lifespan (RLS) and of mtDNA-deficient cells show that the same mutations can increase both RLS and the division rate of yeast lacking the mitochondrial genome. These exciting, parallel findings imply a potential causal relationship between mtDNA mutation and replicative senescence. Furthermore, these results suggest more efficient methods for discovering genes that determine lifespan. Copyright © 2011 WILEY Periodicals, Inc.

DNA replication stress as a hallmark of cancer.

PubMed

Macheret, Morgane; Halazonetis, Thanos D

2015-01-01

Human cancers share properties referred to as hallmarks, among which sustained proliferation, escape from apoptosis, and genomic instability are the most pervasive. The sustained proliferation hallmark can be explained by mutations in oncogenes and tumor suppressors that regulate cell growth, whereas the escape from apoptosis hallmark can be explained by mutations in the TP53, ATM, or MDM2 genes. A model to explain the presence of the three hallmarks listed above, as well as the patterns of genomic instability observed in human cancers, proposes that the genes driving cell proliferation induce DNA replication stress, which, in turn, generates genomic instability and selects for escape from apoptosis. Here, we review the data that support this model, as well as the mechanisms by which oncogenes induce replication stress. Further, we argue that DNA replication stress should be considered as a hallmark of cancer because it likely drives cancer development and is very prevalent.

Conditionally replicative adenovirus for gastrointestinal cancers.

PubMed

Yamamoto, Masato

2004-08-01

The clinical outcome of advanced gastrointestinal (GI) cancers (especially pancreatic and oesophageal cancers) is dismal, despite the advance of conventional therapeutic strategies. Cancer gene therapy is a category of new therapeutics, among which conditionally replicative adenovirus (CRAd) is one promising strategy to overcome existing obstacles of cancer gene therapy. Various CRAds have been developed for GI cancer treatment by taking advantage of the replication biology of adenovirus. Some CRAds have already been tested in clinical trials, but have fallen short of initial expectations. Concerns for clinical applicability include therapeutic potency, replication selectivity and interval end points in clinical trials. In addition, improvement of experimental animal models is needed for a deeper understanding of CRAd biology. Despite these obstacles, CRAds continue to be an exciting area of investigation with great potential for clinical utility. Further virological and oncological research will eventually lead to full realisation of the therapeutic potential of CRAds in the field of GI cancers.

Self-Replicating Cracks: A Collaborative Fracture Mode in Thin Films

NASA Astrophysics Data System (ADS)

Marthelot, Joël; Roman, Benoît; Bico, José; Teisseire, Jérémie; Dalmas, Davy; Melo, Francisco

2014-08-01

Straight cracks are observed in thin coatings under residual tensile stress, resulting into the classical network pattern observed in china crockery, old paintings, or dry mud. Here, we present a novel fracture mechanism where delamination and propagation occur simultaneously, leading to the spontaneous self-replication of an initial template. Surprisingly, this mechanism is active below the standard critical tensile load for channel cracks and selects a robust interaction length scale on the order of 30 times the film thickness. Depending on triggering mechanisms, crescent alleys, spirals, or long bands are generated over a wide range of experimental parameters. We describe with a simple physical model, the selection of the fracture path and provide a configuration diagram displaying the different failure modes.

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.

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.

Selective killing of Kaposi's sarcoma-associated herpesvirus lytically infected cells with a recombinant immunotoxin targeting the viral gpK8.1A envelope glycoprotein

PubMed Central

Chatterjee, Deboeeta; Chandran, Bala

2012-01-01

Kaposi sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) is etiologically associated with three neoplastic syndromes: Kaposi sarcoma and the uncommon HIV-associated B-cell lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman disease. The incidence of the latter B-cell pathology has been increasing in spite of antiretroviral therapy; its association with lytic virus replication has prompted interest in therapeutic strategies aimed at this phase of the virus life cycle. We designed and expressed a recombinant immunotoxin (2014-PE38) targeting the gpK8.1A viral glycoprotein expressed on the surface of the virion and infected cells. We show that this immunotoxin selectively kills KSHV-infected cells in dose-dependent fashion, resulting in major reductions of infectious virus release. The immunotoxin and ganciclovir, an inhibitor of viral DNA replication, showed marked reciprocal potentiation of antiviral activities. These results suggest that the immunotoxin, alone or in combination, may represent a new approach to treat diseases associated with KSHV lytic replication. PMID:22377676

Bifurcation into functional niches in adaptation.

PubMed

White, Justin S; Adami, Christoph

2004-01-01

One of the central questions in evolutionary biology concerns the dynamics of adaptation and diversification. This issue can be addressed experimentally if replicate populations adapting to identical environments can be investigated in detail. We have studied 501 such replicas using digital organisms adapting to at least two fundamentally different functional niches (survival strategies) present in the same environment: one in which fast replication is the way to live, and another where exploitation of the environment's complexity leads to complex organisms with longer life spans and smaller replication rates. While these two modes of survival are closely analogous to those expected to emerge in so-called r and K selection scenarios respectively, the bifurcation of evolutionary histories according to these functional niches occurs in identical environments, under identical selective pressures. We find that the branching occurs early, and leads to drastic phenotypic differences (in fitness, sequence length, and gestation time) that are permanent and irreversible. This study confirms an earlier experimental effort using microorganisms, in that diversification can be understood at least in part in terms of bifurcations on saddle points leading to peak shifts, as in the picture drawn by Sewall Wright.

Lipopolysaccharide-Specific but Not Anti-Flagellar Immunoglobulin A Monoclonal Antibodies Prevent Salmonella enterica Serotype Enteritidis Invasion and Replication within HEp-2 Cell Monolayers

PubMed Central

Iankov, Ianko D.; Petrov, Dragomir P.; Mladenov, Ivan V.; Haralambieva, Iana H.; Mitov, Ivan G.

2002-01-01

The protective potential of immunoglobulin A (IgA) monoclonal antibodies (MAbs) directed against O and H antigens of Salmonella enterica serotype Enteritidis to prevent bacterial adhesion to and invasion of HEp-2 cells was evaluated. Although anti-flagellar IgA MAbs showed strong agglutinating capacities, they did not protect cell monolayers. In contrast, IgA MAbs specific for the O:9 epitope of Salmonella lipopolysaccharide antigen alone prevented S. enterica serotype Enteritidis entry and replication within HEp-2 cells, and the protection was not mediated by direct binding of antibodies to bacterial adhesins or by agglutination of microorganisms. PMID:11854252