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Sample records for polymerase conferring differential

  1. RNA polymerase errors cause splicing defects and can be regulated by differential expression of RNA polymerase subunits

    PubMed Central

    Carey, Lucas B

    2015-01-01

    Errors during transcription may play an important role in determining cellular phenotypes: the RNA polymerase error rate is >4 orders of magnitude higher than that of DNA polymerase and errors are amplified >1000-fold due to translation. However, current methods to measure RNA polymerase fidelity are low-throughout, technically challenging, and organism specific. Here I show that changes in RNA polymerase fidelity can be measured using standard RNA sequencing protocols. I find that RNA polymerase is error-prone, and these errors can result in splicing defects. Furthermore, I find that differential expression of RNA polymerase subunits causes changes in RNA polymerase fidelity, and that coding sequences may have evolved to minimize the effect of these errors. These results suggest that errors caused by RNA polymerase may be a major source of stochastic variability at the level of single cells. DOI: http://dx.doi.org/10.7554/eLife.09945.001 PMID:26652005

  2. International Conference on Multiscale Methods and Partial Differential Equations.

    SciTech Connect

    Thomas Hou

    2006-12-12

    The International Conference on Multiscale Methods and Partial Differential Equations (ICMMPDE for short) was held at IPAM, UCLA on August 26-27, 2005. The conference brought together researchers, students and practitioners with interest in the theoretical, computational and practical aspects of multiscale problems and related partial differential equations. The conference provided a forum to exchange and stimulate new ideas from different disciplines, and to formulate new challenging multiscale problems that will have impact in applications.

  3. Multidrug resistance conferred by novel DNA polymerase mutations in human cytomegalovirus isolates.

    PubMed

    Scott, Gillian M; Weinberg, Adriana; Rawlinson, William D; Chou, Sunwen

    2007-01-01

    The emergence of antiviral-resistant cytomegalovirus (CMV) strains is a continuing clinical problem, with increased numbers of immunocompromised patients given longer-duration antiviral prophylaxis. Two previously unrecognized CMV DNA polymerase mutations (N408K and A834P) identified separately and together in at-risk lung and kidney transplant recipients and a third mutation (L737M) identified in a liver transplant recipient were characterized by marker transfer to antiviral-sensitive laboratory strains AD169 and Towne. Subsequent phenotypic analyses of recombinant strains demonstrated the ability of mutation N408K to confer ganciclovir (GCV) and cidofovir (CDV) resistance and of mutation A834P to confer GCV, foscarnet, and CDV resistance. Mutation L737M did not confer resistance to any of the antiviral agents tested. A recombinant strain containing both N408K and A834P demonstrated increased GCV and CDV resistance compared to the levels of resistance of the virus containing only the A834P mutation. The addition of mutation N408K in combination with A834P also partially reconstituted the replication impairment of recombinant virus containing only A834P. This suggests that perturbation of both DNA polymerization (A834P) and exonuclease (N408K) activities contributes to antiviral resistance and altered replication kinetics in these mutant strains. The identification of these multidrug-resistant CMV strains in at-risk seronegative recipients of organs from seropositive donors suggests that improved prophylactic and treatment strategies are required. The additive effect of multiple mutations on antiviral susceptibility suggests that increasing antiviral-resistant phenotypes can result from different virus-antiviral interactions.

  4. Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases.

    PubMed

    Pavlov, Andrey R; Belova, Galina I; Kozyavkin, Sergei A; Slesarev, Alexei I

    2002-10-15

    Helix-hairpin-helix (HhH) is a widespread motif involved in sequence-nonspecific DNA binding. The majority of HhH motifs function as DNA-binding modules with typical occurrence of one HhH motif or one or two (HhH)(2) domains in proteins. We recently identified 24 HhH motifs in DNA topoisomerase V (Topo V). Although these motifs are dispensable for the topoisomerase activity of Topo V, their removal narrows the salt concentration range for topoisomerase activity tenfold. Here, we demonstrate the utility of Topo V's HhH motifs for modulating DNA-binding properties of the Stoffel fragment of TaqDNA polymerase and Pfu DNA polymerase. Different HhH cassettes fused with either NH(2) terminus or COOH terminus of DNA polymerases broaden the salt concentration range of the polymerase activity significantly (up to 0.5 M NaCl or 1.8 M potassium glutamate). We found that anions play a major role in the inhibition of DNA polymerase activity. The resistance of initial extension rates and the processivity of chimeric polymerases to salts depend on the structure of added HhH motifs. Regardless of the type of the construct, the thermal stability of chimeric Taq polymerases increases under the optimal ionic conditions, as compared with that of TaqDNA polymerase or its Stoffel fragment. Our approach to raise the salt tolerance, processivity, and thermostability of Taq and Pfu DNA polymerases may be applied to all pol1- and polB-type polymerases, as well as to other DNA processing enzymes.

  5. Acanthamoeba can be differentiated by the polymerase chain reaction and simple plating assays.

    PubMed

    Khan, N A; Jarroll, E L; Paget, T A

    2001-09-01

    Acanthamoeba are opportunistic pathogens with invasive and noninvasive species. For clinical purposes it is important to differentiate potentially pathogenic from nonpathogenic isolates. For the rapid and sensitive identification of Acanthamoeba at the genus level, we used a polymerase chain reaction (PCR)-based method which detected as few as five cells. Further, we tested nine isolates of Acanthamoeba for their ability to produce cytopathic effects (CPE) on corneal epithelial cells. On the basis of the results, Acanthamoeba were divided into pathogenic or nonpathogenic groups. However, because CPE assays are not available to every diagnostic laboratory, we developed a simple plating assay based on osmotolerance which correlated well with the CPE assays. Pathogenic Acanthamoeba showed growth on higher osmolarity (agar plates containing one molar mannitol), while growth of nonpathogens was inhibited on these plates. In conclusion, we have developed methods for the rapid identification and differentiation of Acanthamoeba.

  6. Polymerase I and transcript release factor (PTRF) regulates adipocyte differentiation and determines adipose tissue expandability

    PubMed Central

    Perez-Diaz, Sergio; Johnson, Lance A.; DeKroon, Robert M.; Moreno-Navarrete, Jose M.; Alzate, Oscar; Fernandez-Real, Jose M.; Maeda, Nobuyo; Arbones-Mainar, Jose M.

    2014-01-01

    Impaired adipogenesis renders an adipose tissue unable to expand, leading to lipotoxicity and conditions such as diabetes and cardiovascular disease. While factors important for adipogenesis have been studied extensively, those that set the limits of adipose tissue expansion remain undetermined. Feeding a Western-type diet to apolipoprotein E2 knock-in mice, a model of metabolic syndrome, produced 3 groups of equally obese mice: mice with normal glucose tolerance, hyperinsulinemic yet glucose-tolerant mice, and prediabetic mice with impaired glucose tolerance and reduced circulating insulin. Using proteomics, we compared subcutaneous adipose tissues from mice in these groups and found that the expression of PTRF (polymerase I and transcript release factor) associated selectively with their glucose tolerance status. Lentiviral and pharmacologically overexpressed PTRF, whose function is critical for caveola formation, compromised adipocyte differentiation of cultured 3T3-L1cells. In human adipose tissue, PTRF mRNA levels positively correlated with markers of lipolysis and cellular senescence. Furthermore, a negative relationship between telomere length and PTRF mRNA levels was observed in human subcutaneous fat. PTRF is associated with limited adipose tissue expansion underpinning the key role of caveolae in adipocyte regulation. Furthermore, PTRF may be a suitable adipocyte marker for predicting pathological obesity and inform clinical management.—Perez-Diaz, S., Johnson, L. A., DeKroon, R. M., Moreno-Navarrete, J. M., Alzate, O., Fernandez-Real, J. M., Maeda, N., Arbones-Mainar, J. M. Polymerase I and transcript release factor (PTRF) regulates adipocyte differentiation and determines adipose tissue expandability. PMID:24812087

  7. Differential diagnosis of Taenia saginata and Taenia solium infections: from DNA probes to polymerase chain reaction.

    PubMed

    González, Luis Miguel; Montero, Estrella; Sciutto, Edda; Harrison, Leslie J S; Parkhouse, R Michael E; Garate, Teresa

    2002-04-01

    The objective of this work was the rapid and easy differential diagnosis of Taenia saginata and T. solium. First, a T. saginata size-selected genomic deoxyribonucleic acid (gDNA) library was constructed in the vector lambda gt10 using the 2-4 kb fraction from the parasite DNA digested with EcoR1, under 'star' conditions. After differential screening of the library and hybridization analysis with DNA from T. saginata, T. solium, T. taeniaeformis, T. crassiceps, and Echinococcus granulosus (bovine, porcine, and human), 2 recombinant phages were selected. They were designated HDP1 and HDP2. HDP1 reacted specifically with T. saginata DNA, and HDP2 recognized DNA from both T. saginata and T. solium. The 2 DNA probes were then sequenced and further characterized. HDP1 was a repetitive sequence with a 53 bp monomeric unit repeated 24 times in direct tandem along the 1272 bp fragment, while the 3954 bp HDP2 was not a repetitive sequence. Using the sequencing data, oligonucleotides were designed and used in a polymerase chain reaction (PCR). The 2 selected oligonucleotides from probe HDP1 (PTs4F1 and PTs4R1) specifically amplified gDNA from T. saginata, but not T. solium or other related cestodes, with a sensitivity of < 10 pg of T. saginata gDNA, about the quantity of DNA in one taeniid egg. The 3 oligonucleotides selected from the HDP2 sequence (PTs7S35F1, PTs7S35F2, and PTs7S35R1) allowed the differential amplification of gDNA from T. saginata, T. solium and E. granulosus in a multiplex PCR, again with a sensitivity of < 10 pg. These diagnostic tools have immediate application in the differential diagnosis of T. solium and T. saginata in humans and in the diagnosis of dubious cysts in the slaughterhouse. We also hope to apply them to epidemiological surveys of, for example, soil and water in endemic areas.

  8. RNA Polymerase Sigma Factor That Blocks Morphological Differentiation by Streptomyces coelicolor

    PubMed Central

    Gehring, Amy M.; Yoo, Narie J.; Losick, Richard

    2001-01-01

    The filamentous bacterium Streptomyces coelicolor undergoes a complicated process of morphological differentiation that begins with the formation of an aerial mycelium and culminates in sporulation. Genes required for the initiation of aerial mycelium formation have been termed bld (bald), describing the smooth, undifferentiated colonies of mutant strains. By using an insertional mutagenesis protocol that relies on in vitro transposition, we have isolated a bld mutant harboring an insertion in a previously uncharacterized gene, SCE59.12c, renamed here rsuA. The insertion mutant exhibited no measurable growth defect but failed to produce an aerial mycelium and showed a significant delay in the production of the polyketide antibiotic actinorhodin. The rsuA gene encodes an apparent anti-sigma factor and is located immediately downstream of SCE59.13c, renamed here sigU, whose product is inferred to be a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors. The absence of rsuA in a strain that contained sigU caused a block in development, and the overexpression of sigU in an otherwise wild-type strain caused a delay in aerial mycelium formation. However, a strain in which both rsuA and sigU had been deleted was able to undergo morphological differentiation normally. We conclude that the rsuA-encoded anti-sigma factor is responsible for antagonizing the function of the sigma factor encoded by sigU. We also conclude that the sigU-encoded sigma factor is not normally required for development but that its uncontrolled activity obstructs morphological differentiation at an early stage. PMID:11566999

  9. A multiplex real-time polymerase chain reaction assay differentiates between Bolbphorus damnificus and Bolbophorus type II sp

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A duplex quantitative real-time polymerase chain reaction (qPCR) assay was developed to differentiate between Bolbophorus damnificus and Bolbophorus type II species cercariae. Both trematode species are prevalent throughout the commercial catfish industry,.as both infect the ram’s horn snail, Plano...

  10. Respiratory Syncytial Virus Inhibitor AZ-27 Differentially Inhibits Different Polymerase Activities at the Promoter

    PubMed Central

    Noton, Sarah L.; Nagendra, Kartikeya; Dunn, Ewan F.; Mawhorter, Michael E.; Yu, Qin

    2015-01-01

    ABSTRACT Respiratory syncytial virus (RSV) is the leading cause of pediatric respiratory disease. RSV has an RNA-dependent RNA polymerase that transcribes and replicates the viral negative-sense RNA genome. The large polymerase subunit (L) has multiple enzymatic activities, having the capability to synthesize RNA and add and methylate a cap on each of the viral mRNAs. Previous studies (H. Xiong et al., Bioorg Med Chem Lett, 23:6789–6793, 2013, http://dx.doi.org/10.1016/j.bmcl.2013.10.018; C. L. Tiong-Yip et al., Antimicrob Agents Chemother, 58:3867–3873, 2014, http://dx.doi.org/10.1128/AAC.02540-14) had identified a small-molecule inhibitor, AZ-27, that targets the L protein. In this study, we examined the effect of AZ-27 on different aspects of RSV polymerase activity. AZ-27 was found to inhibit equally both mRNA transcription and genome replication in cell-based minigenome assays, indicating that it inhibits a step common to both of these RNA synthesis processes. Analysis in an in vitro transcription run-on assay, containing RSV nucleocapsids, showed that AZ-27 inhibits synthesis of transcripts from the 3′ end of the genome to a greater extent than those from the 5′ end, indicating that it inhibits transcription initiation. Consistent with this finding, experiments that assayed polymerase activity on the promoter showed that AZ-27 inhibited transcription and replication initiation. The RSV polymerase also can utilize the promoter sequence to perform a back-priming reaction. Interestingly, addition of AZ-27 had no effect on the addition of up to three nucleotides by back-priming but inhibited further extension of the back-primed RNA. These data provide new information regarding the mechanism of inhibition by AZ-27. They also suggest that the RSV polymerase adopts different conformations to perform its different activities at the promoter. IMPORTANCE Currently, there are no effective antiviral drugs to treat RSV infection. The RSV polymerase is an

  11. SciCADE 95: International conference on scientific computation and differential equations

    SciTech Connect

    1995-12-31

    This report consists of abstracts from the conference. Topics include algorithms, computer codes, and numerical solutions for differential equations. Linear and nonlinear as well as boundary-value and initial-value problems are covered. Various applications of these problems are also included.

  12. The Teacher and His Staff: Differentiating Teaching Roles. Report of the 1968 Regional TEPS Conferences.

    ERIC Educational Resources Information Center

    National Education Association, Washington, DC. National Commission on Teacher Education and Professional Standards.

    This book contains 10 papers selected from those presented at the 1968 regional conferences of the NEA National Commission on Teacher Education and Professional Standards (NCTEPS) on differentiated staffing: "Teacher Education: Analysis and Recommendations," John Macdonald, chairman, Department of Education, Sir George Williams Univ.; "New…

  13. Rapid and inexpensive species differentiation using a multiplex real-time polymerase chain reaction high-resolution melt assay.

    PubMed

    Elkins, Kelly M; Perez, Anjelica C U; Sweetin, Katherine C

    2016-05-01

    We demonstrate a method for developing real-time polymerase chain reaction (PCR) high-resolution melt (HRM) assays to identify multiple species present in a mixture simultaneously using LCGreen Plus and melt temperatures. Highly specific PCR primers are designed to yield amplicons with different melt temperatures for simple routine species identification compared with differentiating melt curve kinetics traces or difference plots. This method is robust and automatable, and it leads to savings in time and reagent costs, is easily modified to probe any species of interest, eliminates the need for post-PCR gel or capillary electrophoresis in routine assays, and requires no expensive dye-labeled primers.

  14. Differentiation of Giardia duodenalis from other Giardia spp. by using polymerase chain reaction and gene probes.

    PubMed Central

    Mahbubani, M H; Bej, A K; Perlin, M H; Schaefer, F W; Jakubowski, W; Atlas, R M

    1992-01-01

    Giardia spp. are waterborne organisms that are the most commonly identified pathogenic intestinal protozoans in the United States. Current detection techniques for Giardia species in water include microscopy and immunofluorescence techniques. Species of the genus Giardia are classified on the basis of taxonomic criteria, such as cell morphology, and on host specificity. We have developed a polymerase chain reaction- and gene probe-based detection system specific for Giardia spp., which can discriminate between the relevant species of the G. duodenalis type pathogenic to humans and other Giardia species that are not human pathogens. This method can detect a single Giardia cyst and is therefore sensitive enough for environmental monitoring. Images PMID:1734070

  15. Detection and differentiation of filarial parasites by universal primers and polymerase chain reaction-restriction fragment length polymorphism analysis.

    PubMed

    Nuchprayoon, Surang; Junpee, Alisa; Poovorawan, Yong; Scott, Alan L

    2005-11-01

    Filarial nematode parasites are a serious cause of morbidity in humans and animals. Identification of filarial infection using traditional morphologic criteria can be difficult and lead to misdiagnosis. We report on a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based method to detect and differentiate a broad range of filarial species in a single PCR. The first internal transcribed spacer 1 (ITS1) along with the flanking 18S and 5.8S ribosomal DNA (rDNA) were isolated and cloned from Wuchereria bancrofti, Brugia malayi, and Brugia pahangi. Sequence analysis identified conserved sites in the 18S and 5.8S rDNA sequence that could be used as universal priming sites to generate ITS1-distinctive PCR products that were useful for distinguishing filariae at the genus level. The addition of a digestion of the ITS1 PCR product with the restriction endonuclease Ase I generated a fragment profile that allowed differentiation down to the species level for W. bancrofti, B. malayi, B. pahangi, Dirofilaria immitis, and D. repens. The PCR-RFLP of ITS1 rDNA will be useful in diagnosing and differentiating filarial parasites in human, animal reservoir hosts, and mosquito vectors in disease-endemic areas.

  16. Differential diagnosis of Goatpox virus in Taiwan by multiplex polymerase chain reaction assay and high-resolution melt analysis.

    PubMed

    Chan, Kun-Wei; Lee, Ming-Liang; Yang, Wei-Cheng; Wong, Min-Liang; Hsu, Wei-Li; Ho, Chia-Fang; Hsieh, Yao-Ching; Wang, Chi-Young

    2014-03-01

    The A32L gene from a Goatpox virus (GTPV) strain isolated from a goat in Yunlin County (Taiwan) displays several substitutions compared with the sequence of the Kenyan GTPV vaccine strain SGP0240 and the Pellor GTPV strain. Samples from the skin lesions on 6 goats with GTPV infection or from goats with Orf virus (ORFV) infection were tested in a multiplex polymerase chain reaction (PCR) system that used primers GPF, GPR1, and GPR2 as well as previously published primers specific for ORFV. These primers were able to amplify either GTPV or ORFV without cross-reactivity. A high-resolution melt analysis (HRMA) was carried out on amplified DNA from the skin lesions of 6 goats with GTPV infection and with the GTPV SGP0240 strain. The results indicated that the melting temperature profiles amplified from samples with Yunlin GTPV infection can be differentiated from the GTPV SGP0240 strain. The findings showed that a successful differential assay for these GTPVs had been developed. Accordingly, both methods can be used to detect and differentiate GTPV isolated from animals that may have either been vaccinated or been infected with a wild strain. The multiplex PCR and HRMA could be used on skin samples of suspected cases to serve as the front-line and confirmative assays, respectively, which will be beneficial to the eradication of GTPV.

  17. Diagnosis of whooping cough in Switzerland: differentiating Bordetella pertussis from Bordetella holmesii by polymerase chain reaction.

    PubMed

    Pittet, Laure F; Emonet, Stéphane; François, Patrice; Bonetti, Eve-Julie; Schrenzel, Jacques; Hug, Melanie; Altwegg, Martin; Siegrist, Claire-Anne; Posfay-Barbe, Klara M

    2014-01-01

    Bordetella holmesii, an emerging pathogen, can be misidentified as Bordetella pertussis by routine polymerase chain reaction (PCR). In some reports, up to 29% of the patients diagnosed with pertussis have in fact B. holmesii infection and invasive, non-respiratory B. holmesii infections have been reported worldwide. This misdiagnosis undermines the knowledge of pertussis' epidemiology, and may lead to misconceptions on pertussis vaccine's efficacy. Recently, the number of whooping cough cases has increased significantly in several countries. The aim of this retrospective study was to determine whether B. holmesii was contributing to the increase in laboratory-confirmed cases of B. pertussis in Switzerland. A multiplex species-specific quantitative PCR assay was performed on 196 nasopharyngeal samples from Swiss patients with PCR-confirmed Bordetella infection (median age: 6 years-old, minimum 21 days-old, maximum 86 years-old), formerly diagnosed as Bordetella pertussis (IS481+). No B. holmesii (IS481+, IS1001-, hIS1001+) was identified. We discuss whether laboratories should implement specific PCR to recognize different Bordetella species. We conclude that in Switzerland B. holmesii seems to be circulating less than in neighboring countries and that specific diagnostic procedures are not necessary routinely. However, as the epidemiological situation may change rapidly, periodic reevaluation is suggested.

  18. Diagnosis of Whooping Cough in Switzerland: Differentiating Bordetella pertussis from Bordetella holmesii by Polymerase Chain Reaction

    PubMed Central

    Pittet, Laure F.; Emonet, Stéphane; François, Patrice; Bonetti, Eve-Julie; Schrenzel, Jacques; Hug, Melanie; Altwegg, Martin; Siegrist, Claire-Anne; Posfay-Barbe, Klara M.

    2014-01-01

    Bordetella holmesii, an emerging pathogen, can be misidentified as Bordetella pertussis by routine polymerase chain reaction (PCR). In some reports, up to 29% of the patients diagnosed with pertussis have in fact B. holmesii infection and invasive, non-respiratory B. holmesii infections have been reported worldwide. This misdiagnosis undermines the knowledge of pertussis' epidemiology, and may lead to misconceptions on pertussis vaccine's efficacy. Recently, the number of whooping cough cases has increased significantly in several countries. The aim of this retrospective study was to determine whether B. holmesii was contributing to the increase in laboratory-confirmed cases of B. pertussis in Switzerland. A multiplex species-specific quantitative PCR assay was performed on 196 nasopharyngeal samples from Swiss patients with PCR-confirmed Bordetella infection (median age: 6 years-old, minimum 21 days-old, maximum 86 years-old), formerly diagnosed as Bordetella pertussis (IS481+). No B. holmesii (IS481+, IS1001−, hIS1001+) was identified. We discuss whether laboratories should implement specific PCR to recognize different Bordetella species. We conclude that in Switzerland B. holmesii seems to be circulating less than in neighboring countries and that specific diagnostic procedures are not necessary routinely. However, as the epidemiological situation may change rapidly, periodic reevaluation is suggested. PMID:24586447

  19. Differential detection of shrimp and crab for food labeling using polymerase chain reaction.

    PubMed

    Taguchi, Hiromu; Watanabe, Satoshi; Temmei, Yusuke; Hirao, Takashi; Akiyama, Hiroshi; Sakai, Shinobu; Adachi, Reiko; Sakata, Kozue; Urisu, Atsuo; Teshima, Reiko

    2011-04-27

    Shrimp and crab are well-known as allergenic ingredients. According to Japanese food allergy labeling regulations, shrimp species (including prawns, crayfishes, and lobsters) and crab species must be differentially declared when ≥10 ppm (total protein) of an allergenic ingredient is present. However, the commercial ELISA tests for the detection of crustacean proteins cannot differentiate between shrimp and crab. Therefore, two methods were developed to discriminate shrimp and crab: a shrimp-PCR method with postamplification digestion and a crab-PCR method that specifically amplifies a fragment of the 16S rRNA gene. The sensitivity and specificity of both PCR methods were verified by experiments using DNA extracted from 15 shrimp species, 13 crab species, krill, mysid, mantis shrimp, other food samples (cephalopod, shellfish, and fish), incurred foods, and commercial food products. Both PCR methods could detect 5 pg of DNA extracted from target species and 50 ng of genomic DNA extracted from incurred foods containing 10 ppm (μg/g) total protein of shrimp or crab. The two PCR methods were considered to be specific enough to separately detect species belonging to shrimp and crab. Although false-positive and false-negative results were obtained from some nontarget crustacean species, the proposed PCR methods, when used in conjunction with ELISA tests, would be a useful tool for confirmation of the validity of food allergy labeling and management of processed food safety for allergic patients.

  20. Myogenic differentiation of L6 rat myoblasts: evidence for pleiotropic effects on myogenesis by RNA polymerase II mutations to alpha-amanitin resistance.

    PubMed Central

    Crerar, M M; Leather, R; David, E; Pearson, M L

    1983-01-01

    To assess the functional role of RNA polymerase II in the regulation of transcription during muscle differentiation, we isolated and characterized a large number of independent alpha-amanitin-resistant (AmaR) mutants of L6 rat myoblasts that express both wild-type and altered RNA polymerase II activities. We also examined their myogenic (Myo) phenotype by determining their ability to develop into mature myotubes, to express elevated levels of muscle creatine kinase, and to synthesize muscle-characteristic proteins as detected by two-dimensional polyacrylamide gel electrophoresis. We found a two- to threefold increase in the frequency of clones with a myogenic-defective phenotype in the AmaR (RNA polymerase II) mutants as compared to control ethyl methane sulfonate-induced, 6-thioguanine-resistant (hypoxanthine, guanine phosphoribosyl transferase) mutants or to unselected survivors also exposed to ethyl methane sulfonate. Subsequent analysis showed that about half of these myogenic-defective AmaR mutants had a conditional Myo(ama) phenotype; when cultured in the presence of amanitin, they exhibited a Myo- phenotype; in its absence they exhibited a Myo+ phenotype. This conditional Myo(ama) phenotype is presumably caused by the inactivation by amanitin of the wild-type amanitin-sensitive RNA polymerase II activity and the subsequent rise in the level of mutant amanitin-resistant RNA polymerase II activity. In these Myo(ama) mutants, the wild-type RNA polymerase II is normally dominant with respect to the Myo+ phenotype, whereas the mutant RNA polymerase II is recessive and results in a Myo- phenotype only when the wild-type enzyme is inactivated. These findings suggest that certain mutations in the amaR structural gene for the amanitin-binding subunit of RNA polymerase II can selectively impair the transcription of genes specific for myogenic differentiation but not those specific for myoblast proliferation. Images PMID:6865946

  1. Identification of nickel response genes in abnormal early developments of sea urchin by differential display polymerase chain reaction.

    PubMed

    Ryu, Tae Kwon; Lee, Gunsup; Rhee, Yong; Park, Heung-Sik; Chang, Man; Lee, Sukchan; Lee, Jaean; Lee, Taek-Kyun

    2012-10-01

    Bioassays and biomarkers have been previously developed to assess the effects of heavy metal contaminants on the early life stages of the sea urchin. In this study, malformation in the early developmental processes was observed in sea urchin (Strongylocentrotus intermedius) larvae exposed to 10 ppm Ni for over 30 h. The most critical stage at which the triggering of nickel effects takes place is thought to be the blastula stage, which occurs after fertilization in larval development. To investigate the molecular-level responses of sea urchin exposed to heavy metal stress and to explore the differentially expressed genes that are induced or repressed by nickel, differential display polymerase chain reaction (DD-PCR) was used with sea urchin mRNAs. The malformation-related genes expressed in the early life stages of the sea urchin were cloned from larvae exposed to 10 ppm of nickel for 15 h, and accessed via DD-PCR. Sequence analysis results revealed that each of the genes evidenced high homology with EGF2, PCSK9, serine/threonine protein kinase, apolipophorin precursor protein, and MGC80921 protein/transcript variant 2. This result may prove useful in the development of novel biomarkers for the assessment of heavy metal stresses on sea urchin embryos.

  2. Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens

    PubMed Central

    Sexton, Nicole R.; Smith, Everett Clinton; Blanc, Hervé; Vignuzzi, Marco; Peersen, Olve B.

    2016-01-01

    ABSTRACT Positive-sense RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it on solved picornaviral RdRp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(−)] ExoN activity. Using this method, we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I, we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and a decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. The results also indicate that, with regard to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multiprotein replicase-proofreading complex. IMPORTANCE RNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity

  3. Differential diagnosis of fowlpox and infectious laryngotracheitis viruses in chicken diphtheritic manifestations by mono and duplex real-time polymerase chain reaction.

    PubMed

    Davidson, Irit; Raibstein, Israel; Altory, Amira

    2015-01-01

    Infectious laryngotracheitis virus (ILTV) and fowlpox virus (FPV) cause diphtheritic lesions in chicken tracheas and can simultaneously infect the same bird. A differential molecular diagnostic test, the duplex real-time polymerase chain reaction, is now reported using ILTV and FPV vaccine viruses and clinical samples from chickens, either uninfected or naturally infected with ILTV or FPV, or with both viruses. The dual virus amplification by real-time polymerase chain reaction was demonstrated to behave similarly to monoplex amplification, in spite of the fact that the real-time exponential amplification plots of the vaccine viruses were more illustrative than those of the clinical samples.

  4. Detection of nonauthorized genetically modified organisms using differential quantitative polymerase chain reaction: application to 35S in maize.

    PubMed

    Cankar, Katarina; Chauvensy-Ancel, Valérie; Fortabat, Marie-Noelle; Gruden, Kristina; Kobilinsky, André; Zel, Jana; Bertheau, Yves

    2008-05-15

    Detection of nonauthorized genetically modified organisms (GMOs) has always presented an analytical challenge because the complete sequence data needed to detect them are generally unavailable although sequence similarity to known GMOs can be expected. A new approach, differential quantitative polymerase chain reaction (PCR), for detection of nonauthorized GMOs is presented here. This method is based on the presence of several common elements (e.g., promoter, genes of interest) in different GMOs. A statistical model was developed to study the difference between the number of molecules of such a common sequence and the number of molecules identifying the approved GMO (as determined by border-fragment-based PCR) and the donor organism of the common sequence. When this difference differs statistically from zero, the presence of a nonauthorized GMO can be inferred. The interest and scope of such an approach were tested on a case study of different proportions of genetically modified maize events, with the P35S promoter as the Cauliflower Mosaic Virus common sequence. The presence of a nonauthorized GMO was successfully detected in the mixtures analyzed and in the presence of (donor organism of P35S promoter). This method could be easily transposed to other common GMO sequences and other species and is applicable to other detection areas such as microbiology.

  5. Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate ompA DNA of Chlamydia spp.

    PubMed Central

    Kaltenboeck, B; Kousoulas, K G; Storz, J

    1992-01-01

    Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis. Images PMID:1349899

  6. Natural Polymorphisms Conferring Resistance to HCV Protease and Polymerase Inhibitors in Treatment-Naïve HIV/HCV Co-Infected Patients in China

    PubMed Central

    Wang, Charles; Hu, Fengyu; Ning, Chuanyi; Lan, Yun; Tang, Xiaoping; Tucker, Joseph D.; Cai, Weiping

    2016-01-01

    Background The advent of direct-acting agents (DAAs) has improved treatment of HCV in HIV co-infection, but may be limited by primary drug resistance. This study reports the prevalence of natural polymorphisms conferring resistance to NS3/4A protease inhibitors and NS5B polymerase inhibitors in treatment-naïve HIV/HCV co-infected individuals in China. Methods Population based NS3/4A sequencing was completed for 778 treatment-naïve HIV/HCV co-infected patients from twelve provinces. NS3 sequences were amplified by nested PCR using in-house primers for genotypes 1–6. NS5B sequencing was completed for genotyping in 350 sequences. Resistance-associated variants (RAVs) were identified in positions associated with HCV resistance. Results Overall, 72.8% (566/778) of all HCV sequences had at least one RAV associated with HCV NS3/4A protease inhibitor resistance. Variants were found in 3.6% (7/193) of genotype 1, 100% (23/23) of genotype 2, 100% (237/237) of genotype 3 and 92% (299/325) of genotype 6 sequences. The Q80K variant was present in 98.4% of genotype 6a sequences. High-level RAVs were rare, occurring in only 0.8% of patients. 93% (64/69) patients with genotype 1b also carried the C316N variant associated with NS5B low-level resistance. Conclusions The low frequency of high-level RAVs associated with primary HCV DAA resistance among all genotypes in HIV/HCV co-infected patients is encouraging. Further phenotypic studies and clinical research are needed. PMID:27341031

  7. Directed Polymerase Evolution

    PubMed Central

    Chen, Tingjian; Romesberg, Floyd E.

    2014-01-01

    Polymerases evolved in nature to synthesize DNA and RNA, and they underlie the storage and flow of genetic information in all cells. The availability of these enzymes for use at the bench has driven a revolution in biotechnology and medicinal research; however, polymerases did not evolve to function efficiently under the conditions required for some applications and their high substrate fidelity precludes their use for most applications that involve modified substrates. To circumvent these limitations, researchers have turned to directed evolution to tailor the properties and/or substrate repertoire of polymerases for different applications, and several systems have been developed for this purpose. These systems draw on different methods of creating a pool of randomly mutated polymerases and are differentiated by the process used to isolate the most fit members. A variety of polymerases have been evolved, providing new or improved functionality, as well as interesting new insight into the factors governing activity. PMID:24211837

  8. Neurogenic differentiation factor NeuroD confers protection against radiation-induced intestinal injury in mice

    PubMed Central

    Li, Ming; Du, Aonan; Xu, Jing; Ma, Yanchao; Cao, Han; Yang, Chao; Yang, Xiao-Dong; Xing, Chun-Gen; Chen, Ming; Zhu, Wei; Zhang, Shuyu; Cao, Jianping

    2016-01-01

    The gastrointestinal tract, especially the small intestine, is particularly sensitive to radiation, and is prone to radiation-induced injury as a result. Neurogenic differentiation factor (NeuroD) is an evolutionarily-conserved basic helix-loop-helix (bHLH) transcription factor. NeuroD contains a protein transduction domain (PTD), which allows it to be exogenously delivered across the membrane of mammalian cells, whereupon its transcription activity can be unleashed. Whether NeuroD has therapeutic effects for radiation-induced injury remains unclear. In the present study, we prepared a NeuroD-EGFP recombinant protein, and explored its protective effects on the survival and intestinal damage induced by ionizing radiation. Our results showed that NeuroD-EGFP could be transduced into small intestine epithelial cells and tissues. NeuroD-EGFP administration significantly increased overall survival of mice exposed to lethal total body irradiation (TBI). This recombinant NeuroD also reduced radiation-induced intestinal mucosal injury and apoptosis, and improved crypt survival. Expression profiling of NeuroD-EGFP-treated mice revealed upregulation of tissue inhibitor of metalloproteinase 1 (TIMP-1), a known inhibitor of apoptosis in mammalian cells. In conclusion, NeuroD confers protection against radiation-induced intestinal injury, and provides a novel therapeutic clinical option for the prevention of intestinal side effects of radiotherapy and the treatment of victims of incidental exposure. PMID:27436572

  9. Differential furanose selection in the active sites of archaeal DNA polymerases probed by fixed-conformation nucleotide analogues

    PubMed Central

    Ketkar, Amit; Zafar, Maroof K.; Banerjee, Surajit; Marquez, Victor E.; Egli, Martin; Eoff, Robert L.

    2012-01-01

    DNA polymerases select for the incorporation of deoxyribonucleotide triphosphates (dNTPs) using amino acid side-chains that act as a “steric-gate” to bar improper incorporation of rNTPs. An additional factor in the selection of nucleotide substrates resides in the preferred geometry for the furanose moiety of the incoming nucleotide triphosphate. We have probed the role of sugar geometry during nucleotide selection by model DNA polymerases from Sulfolobus solfataricus using fixed conformation nucleotide analogues. North-methanocarba-dATP (N-MC-dATP) locks the central ring into a RNA-type (C2′-exo, North) conformation near a C3′-endo pucker and South-methanocarba-dATP (S-MC-dATP) locks the central ring system into a (C3′-exo, South) conformation near a C2′-endo pucker. Dpo4 preferentially inserts N-MC-dATP and in the crystal structure of Dpo4 in complex with N-MC-dAMP, the nucleotide analogue superimposes almost perfectly with Dpo4 bound to unmodified dATP. Biochemical assays indicate that the S. solfataricus B-family DNA polymerase Dpo1 can insert and extend from both N-MC-dATP and S-MC-dATP. In this respect, Dpo1 is unexpectedly more tolerant of substrate conformation than Dpo4. The crystal structure of Dpo4 bound to S-MC-dADP shows that poor incorporation of the Southern pucker by the Y-family polymerase results from a hydrogen bond between the 3′-OH group of the nucleotide analogue and the OH group of the steric gate residue, Tyr12, shifting the S-MC-dADP molecule away from the dNTP binding pocket and distorting the base pair at the primer-template junction. These results provide insights into substrate specificity of DNA polymerases, as well as molecular mechanisms that act as a barrier against insertion of rNTPs. PMID:23050956

  10. Detection and differentiation of wild-type and vaccine strains of canine distemper virus by a duplex reverse transcription polymerase chain reaction

    PubMed Central

    Dong, X. Y.; Li, W. H.; Zhu, J. L.; Liu, W. J.; Zhao, M. Q.; Luo, Y. W.; Chen, J. D.

    2015-01-01

    Canine distemper virus (CDV) is the cause of canine distemper (CD) which is a severe and highly contagious disease in dogs. In the present study, a duplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of CDV. Four primers were designed to detect and discriminate the two viruses by generating 638- and 781-bp cDNA products, respectively. Furthermore, the duplex RT-PCR method was used to detect 67 field samples suspected of CD from Guangdong province in China. Results showed that, 33 samples were to be wild-type-like. The duplex RT-PCR method exhibited high specificity and sensitivity which could be used to effectively detect and differentiate wild-type and vaccine CDV, indicating its use for clinical detection and epidemiological surveillance. PMID:27175171

  11. An oligonucleotide-ligation assay for the differentiation between Cyclospora and Eimeria spp. polymerase chain reaction amplification products.

    PubMed

    Jinneman, K C; Wetherington, J H; Hill, W E; Omiescinski, C J; Adams, A M; Johnson, J M; Tenge, B J; Dang, N L; Wekell, M M

    1999-06-01

    An oligonucleotide-ligation assay (OLA) was developed and compared to a restriction fragment length polymorphism (RFLP) test for distinguishing between 294-bp polymerase chain reaction (PCR) amplification products of the 18S rRNA gene from Cyclospora and Eimeria spp. The PCR/OLA correctly distinguished between three Cyclospora, three E. tenella, and one E. mitis strains and the ratio of positive to negative spectrophotometric absorbance (A490) values for each strain ranged from 4.086 to 15.280 (median 9.5). PCR/OLA provides a rapid, reliable, spectrophotometric alternative to PCR/RFLP.

  12. Epidermal loss of Gαq confers a migratory and differentiation defect in keratinocytes

    PubMed Central

    Callejas-Valera, Juan Luis; Hansen, Karina K.; Molinolo, Alfredo A.; Inoue, Asuka; Offermanns, Stefan; Gutkind, J. Silvio

    2017-01-01

    G-protein coupled receptors (GPCRs), which activate heterotrimeric G proteins, are an essential class of transmembrane receptors that are responsible for a myriad of signaling events in normal and pathologic conditions. Two members of the G protein family, Gαq and Gα11, activate one of the main GPCR pathways and function as oncogenes by integrating mitogen-stimulated signaling cascades that are active under malignant conditions. Recently, it has been shown that targeted deletion of Gα11 and Gαq from endothelial cells impairs the Rho-mediated formation of focal adherens junctions, suggesting that Gα11/q signaling may also play a significant role in cytoskeletal-mediated cellular responses in epithelial cells. Indeed, combined deletion of Gα11 and Gαq confers a significant migratory defect in keratinocytes that delays cutaneous wound healing in an in vivo setting. This delay can be attributed to a defect during the reepithelialization phase due to significantly attenuated migratory capacity of Gαq-null keratinocytes under combined Gα11 deficiency. In fact, cells lacking Gα11/q demonstrate a severely reduced ability to respond to mitogenic and migratory signals in the microenvironment, leading to inappropriate and premature terminal differentiation. These results suggest that Gα11/q signaling pathways may be critical for integrating mitogenic signals and cytoskeletal function to achieve normal physiological responses. Emergence of a malignant phenotype may therefore arise from both under- and overexpression of Gα11/q signaling, implicating its upstream regulation as a potential therapeutic target in a host of pathologic conditions. PMID:28301547

  13. Polymerase chain reaction assay based on ratA gene allows differentiation between Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum.

    PubMed

    Batista, Diego Felipe Alves; de Freitas Neto, Oliveiro Caetano; Lopes, Priscila Diniz; de Almeida, Adriana Maria; Barrow, Paul Andrew; Berchieri, Angelo

    2013-03-01

    Salmonella Pullorum and Salmonella Gallinarum are classified as biovars of Salmonella enterica subsp. enterica serovar Gallinarum. These salmonellae are the causative agents of Pullorum disease and fowl typhoid, respectively, and are widely distributed throughout the world. Although many developed countries have eradicated these diseases from commercial poultry, they are still the cause of significant economic loss in developing countries. When serovar Gallinarum is isolated, it is difficult to immediately differentiate between biovars because they are antigenically identical by serotyping. However, they cause distinct diseases with different epidemiology, and therefore it is important to differentiate them. This may be done biochemically but takes 2 to 3 days. In the present study, S. Pullorum and S. Gallinarum whole genomes were compared, and 1 genomic region of difference, which is part of the ratA gene, was chosen as a molecular marker for a polymerase chain reaction assay to differentiate rapidly between these organisms. In all, 26 strains of S. Gallinarum and 17 S. Pullorum strains were tested and successfully differentiated by the assay.

  14. Use of genetic polymorphisms detected by the random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) for differentiation and identification of Aedes aegypti subspecies and populations.

    PubMed

    Ballinger-Crabtree, M E; Black, W C; Miller, B R

    1992-12-01

    Amplification of random regions of genomic DNA using 10-base primers in the random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to differentiate and identify mosquito populations based on genetic variation. Genomic DNA was extracted from individual mosquitoes from 11 geographic populations of Aedes aegypti and amplified in PCR reactions using single primers of arbitrary nucleotide sequence. Discriminant analysis of the population frequencies of RAPD fragments produced using three different primers allowed accurate discrimination between the geographic populations in 89% of individuals and between subspecies (Ae. aegypti aegypti versus Ae. aegypti formosus) in 100% of mosquitoes tested. The genetic relatedness of the populations was estimated using three different statistical methods, and unknown populations were correctly classified in a blind test. These results indicate that the RAPD-PCR technique will be useful in studies of arthropod molecular taxonomy and in epidemiologic studies of the relatedness of geographic populations and vector movement.

  15. Development and evaluation of a 1-step duplex reverse transcription polymerase chain reaction for differential diagnosis of chikungunya and dengue infection.

    PubMed

    Dash, Paban Kumar; Parida, Manmohan; Santhosh, S R; Saxena, Parag; Srivastava, Ambuj; Neeraja, Mamidi; Lakshmi, V; Rao, P V Lakshmana

    2008-09-01

    Dengue (DEN) and chikungunya (CHIK) have emerged as the 2 most important arboviral infections of global significance. The similarities in clinical presentations, their circulation in the same geographic area, and the transmission through the same vector necessitate an urgent need for the differential diagnosis of these 2 infections. So far, no single assay is reported for differential diagnosis of these 2 infections. In this study, we report the development and evaluation of a 1-step single-tube duplex reverse transcription polymerase chain reaction (D-RT-PCR) assay by targeting E1 gene of CHIK and C-prM gene junction of DEN virus (DENV), respectively. The sensitivity of this assay was found to be better than conventional virus isolation and could detect as low as 100 copies of genomic RNA, which is equivalent to respective virus-specific RT-PCR. The evaluation was carried out with 360 clinical samples from recent CHIK and DEN outbreaks in India. This assay could also be able to detect dual infection of CHIK and DEN in 3 patients. The phylogenetic analysis based on the nucleotide sequencing of D-RT-PCR amplicon could precisely identify the genotypes of all the serotypes of DENV and CHIK viruses (CHIKV). These findings demonstrate the potential clinical and epidemiologic application of D-RT-PCR for rapid sensitive detection, differentiation, and genotyping of DENV and CHIKV in clinical samples.

  16. Polymerase chain reaction-hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation.

    PubMed

    Xu, Chen; Zhang, Nan; Huo, Qianyu; Chen, Minghui; Wang, Rengfeng; Liu, Zhili; Li, Xue; Liu, Yunde; Bao, Huijing

    2016-04-15

    In this article, we discuss the polymerase chain reaction (PCR)-hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA-BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase-streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR-hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR-hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications.

  17. Influenza virus polymerase confers independence of the cellular cap-binding factor eIF4E for viral mRNA translation

    PubMed Central

    Yángüez, Emilio; Rodriguez, Paloma; Goodfellow, Ian; Nieto, Amelia

    2012-01-01

    The influenza virus mRNAs are structurally similar to cellular mRNAs nevertheless; the virus promotes selective translation of viral mRNAs despite the inhibition of host cell protein synthesis. The infection proceeds normally upon functional impairment of eIF4E cap-binding protein, but requires functional eIF4A helicase and eIF4G factor. Here, we have studied whether the presence of cis elements in viral mRNAs or the action of viral proteins are responsible for this eIF4E-independence. The eIF4E protein is required for viral mRNAs translation in vitro, indicating that cis-acting RNA sequences are not involved in this process. We also show that PB2 viral polymerase subunit interacts with the eIF4G protein. In addition, a chimeric mRNA containing viral UTRs sequences transcribed by the viral polymerase out of the infection is successfully translated independently of an impaired eIF4E factor. These data support that the viral polymerase is responsible for the eIF4E independence of influenza virus mRNAs translation. PMID:22112850

  18. TATA box-binding protein (TBP) is a constituent of the polymerase I-specific transcription initiation factor TIF-IB (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II, and III transcription factors.

    PubMed Central

    Radebaugh, C A; Matthews, J L; Geiss, G K; Liu, F; Wong, J M; Bateman, E; Camier, S; Sentenac, A; Paule, M R

    1994-01-01

    The role of the Acanthamoeba castellanii TATA-binding protein (TBP) in transcription was examined. Specific antibodies against the nonconserved N-terminal domain of TBP were used to verify the presence of TBP in the fundamental transcription initiation factor for RNA polymerase I, TIF-IB, and to demonstrate that TBP is part of the committed initiation complex on the rRNA promoter. The same antibodies inhibit transcription in all three polymerase systems, but they do so differentially. Oligonucleotide competitors were used to evaluate the accessibility of the TATA-binding site in TIF-IB, TFIID, and TFIIIB. The results suggest that insertion of TBP into the polymerase II and III factors is more similar than insertion into the polymerase I factor. Images PMID:8264628

  19. TATA box-binding protein (TBP) is a constituent of the polymerase I-specific transcription initiation factor TIF-IB (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II, and III transcription factors.

    PubMed

    Radebaugh, C A; Matthews, J L; Geiss, G K; Liu, F; Wong, J M; Bateman, E; Camier, S; Sentenac, A; Paule, M R

    1994-01-01

    The role of the Acanthamoeba castellanii TATA-binding protein (TBP) in transcription was examined. Specific antibodies against the nonconserved N-terminal domain of TBP were used to verify the presence of TBP in the fundamental transcription initiation factor for RNA polymerase I, TIF-IB, and to demonstrate that TBP is part of the committed initiation complex on the rRNA promoter. The same antibodies inhibit transcription in all three polymerase systems, but they do so differentially. Oligonucleotide competitors were used to evaluate the accessibility of the TATA-binding site in TIF-IB, TFIID, and TFIIIB. The results suggest that insertion of TBP into the polymerase II and III factors is more similar than insertion into the polymerase I factor.

  20. Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains.

    PubMed

    Wilkes, Rebecca P; Sanchez, Elena; Riley, Matthew C; Kennedy, Melissa A

    2014-01-01

    Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors' laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10(-1) TCID(50)). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.

  1. Taenia saginata: differential diagnosis of human taeniasis by polymerase chain reaction-restriction fragment length polymorphism assay.

    PubMed

    Nunes, Cáris Maroni; Dias, Ana Karina Kerche; Dias, Francisca Elda Ferreira; Aoki, Sérgio Moraes; de Paula, Henrique Borges; Lima, Luis Gustavo Ferraz; Garcia, José Fernando

    2005-08-01

    Speciation of Taenia in human stool is important because of their different clinical and epidemiological features. DNA analysis has recently become possible which overcomes the problems of differentiating human taeniid cestodes morphologically. In the present study, we evaluated PCR coupled to restriction fragment length polymorphism to differentiate Taenia solium from Taenia saginata eggs present in fecal samples from naturally infected patients. A different DraI-RFLP pattern: a two-band pattern (421 and 100 bp) for T. saginata and a three-band pattern (234, 188, and 99 bp) for T. solium was observed allowing the two species to be separated. The lower detection limit of the PCR-RFLP using a non-infected fecal sample prepared with a given number of T. saginata eggs was 34 eggs in 2 g stool sediment. The 521 bp mtDNA fragment was detected in 8 out of 12 Taenia sp. carriers (66.6%). Of these, three showed a T. solium pattern and five a T. saginata pattern.

  2. Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities

    PubMed Central

    Khadka, Prabhat; Hsu, Joseph K.; Veith, Sebastian; Tadokoro, Takashi; Shamanna, Raghavendra A.; Mangerich, Aswin; Croteau, Deborah L.

    2015-01-01

    Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs and PAR is poorly understood. Here we report that all human RecQ helicases interact with PAR noncovalently. Furthermore, we define the effects that PARP1, PARylated PARP1, and PAR have on RECQL5 and WRN, using both in vitro and in vivo assays. We show that PARylation is involved in the recruitment of RECQL5 and WRN to laser-induced DNA damage and that RECQL5 and WRN have differential responses to PARylated PARP1 and PAR. Furthermore, we show that the loss of RECQL5 or WRN resulted in increased sensitivity to PARP inhibition. In conclusion, our results demonstrate that PARP1 and PAR actively, and in some instances differentially, regulate the activities and cellular localization of RECQL5 and WRN, suggesting that PARylation acts as a fine-tuning mechanism to coordinate their functions in time and space during the genotoxic stress response. PMID:26391948

  3. Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

    PubMed Central

    Gomes, Thiago dos Santos; Garcia, Mariana Coimbra; de Souza Cunha, Flavia; Peralta, José Mauro; Peralta, Regina Helena Saramago

    2014-01-01

    Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (Tm) was 73°C and 70°C, respectively. For E. hartmanni, the Tm was 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries. PMID:24693242

  4. [Detection and differentiation of Entamoeba histolytica and Entamoeba dispar by polymerase chain reaction in a community in Zulia State, Venezuela].

    PubMed

    Rivero, Zulbey; Bracho, Angela; Calchi, Marinella; Díaz, Iris; Acurero, Ellen; Maldonado, Adriana; Chourio, Glenis; Arráiz, Nailet; Corzo, Gilbert

    2009-01-01

    Differential identification of Entamoeba histolytica and Entamoeba dispar is essential for both appropriate patient treatment and epidemiological purposes. To determine the prevalence of these amoeba infections in Santa Rosa de Agua (Maracaibo, Zulia State, Venezuela), a PCR assay using specific primers for each species was standardized and applied. 204 stool samples were analyzed through direct microscopic examination with SSF (0.85%) and lugol, formol-ether concentration, and PCR. Under direct microscopy, 42 individuals (20.58%) presented the E. histolytica/E. dispar complex. Meanwhile PCR showed 47 positive cases for these amoebas: 22 E. histolytica (10.78%), 16 E. dispar (7.84%), and 9 (4.41%) mixed infections. There was no significant difference in the presence of E. histolytica and/or E. dispar according to either gender or age. There were no cases of these amoebas in children under 2 years of age. Observed frequency of E. histolytica (31/204) shows the endemic nature of amoeba infection in this community.

  5. Heavy transcription of yeast genes correlates with differential loss of histone H2B relative to H4 and queued RNA polymerases

    PubMed Central

    Cole, Hope A.; Ocampo, Josefina; Iben, James R.; Chereji, Răzvan V.; Clark, David J.

    2014-01-01

    Eukaryotic chromatin is composed of nucleosomes, which contain nearly two coils of DNA wrapped around a central histone octamer. The octamer contains an H3-H4 tetramer and two H2A-H2B dimers. Gene activation is associated with chromatin disruption: a wider nucleosome-depleted region (NDR) at the promoter and reduced nucleosome occupancy over the coding region. Here, we examine the nature of disrupted chromatin after induction, using MNase-seq to map nucleosomes and subnucleosomes, and a refined high-resolution ChIP-seq method to map H4, H2B and RNA polymerase II (Pol II) genome-wide. Over coding regions, induced genes show a differential loss of H2B relative to H4, which correlates with Pol II density and the appearance of subnucleosomes. After induction, Pol II is surprisingly low at the promoter, but accumulates on the gene and downstream of the termination site, implying that dissociation is very slow. Thus, induction-dependent chromatin disruption reflects both eviction of H2A-H2B dimers and the presence of queued Pol II elongation complexes. We propose that slow Pol II dissociation after transcription is a major factor in chromatin disruption and that it may be of critical importance in gene regulation. PMID:25348398

  6. A simple and rapid nested polymerase chain reaction-restriction fragment length polymorphism technique for differentiation of pathogenic and nonpathogenic Leptospira spp.

    PubMed

    Djadid, Navid Dinparast; Ganji, Zahra Faghanzadeh; Gouya, Mohammad Mehdi; Rezvani, Mahmood; Zakeri, Sedigheh

    2009-03-01

    A rapid and specific nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to detect and differentiate pathogenic and nonpathogenic Leptospira spp. Leptospiral genomic DNA was extracted from suspected human sera using an improved method of standard phenol-chloroform, and specific primers have been used to amplify 16S ribosomal RNA from all pathogenic and nonpathogenic Leptospira spp. The PCR products of all nonpathogenic species were digested with ApoI enzyme, but not pathogenic. To evaluate this assay, we analyzed 283 serum samples collected from suspected patients with leptospirosis. Nested PCR assay confirmed that 42 (14.8%) of 283 samples harbored Leptospira infection, and RFLP assay confirmed 38 (90.5%) of 42 and 4 (9.5%) of 42 positive cases had pathogenic and nonpathogenic Leptospira spp., respectively. Based on sequencing results, Leptospira interrogans, Leptospira kirschneri, and Leptospira wolffii and nonpathogenic Leptospira biflexa and Leptospira genomospecies 3 have been detected among analyzed samples. The nested PCR-RFLP assay developed in this study fulfills this requirement in the early stage of infection.

  7. Detection of monoclonality in intestinal lymphoma with polymerase chain reaction for antigen receptor gene rearrangement analysis to differentiate from enteritis in dogs.

    PubMed

    Ohmura, S; Leipig, M; Schöpper, I; Hergt, F; Weber, K; Rütgen, B C; Tsujimoto, H; Hermanns, W; Hirschberger, J

    2017-03-01

    The diagnosis of canine intestinal lymphoma by morphological examination is challenging, especially when endoscopic tissue specimens are used. The utility of detection of antigen receptor gene rearrangement by polymerase chain reaction (PARR) in canine lymphoma has been well established, but its usefulness to distinguish enteritis and intestinal lymphoma remains unclear. In this retrospective study we assessed clonality of 29 primary canine intestinal lymphoma, 14 enteritis and 15 healthy control cases by PARR analysis, using formalin-fixed, paraffin-embedded full-thickness tissue specimens. We could detect monoclonal rearrangements in 22 of 29 canine intestinal lymphomas [76%; 95% confidence interval (CI) 56-90%] and polyclonal rearrangements in all of the enteritis and healthy control cases (100%; CI 88-100%). We revealed a predominance of T-cell phenotype compared to B-cell phenotype (85%; CI 65-96% and 15%; CI 4-35%, respectively). We showed that PARR analysis contributes to differentiation of canine intestinal lymphoma from enteritis and to phenotyping of lymphomas.

  8. Differential Mutation Patterns in Thymidine Kinase and DNA Polymerase Genes of Herpes Simplex Virus Type 1 Clones Passaged in the Presence of Acyclovir or Penciclovir

    PubMed Central

    Suzutani, Tatsuo; Ishioka, Ken; De Clercq, Erik; Ishibashi, Kei; Kaneko, Hisatoshi; Kira, Toshihiko; Hashimoto, Koh-ichi; Ogasawara, Masahiro; Ohtani, Katsuki; Wakamiya, Nobutaka; Saijo, Masayuki

    2003-01-01

    A total of 21 clones of acyclovir (ACV)-resistant (ACVr) herpes simplex virus type 1 (HSV-1) and 23 clones of penciclovir (PCV)-resistant (PCVr) HSV-1, emerging during serial passages in the presence of ACV or PCV, were isolated under conditions excluding contamination of resistant mutants in the starting virus culture, and their mutations in the thymidine kinase (TK) and DNA polymerase (DNA Pol) genes were analyzed comparatively. Mutations in the TK genes from ACVr mutants consisted of 50% single nucleotide substitutions and 50% frameshift mutations, while the corresponding figures for the PCVr mutants were 4 and 96%, respectively (P < 0.001). Eight of the 21 ACVr clones, but none of the 23 PCVr clones, had mutations in DNA Pol. Only nucleotide substitution(s) could be detected in the DNA Pol gene, as the gene is essential for virus replication. Therefore, the results for the DNA Pol mutants are concordant with those for the TK mutants in that a single nucleotide substitution was commonly observed in the ACVr, but not in the PCVr, mutants. These results clearly point to differential mutation patterns between ACVr and PCVr HSV-1 clones. PMID:12709344

  9. A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus.

    PubMed

    Si, Wei; Zhou, Shun; Wang, Zhao; Cui, Shang-jin

    2010-05-01

    A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV). A pair of primers (P1 and P4) specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV), canine parvovirus (CPV), canine coronavirus (CCV), rabies virus (RV), or canine adenovirus (CAV). The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance.

  10. A PB1 T296R substitution enhance polymerase activity and confer a virulent phenotype to a 2009 pandemic H1N1 influenza virus in mice.

    PubMed

    Yu, Zhijun; Cheng, Kaihui; Sun, Weiyang; Zhang, Xinghai; Li, Yuanguo; Wang, Tiecheng; Wang, Hualei; Zhang, Qianyi; Xin, Yue; Xue, Li; Zhang, Kun; Huang, Jing; Yang, Songtao; Qin, Chuan; Wilker, Peter R; Yue, Donghui; Chen, Hualan; Gao, Yuwei; Xia, Xianzhu

    2015-12-01

    While the 2009 pandemic H1N1 virus has become established in the human population as a seasonal influenza virus, continued adaptation may alter viral virulence. Here, we passaged a 2009 pandemic H1N1 virus (A/Changchun/01/2009) in mice. Serial passage in mice generated viral variants with increased virulence. Adapted variants displayed enhanced replication kinetics in vitro and vivo. Analysis of the variants genomes revealed 6 amino acid changes in the PB1 (T296R), PA (I94V), HA (H3 numbering; N159D, D225G, and R226Q), and NP (D375N). Using reverse genetics, we found that a PB1-T296R substitution found in all adapted viral variants enhanced viral replication kinetics in vitro and vivo, increased viral polymerase activity in human cells, and was sufficient for enhanced virulence of the 2009 pandemic H1N1 virus in mice. Therefore, we defined a novel influenza pathogenic determinant, providing further insights into the pathogenesis of influenza viruses in mammals.

  11. Antisense myb inhibition of purified erythroid progenitors in development and differentiation is linked to cycling activity and expression of DNA polymerase alpha

    SciTech Connect

    Valtieri, M.; Venturelli, D.; Care, A.; Fossati, C.; Pelosi, E.; Labbaye, C.; Mattia, G.; Gewirtz, A.M.; Calabretta, B.; Peschle, C. )

    1991-03-15

    These studies aimed to determine the expression and functional role of c-myb in erythroid progenitors with different cycling activities. In the first series of experiments the erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU-E) populations from adult peripheral blood (PB), bone marrow (BM), and embryonic-fetal liver (FL) were treated with either c-myb antisense oligomers or 3H-thymidine (3H-TdR). A direct correlation was always observed between the inhibitory effect of anti-myb oligomers and the level of cycling activity. Thus, the inhibitory effect of antisense c-myb on the number of BFU-E colonies was 28.3% +/- 15.8% in PB, 53.4% +/- 9.3% in BM, and 68.2% +/- 24.5% in FL. Both adult and embryonic CFU-E were markedly inhibited. Using purified PB progenitors, we observed a similar pattern, although with slightly lower inhibitory effects. In the 3H-TdR suicide assay the killing index of BFU-E was 8.9% +/- 4.2% in PB, 29.4% +/- 6.5% in BM, and 40.1% +/- 9.6% in FL. The values for adult and embryonic CFU-E were 55.7% +/- 7.9% and 60.98% +/- 6.6%, respectively. We then investigated the kinetics of c-myb mRNA level during the erythroid differentiation of purified adult PB and FL BFU-E, as evaluated in liquid-phase culture by reverse transcription-polymerase chain reaction. Adult erythroid precursors showed a gradual increase of c-myb mRNA from day 4 through day 8 of culture and a sharp decrease at later times, whereas the expression of c-myb mRNA and protein in differentiation embryonic precursors peaked 2 days earlier. In both cases, c-myb mRNA level peaked at the CFU-E stage of differentiation. Finally, highly purified adult PB BFU-E were stimulated into cycling by a 3-day treatment with interleukin-3 in liquid phase: both the sensitivity to c-myb antisense oligomers and the 3H-TdR suicide index showed a gradual, strictly parallel increase.

  12. Development of a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction assay for the differential diagnosis of Feline leukemia virus vaccine and wild strains.

    PubMed

    Ho, Chia-Fang; Chan, Kun-Wei; Yang, Wei-Cheng; Chiang, Yu-Chung; Chung, Yang-Tsung; Kuo, James; Wang, Chi-Young

    2014-07-01

    A multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) was developed for the differential diagnosis of Feline leukemia virus (FeLV) vaccine and wild-type strains based on a point mutation between the vaccine strain (S) and the wild-type strain (T) located in the p27 gene. This system was further upgraded to obtain a real-time ARMS RT-PCR (ARMS qRT-PCR) with a high-resolution melt analysis (HRMA) platform. The genotyping of various strains of FeLV was determined by comparing the HRMA curves with the defined wild-type FeLV (strain TW1), and the results were expressed as a percentage confidence. The detection limits of ARMS RT-PCR and ARMS qRT-PCR combined with HRMA were 100 and 1 copies of transcribed FeLV RNA per 0.5 ml of sample, respectively. No false-positive results were obtained with 6 unrelated pathogens and 1 feline cell line. Twelve FeLV Taiwan strains were correctly identified using ARMS qRT-PCR combined with HRMA. The genotypes of the strains matched the defined FeLV wild-type strain genotype with at least 91.17% confidence. A higher degree of sequence polymorphism was found throughout the p27 gene compared with the long terminal repeat region. In conclusion, the current study describes the phylogenetic relationship of the FeLV Taiwan strains and demonstrates that the developed ARMS RT-PCR assay is able to be used to detect the replication of a vaccine strain that has not been properly inactivated, thus acting as a safety check for the quality of FeLV vaccines.

  13. Differential transactivation by orphan nuclear receptor NOR1 and its fusion gene product EWS/NOR1: possible involvement of poly(ADP-ribose) polymerase I, PARP-1.

    PubMed

    Ohkura, Naganari; Nagamura, Yuko; Tsukada, Toshihiko

    2008-10-15

    In extraskeletal myxoid chondrosarcoma, a chromosomal translocation creates a gene fusion between EWS and an orphan nuclear receptor, NOR1. The resulting fusion protein EWS/NOR1 has been believed to lead to malignant transformation by functioning as a transactivator for NOR1-target genes. By comparing the gene expression profiles of NOR1- and EWS/NOR1-overexpressing cells, we found that they largely shared up-regulated genes, but no significant correlation was observed with respect to the transactivation levels of each gene. In addition, the proteins associated with NOR1 and EWS/NOR1 were mostly the same in these cells. The results suggest that these proteins differentially transactivate overlapping target genes through a similar transcriptional machinery. To clarify the mechanisms underlying the transcriptional divergence between NOR1 and EWS/NOR1, we searched for alternatively associated proteins, and identified poly(ADP-ribose) polymerase I (PARP-1) as an NOR1-specific binding protein. Consistent with its binding properties, PARP-1 acted as a transcriptional repressor of NOR1, but not EWS/NOR1, in a luciferase reporter assay employing PARP-1(-/-) fibroblasts. Interestingly, suppressive activity of PARP-1 was observed in a DNA response element-specific manner, and in a subtype-specific manner toward the NR4A family (Nur77, Nurr1, and NOR1), suggesting that PARP-1 plays a role in the diversity of transcriptional regulation mediated by the NR4A family in normal cells. Altogether, our findings suggest that NOR1 and EWS/NOR1 regulate overlapping target genes differently by utilizing associated proteins, including PARP-1; and that EWS/NOR1 may acquire oncogenic activities by avoiding (or gaining) transcription factor-specific modulation by the associated proteins.

  14. Rapid Differentiation and Identification of Potential Severe Strains of Citrus tristeza Virus by Real-Time Reverse Transcription Polymerase Chain Reaction Assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex Taqman®-based real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to detect all strains of Citrus tristeza virus (CTV) and to identify potentially severe strains of the virus. A CTV TaqMan probe (CTV-CY5) based on the coat protein (CP) gene sequences...

  15. T7-RNA Polymerase

    NASA Technical Reports Server (NTRS)

    1997-01-01

    T7-RNA Polymerase grown on STS-81. Structure-Function Relationships of RNA Polymerase: DNA-dependent RNA polymerase is the key enzyme responsible for the biosynthesis of RNA, a process known as transcription. Principal Investigator's include Dr. Dan Carter, Dr. B.C. Wang, and Dr. John Rose of New Century Pharmaceuticals.

  16. Difference in root K+ retention ability and reduced sensitivity of K+-permeable channels to reactive oxygen species confer differential salt tolerance in three Brassica species

    PubMed Central

    Chakraborty, Koushik; Bose, Jayakumar; Shabala, Lana; Shabala, Sergey

    2016-01-01

    Brassica species are known to possess significant inter and intraspecies variability in salinity stress tolerance, but the cell-specific mechanisms conferring this difference remain elusive. In this work, the role and relative contribution of several key plasma membrane transporters to salinity stress tolerance were evaluated in three Brassica species (B. napus, B. juncea, and B. oleracea) using a range of electrophysiological assays. Initial root growth assay and viability staining revealed that B. napus was most tolerant amongst the three species, followed by B. juncea and B. oleracea. At the mechanistic level, this difference was conferred by at least three complementary physiological mechanisms: (i) higher Na+ extrusion ability from roots resulting from increased expression and activity of plasma membrane SOS1-like Na+/H+ exchangers; (ii) better root K+ retention ability resulting from stress-inducible activation of H+-ATPase and ability to maintain more negative membrane potential under saline conditions; and (iii) reduced sensitivity of B. napus root K+-permeable channels to reactive oxygen species (ROS). The last two mechanisms played the dominant role and conferred most of the differential salt sensitivity between species. Brassica napus plants were also more efficient in preventing the stress-induced increase in GORK transcript levels and up-regulation of expression of AKT1, HAK5, and HKT1 transporter genes. Taken together, our data provide the mechanistic explanation for differential salt stress sensitivity amongst these species and shed light on transcriptional and post-translational regulation of key ion transport systems involved in the maintenance of the root plasma membrane potential and cytosolic K/Na ratio as a key attribute for salt tolerance in Brassica species. PMID:27340231

  17. Polymerase chain reaction-restriction fragment length polymorphism of the rpoB gene for identification of Mycobacterium avium subsp. paratuberculosis and differentiation of Mycobacterium avium subspecies.

    PubMed

    Whang, Jake; Lee, Byung Soo; Choi, Go-Eun; Cho, Sang-Nae; Kil, Park Young; Collins, Michael T; Shin, Sung Jae

    2011-05-01

    Mycobacterial speciation by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) of the rpoB gene was evaluated for identification of Mycobacterium avium subsp. paratuberculosis (MAP) and other Mycobacterium avium complex (MAC) members to the species or subspecies level by comparison with conventional methods including hsp65 sequencing, high-performance liquid chromatography, and PCR for accepted species- or subspecies-specific genomic targets. A total of 185 type and clinical mycobacterial strains from humans, animals, and environments were tested. A 360-bp PCR product was subsequently digested with MspI, HaeIII, and SmaI restriction enzymes. The PRA using SmaI restriction showed a unique digestion pattern for MAP distinguishing it from other MAC members and other Mycobacterium spp. Moreover, HaeIII and MspI restriction of the rpoB gene enabled MAC-species and -subspecies discrimination. The rpoB-PRA using SmaI or MspI and HaeIII restriction of the rpoB gene is a simple, convenient, and reliable confirmatory assay for simultaneous identification of MAP and other MAC members.

  18. The rpoZ Gene, Encoding the RNA Polymerase Omega Subunit, Is Required for Antibiotic Production and Morphological Differentiation in Streptomyces kasugaensis

    PubMed Central

    Kojima, Ikuo; Kasuga, Kano; Kobayashi, Masayuki; Fukasawa, Akira; Mizuno, Satoshi; Arisawa, Akira; Akagawa, Hisayoshi

    2002-01-01

    The occurrence of pleiotropic mutants that are defective in both antibiotic production and aerial mycelium formation is peculiar to streptomycetes. Pleiotropic mutant KSB was isolated from wild-type Streptomyces kasugaensis A1R6, which produces kasugamycin, an antifungal aminoglycoside antibiotic. A 9.3-kb DNA fragment was cloned from the chromosomal DNA of strain A1R6 by complementary restoration of kasugamycin production and aerial hypha formation to mutant KSB. Complementation experiments with deletion plasmids and subsequent DNA analysis indicated that orf5, encoding 90 amino acids, was responsible for the restoration. A protein homology search revealed that orf5 was a homolog of rpoZ, the gene that is known to encode RNA polymerase subunit omega (ω), thus leading to the conclusion that orf5 was rpoZ in S. kasugaensis. The pleiotropy of mutant KSB was attributed to a 2-bp frameshift deletion in the rpoZ region of mutant KSB, which probably resulted in a truncated, incomplete ω of 47 amino acids. Furthermore, rpoZ-disrupted mutant R6D4 obtained from strain A1R6 by insertion of Tn5 aphII into the middle of the rpoZ-coding region produced neither kasugamycin nor aerial mycelia, similar to mutant KSB. When rpoZ of S. kasugaensis and Streptomyces coelicolor, whose deduced products differed in the sixth amino acid residue, were introduced into mutant R6D4 via a plasmid, both transformants produced kasugamycin and aerial hyphae without significant differences. This study established that rpoZ is required for kasugamycin production and aerial mycelium formation in S. kasugaensis and responsible for pleiotropy. PMID:12426327

  19. Differentiation of toxigenic Staphylococcus aureus in staphylococcal isolates from prepared and frozen foods by combined arbitrarily primed polymerase chain reaction and DNA probe.

    PubMed

    Córdoba, Maria G; Jordano, Rafael; Aranda, Emilio; Benito, Maria J; Córdoba, Juan J

    2003-06-01

    In prepared and frozen flamenquín and hake fish fingers Staphylococcus aureus as sanitary hazards have been detected. In the present work, a combined method that includes an arbitrarily primed PCR (AP-PCR) and a mixed DNA probe hybridisation designed for the enterotoxigenic genes sea, seb, sec, and sed will be assayed to differentiate enterotoxigenic S. aureus from other staphylococcal species isolated during the processing of prepared and frozen foods. From the protocols tested for the AP-PCR, the highest number of amplification bands showing the best resolution was achieved at 30 degrees C annealing and 35 degrees C extension temperatures. Several staphylococci identified by a biochemical test as S. aureus showed in the AP-PCR analysis different banding patterns to the references S. aureus. The isolates, were investigated by slot blot hybridisation for genes encoding A, B, C, and D staphylococcal enterotoxins to determine their enterotoxigenic potential. Several isolates characterised by the AP-PCR analysis as S. aureus hybridised with the DNA probe mixture. The combined AP-PCR and DNA probe hybridisation assayed was able to differentiate toxigenic S. aureus from other staphylococcal species from prepared and frozen foods. This method could be considered as microbial quality assurance in these products.

  20. Differentiation of canine distemper virus isolates in fur animals from various vaccine strains by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism according to phylogenetic relations in china.

    PubMed

    Wang, Fengxue; Yan, Xijun; Chai, Xiuli; Zhang, Hailing; Zhao, Jianjun; Wen, Yongjun; Wu, Wei

    2011-02-27

    In order to effectively identify the vaccine and field strains of Canine distemper virus (CDV), a new differential diagnostic test has been developed based on reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). We selected an 829 bp fragment of the nucleoprotein (N) gene of CDV. By RFLP analysis using BamHI, field isolates were distinguishable from the vaccine strains. Two fragments were obtained from the vaccine strains by RT-PCR-RFLP analysis while three were observed in the field strains. An 829 nucleotide region of the CDV N gene was analyzed in 19 CDV field strains isolated from minks, raccoon dogs and foxes in China between 2005 and 2007. The results suggest this method is precise, accurate and efficient. It was also determined that three different genotypes exist in CDV field strains in fur animal herds of the north of China, most of which belong to Asian type. Mutated field strains, JSY06-R1, JSY06-R2 and JDH07-F1 also exist in Northern China, but are most closely related to the standard virulent strain A75/17, designated in Arctic and America-2 genetype in the present study, respectively.

  1. Differential role for competitive reverse transcriptase-polymerase chain reaction and intracellular cytokine staining as diagnostic tools for the assessment of intragraft cytokine profiles in rejecting and nonrejecting heart allografts.

    PubMed

    Spriewald, B M; Hara, M; Bushell, A; Jenkins, S; Morris, P J; Wood, K J

    2000-11-01

    The early and reliable diagnosis of allograft rejection is a difficult task and the assessment of cytokine expression in the grafts can be a helpful parameter. We have compared competitive reverse transcriptase-polymerase chain reaction (RT-PCR) with intracellular cytokine staining by flow cytometry as tools to measure cytokine expression in rejecting and nonrejecting murine cardiac allografts. Both techniques gave comparable results for cytokine expression in rejecting allografts and syngeneic controls. Grafts from mice pretreated with anti-CD4 antibody and donor-specific blood transfusion showed a marked reduction in cytokine expression, as assessed by competitive RT-PCR, even though a cellular infiltrate was present in the graft. In contrast, the cytokine production measured by intracellular cytokine staining of the isolated graft-infiltrating cells was high and exceeded even that of the rejecting allografts. We conclude that intracellular cytokine staining of graft-infiltrating leukocytes by flow cytometry does not necessarily reflect accurately the cytokine milieu in the graft. This technique might therefore have a limited clinical application in contrast to competitive RT-PCR for the differentiation between graft acceptance and graft rejection.

  2. The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

    PubMed Central

    Barbedo, Leonardo Silva; Figueiredo-Carvalho, Maria Helena Galdino; Muniz, Mauro de Medeiros; Zancopé-Oliveira, Rosely Maria

    2016-01-01

    Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories. PMID:27074256

  3. Development and use of a multiplex real-time quantitative polymerase chain reaction assay for detection and differentiation of Porcine circovirus-2 genotypes 2a and 2b in an epidemiological survey.

    PubMed

    Gagnon, Carl A; del Castillo, Jérome R E; Music, Nedzad; Fontaine, Guy; Harel, Josée; Tremblay, Donald

    2008-09-01

    By the end of 2004, the Canadian swine population had experienced a severe increase in the incidence of Porcine circovirus-associated disease (PCVAD), a problem that was associated with the emergence of a new Porcine circovirus-2 genotype (PCV-2b), previously unrecovered in North America. Thus, it became important to develop a diagnostic tool that could differentiate between the old and new circulating genotypes (PCV-2a and PCV-2b, respectively). Consequently, a multiplex real-time quantitative polymerase chain reaction (mrtqPCR) assay that could sensitively and specifically identify and differentiate PCV-2 genotypes was developed. A retrospective epidemiologic survey that used the mrtqPCR assay was performed to determine if cofactors could affect the risk of PCVAD. From 121 PCV-2-positive cases gathered for this study, 4.13%, 92.56%, and 3.31% were positive for PCV-2a, PCV-2b, and both genotypes, respectively. In a data analysis using univariate logistic regressions, the PCVAD-compatible (PCVAD/c) score was significantly associated with the presence of Porcine reproductive and respiratory syndrome virus (PRRSV), PRRSV viral load, PCV-2 viral load, and PCV-2 immunohistochemistry (IHC) results. Polytomous logistic regression analysis revealed that PCVAD/c score was affected by PCV-2 viral load (P = 0.0161) and IHC (P = 0.0128), but not by the PRRSV variables (P > 0.9), which suggests that mrtqPCR in tissue is a reliable alternative to IHC. Logistic regression analyses revealed that PCV-2 increased the odds ratio of isolating 2 major swine pathogens of the respiratory tract, Actinobacillus pleuropneumoniae and Streptococcus suis serotypes 1/2, 1, 2, 3, 4, and 7, which are serotypes commonly associated with clinical diseases.

  4. Does the oxytocin receptor (OXTR) polymorphism (rs2254298) confer 'vulnerability' for psychopathology or 'differential susceptibility'? Insights from evolution.

    PubMed

    Brüne, Martin

    2012-04-17

    The diathesis-stress model of psychiatric conditions has recently been challenged by the view that it might be more accurate to speak of 'differential susceptibility' or 'plasticity' genes, rather than one-sidedly focusing on individual vulnerability. That is, the same allelic variation that predisposes to a psychiatric disorder if associated with (developmentally early) environmental adversity may lead to a better-than-average functional outcome in the same domain under thriving (or favourable) environmental conditions. Studies of polymorphic variations of the serotonin transporter gene, the monoamino-oxidase-inhibitor A coding gene or the dopamine D4 receptor gene indicate that the early environment plays a crucial role in the development of favourable versus unfavourable outcomes. Current evidence is limited, however, to establishing a link between genetic variation and behavioural phenotypes. In contrast, little is known about how plasticity may be expressed at the neuroanatomical level as a 'hard-wired' correlate of observable behaviour. The present review article seeks to further strengthen the argument in favour of the differential susceptibility theory by incorporating findings from behavioural and neuroanatomical studies in relation to genetic variation of the oxytocin receptor gene. It is suggested that polymorphic variation at the oxytocin receptor gene (rs2254298) is associated with sociability, amygdala volume and differential risk for psychiatric conditions including autism, depression and anxiety disorder, depending on the quality of early environmental experiences. Seeing genetic variation at the core of developmental plasticity can explain, in contrast to the diathesis-stress perspective, why evolution by natural selection has maintained such 'risk' alleles in the gene pool of a population.

  5. The expanding polymerase universe.

    PubMed

    Goodman, M F; Tippin, B

    2000-11-01

    Over the past year, the number of known prokaryotic and eukaryotic DNA polymerases has exploded. Many of these newly discovered enzymes copy aberrant bases in the DNA template over which 'respectable' polymerases fear to tread. The next step is to unravel their functions, which are thought to range from error-prone copying of DNA lesions, somatic hypermutation and avoidance of skin cancer, to restarting stalled replication forks and repairing double-stranded DNA breaks.

  6. Identification of differentially expressed genes that potentially confer pest resistance in transgenic ChIFN-γ tobacco.

    PubMed

    Wu, Yong-Jun; Wu, Yu-Jun; Luo, Xi; Shen, Xi-Long; Zhao, De-Gang

    2014-06-15

    Chicken interferon-γ (ChIFN-γ) is both an inhibitor of viral replication and a regulator of numerous immunological functions. However, since little is known about the mechanisms underlying the insect-resistance of transgenic ChIFN-γ, a transgenic ChIFN-γ tobacco line was employed in the present study to explore this mechanism. A cDNA microarray (with 43,760 unigenes) was used to analyze the gene expression profiles of transgenic and wild-type (WT) tobacco leaves at two different growth stages. Compared with the WT, 1529 and 405 expressed sequence tags were significantly up- or downregulated on days 119 and 147, respectively. The differentially expressed genes (DEGs) are involved in metabolic regulation, cell division and differentiation, material synthesis and transport, signal transduction, and protein synthesis and degradation. Candidate genes that may increase cell density, thicken cell walls, promote secondary metabolite synthesis, and mediate plant hormone-induced resistance responses were used to identify the ChIFN-γ-mediated insect-resistance mechanisms. The insect-resistance of transgenic ChIFN-γ tobacco possibly involves unknown signaling pathways, which may directly or indirectly affect DEG expression-mediating genes. The degree of pest resistance increased as the plants grew. Three genes likely to be related to jasmonic acid- or salicylic acid-dependent plant defense responses, including CAF 1, Cop 8/CSN, and HD, are implicated in the insect-resistance of the transgenic plants. The mechanism of transgenic ChIFN-γ tobacco resistance also involves RPS20 and other genes that induce microRNA-based gene regulation. The ChIFN-γ-mediated DGEs contribute to insect-resistance in transgenic ChIFN-γ tobacco, which provides new insight into the role of ChIFN-γ.

  7. Differentiation of bacterial versus viral otitis media using a combined Raman scattering spectroscopy and low coherence interferometry probe (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Zhao, Youbo; Shelton, Ryan L.; Tu, Haohua; Nolan, Ryan M.; Monroy, Guillermo L.; Chaney, Eric J.; Boppart, Stephen A.

    2016-02-01

    Otitis media (OM) is a highly prevalent disease that can be caused by either a bacterial or viral infection. Because antibiotics are only effective against bacterial infections, blind use of antibiotics without definitive knowledge of the infectious agent, though commonly practiced, can lead to the problems of potential harmful side effects, wasteful misuse of medical resources, and the development of antimicrobial resistance. In this work, we investigate the feasibility of using a combined Raman scattering spectroscopy and low coherence interferometry (LCI) device to differentiate OM infections caused by viruses and bacteria and improve our diagnostic ability of OM. Raman spectroscopy, an established tool for molecular analysis of biological tissue, has been shown capable of identifying different bacterial species, although mostly based on fixed or dried sample cultures. LCI has been demonstrated recently as a promising tool for determining tympanic membrane (TM) thickness and the presence and thickness of middle-ear biofilm located behind the TM. We have developed a fiber-based ear insert that incorporates spatially-aligned Raman and LCI probes for point-of-care diagnosis of OM. As shown in human studies, the Raman probe provides molecular signatures of bacterial- and viral-infected OM and normal middle-ear cavities, and LCI helps to identify depth-resolved structural information as well as guide and monitor positioning of the Raman spectroscopy beam for relatively longer signal acquisition time. Differentiation of OM infections is determined by correlating in vivo Raman data collected from human subjects with the Raman features of different bacterial and viral species obtained from cultured samples.

  8. A differential absorption lidar instrument for the measurment of carbon dioxide and methane in the lower troposphere (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Budinov, Daniel; Clements, Robert; Rae, Cameron F.; Moncrieff, John B.; Jack, James W.

    2016-12-01

    Developments in the remote detection of trace gases in the atmosphere using Differential Absorption Lidar have been driven largely by improvements in two key technologies: lasers and detectors. We have designed and built a narrow linewidth pulsed laser source with a well-controlled output wavelength and sufficient pulse energy to measure the concentration profile of CO2 and CH4 to a range in excess of 4km. We describe here the initial measurements of concentration profiles recorded with this instrument. The system is built around a custom-designed Newtonian telescope with a 40cm diameter primary mirror. Laser sources and detectors attach directly to the side of the telescope allowing for flexible customization with a range of additional equipment. The instrument features an all-solid-state laser source based on an optical parametric oscillator (OPO) pumped by an YLF based diode-laser pumped solid-state laser and seeded by a tuned DFB seed. This provides a range of available wavelengths suitable for DIAL within the 1.5-1.6 μm spectral region. The output of the OPO is beam expanded and transmitted coaxially from the receiver telescope. A gas cell within the laser source controls the seed wavelength and allows the wavelength to be tuned to match a specific absorption feature of the selected gas species. The source can be rapidly tuned between the on-line and off-line wavelengths to make a DIAL measurement of either CO2 or CH4 The receiver is based on an InGaAs avalanche photodetector. Whilst photodiode detectors are a low-cost solution their limited sensitivity restricts the maximum range over which a signal can be detected. The receiver signal is digitised for subsequent processing to produce a sightline concentration profile. The instrument is mounted on a robust gimballed mount providing full directional movement within the upper hemisphere. Both static pointing and angular scan modes are available. Accurate angular position is available giving the sightline

  9. The polymerase chain reaction.

    PubMed

    Welch, Hazel M

    2012-01-01

    The polymerase chain reaction (PCR) has had a significant impact on all aspects of the molecular biosciences, from cancer research to forensic science. The sensitivity and specificity inherent in the technique allow minute quantities of genetic material to be detected while the unique properties of thermostable DNA polymerase ensure that abundant copies are reliably reproduced to levels that can be visualized and/or used for further applications. This chapter describes applications of PCR and PCR-RT to investigate primary cancer and metastatic disease at both the DNA and mRNA expression levels.

  10. Polymerase chain reaction system

    DOEpatents

    Benett, William J.; Richards, James B.; Stratton, Paul L.; Hadley, Dean R.; Milanovich, Fred P.; Belgrader, Phil; Meyer, Peter L.

    2004-03-02

    A portable polymerase chain reaction DNA amplification and detection system includes one or more chamber modules. Each module supports a duplex assay of a biological sample. Each module has two parallel interrogation ports with a linear optical system. The system is capable of being handheld.

  11. Selenotrisulfide inhibits initiation by RNA polymerase II but not elongation

    SciTech Connect

    Frenkel, G.D.; Falvey, D.

    1989-03-01

    We previously reported that RNA polymerase II (purified from wheat germ) is inhibited by selenotrisulfides, the products of the reaction of selenite with sulfhydryl compounds. We have now found that the initiation stage of the reaction is inhibited by selenotrisulfide but the elongation stage of the reaction is not. The actual start of the RNA chain is not inhibited by the selenotrisulfide, but rather the formation of the enzyme-DNA binary complex. Selenotrisulfide has a similar differential effect on initiation and elongation by RNA polymerase II from HeLa cells; in contrast, with E. coli RNA polymerase, it inhibits elongation as well.

  12. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase

    DOE PAGES

    McInerney, Peter; Adams, Paul; Hadi, Masood Z.

    2014-01-01

    As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Errormore » rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu , Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.« less

  13. Conference Resolution

    NASA Astrophysics Data System (ADS)

    2009-04-01

    Since the first IUPAP International Conference on Women in Physics (Paris, March 2002) and the Second Conference (Rio de Janeiro, May 2005), progress has continued in most countries and world regions to attract girls to physics and advance women into leadership roles, and many working groups have formed. The Third Conference (Seoul, October 2008), with 283 attendees from 57 countries, was dedicated to celebrating the physics achievements of women throughout the world, networking toward new international collaborations, building each participant's capacity for career success, and aiding the formation of active regional working groups to advance women in physics. Despite the progress, women remain a small minority of the physics community in most countries.

  14. Exploiting polymerase promiscuity: A simple colorimetric RNA polymerase assay.

    PubMed

    Vassiliou, W; Epp, J B; Wang, B B; Del Vecchio, A M; Widlanski, T; Kao, C C

    2000-09-01

    We developed a convenient colorimetric assay for monitoring RNA synthesis from DNA-dependent RNA polymerases (DdRp) and viral RNA-dependent RNA polymerases (RdRp). ATP and GTP with a p-nitrophenyl moiety attached to the gamma-phosphate were synthesized (PNP-NTPs). These PNP-NTPs can be used for RNA synthesis by several RNA polymerases, including the RdRps from brome mosaic virus and bovine viral diarrhea virus and the DdRps from bacteriophage T7 and SP6. When the polymerase reactions were performed in the presence of alkaline phosphatase, which digests the p-nitrophenylpyrophosphate side-product of phosphoryl transfer to the chromogenic p-nitrophenylate, an increase in absorbence at 405 nm was observed. These nucleotide analogues were used in continuous colorimetric monitoring of polymerase activity. Furthermore, the PNP-NTPs were found to be stable and utilized by RNA polymerases in the presence of human plasma. This simple colorimetric polymerase assay can be performed in a standard laboratory spectrophotometer and will be useful in screens for inhibitors of viral RNA synthesis.

  15. Adaptive strategies of the influenza virus polymerase for replication in humans.

    PubMed

    Mehle, Andrew; Doudna, Jennifer A

    2009-12-15

    Transmission of influenza viruses into the human population requires surmounting barriers to cross-species infection. Changes in the influenza polymerase overcome one such barrier. Viruses isolated from birds generally contain polymerases with the avian-signature glutamic acid at amino acid 627 in the PB2 subunit. These polymerases display restricted activity in human cells. An adaptive change in this residue from glutamic acid to the human-signature lysine confers high levels of polymerase activity in human cells. This mutation permits escape from a species-specific restriction factor that targets polymerases from avian viruses. A 2009 swine-origin H1N1 influenza A virus recently established a pandemic infection in humans, even though the virus encodes a PB2 with the restrictive glutamic acid at amino acid 627. We show here that the 2009 H1N1 virus has acquired second-site suppressor mutations in its PB2 polymerase subunit that convey enhanced polymerase activity in human cells. Introduction of this polymorphism into the PB2 subunit of a primary avian isolate also increased polymerase activity and viral replication in human and porcine cells. An alternate adaptive strategy has also been identified, whereby introduction of a human PA subunit into an avian polymerase overcomes restriction in human cells. These data reveal a strategy used by the 2009 H1N1 influenza A virus and identify other pathways by which avian and swine-origin viruses may evolve to enhance replication, and potentially pathogenesis, in humans.

  16. Conference Space

    ERIC Educational Resources Information Center

    Tillett, Wade

    2016-01-01

    The following is an exploration of the spatial configurations (and their implications) within a typical panel session at an academic conference. The presenter initially takes up different roles and hyperbolically describes some possible messages that the spatial arrangement sends. Eventually, the presenter engages the audience members in atypical…

  17. Development and Validation of TaqMan Real-Time Polymerase Chain Reaction Assays for the Quantitative and Differential Detection of Wild-Type Infectious Laryngotracheitis Viruses from a Glycoprotein G-Deficient Candidate Vaccine Strain.

    PubMed

    Shil, Niraj K; Legione, Alistair R; Markham, Philip F; Noormohammadi, Amir H; Devlin, Joanne M

    2015-03-01

    Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens with a worldwide distribution. Differentiating between wild-type and vaccine strains of ILT virus (ILTV) would be useful for enhancing disease control, and in the early stages of a disease outbreak molecular diagnostic tools for the detection and differentiation of the circulating virus could be applied. This study developed TaqMan real-time PCR (qPCR) assays to detect and differentiate the glycoprotein G (gG)-deficient (ΔgG) ILTV candidate vaccine strain of ILTV from ILTV strains that contain the gG gene. The gG+ve and gG-ve ILTV TaqMan assays were used in individual and multiplex format to detect, differentiate, and quantitate ILTV DNA in laboratory and clinical samples. The assays were highly sensitive and highly specific, with a detection limit of 10 viral template copies for each assay. Low interassay coefficients of variation were recorded (0.021-0.042 and 0.013-0.039) for gG+ve and gG-ve TaqMan assays, respectively. The multiplex assay was successfully used to examine the replication kinetics of wild-type and ΔgG strains of ILTV in cultured leghorn male hepatoma cells and embryonated hen eggs under coinfection conditions. The results showed that the TaqMan qPCR assay, along with the ΔgG ILTV vaccine, has the potential to be used in a "Differentiating Infected from Vaccinated Animals" strategy for the control and eradication of ILT.

  18. Genetic structure of soil population of fungus Fusarium oxysporum Schlechtend.: Fr.: Molecular reidentification of the species and genetic differentiation of isolates using polymerase chain reaction technique with universal primers (UP-PCR)

    SciTech Connect

    Bulat, S.A.; Mironenko, N.V.; Zholkevich, Yu.G.

    1995-07-01

    The genetic structure of three soil populations of fungus Fusarium oxysporum was analyzed using polymerase chain reaction with universal primers (UP-PCR). Distinct UP-PCR variants revealed by means of cross-dot hybridization of amplified DNA and restriction analysis of nuclear ribosomal DNA represent subspecies or sibling species of F. oxysporum. The remaining isolates of F. oxysporum showed moderate UP-PCR polymorphism characterized by numerous types, whose relatedness was analyzed by computer treatment of the UP-PCR patterns. The genetic distance trees based on the UP-PCR patterns, which were obtained with different universal primers, demonstrated similar topology. This suggests that evolutionarily important genome rearrangements correlatively occur within the entire genome. Isolates representing different UP-PCR polymorphisms were encountered in all populations, being distributed asymmetrically in two of these. In general, soil populations of F. oxysporum were represented by numerous genetically isolated groups with a similar genome structure. The genetic heterogeneity of the isolates within these groups is likely to be caused by the parasexual process. The usefulness of the UP-PCR technique for population studies of F. oxysporum was demonstrated. 39 refs., 7 figs., 2 tabs.

  19. Thirteenth International Laser Radar Conference

    NASA Technical Reports Server (NTRS)

    1986-01-01

    One hundred fifteen papers were presented in both oral and poster sessions. The topics of the conference sessions were: spaceborne lidar applications; extinction/visibility; differential absorption lidar; winds and tropospheric studies; middle atmosphere; clouds and multiple scattering; pollution studies; and new systems.

  20. Altering the interaction between σ70 and RNA polymerase generates complexes with distinct transcription-elongation properties

    PubMed Central

    Berghöfer-Hochheimer, Yvonne; Lu, Chi Zen; Gross, Carol A.

    2005-01-01

    We compare the elongation behavior of native Escherichia coli RNA polymerase holoenzyme assembled in vivo, holoenzyme reconstituted from σ70 and RNA polymerase in vitro, and holoenzyme with a specific alteration in the interface between σ70 and RNA polymerase. Elongating RNA polymerase from each holoenzyme has distinguishable properties, some of which cannot be explained by differential retention or rebinding of σ70 during elongation, or by differential presence of elongation factors. We suggest that interactions between RNA polymerase and σ70 may influence the ensemble of conformational states adopted by RNA polymerase during initiation. These states, in turn, may affect the conformational states adopted by the elongating enzyme, thereby physically and functionally imprinting RNA polymerase. PMID:15650048

  1. Human placental eXpanded (PLX) mesenchymal-like adherent stromal cells confer neuroprotection to nerve growth factor (NGF)-differentiated PC12 cells exposed to ischemia by secretion of IL-6 and VEGF.

    PubMed

    Lahiani, Adi; Zahavi, Efrat; Netzer, Nir; Ofir, Racheli; Pinzur, Lena; Raveh, Shani; Arien-Zakay, Hadar; Yavin, Ephraim; Lazarovici, Philip

    2015-02-01

    Mesenchymal stem cells are potent candidates in stroke therapy due to their ability to secrete protective anti-inflammatory cytokines and growth factors. We investigated the neuroprotective effects of human placental mesenchymal-like adherent stromal cells (PLX) using an established ischemic model of nerve growth factor (NGF)-differentiated pheochromocytoma PC12 cells exposed to oxygen and glucose deprivation (OGD) followed by reperfusion. Under optimal conditions, 2 × 10⁵ PLX cells, added in a trans-well system, conferred 30-60% neuroprotection to PC12 cells subjected to ischemic insult. PC12 cell death, measured by LDH release, was reduced by PLX cells or by conditioned medium derived from PLX cells exposed to ischemia, suggesting the active release of factorial components. Since neuroprotection is a prominent function of the cytokine IL-6 and the angiogenic factor VEGF165, we measured their secretion using selective ELISA of the cells under ischemic or normoxic conditions. IL-6 and VEGF165 secretion by co-culture of PC12 and PLX cells was significantly higher under ischemic compared to normoxic conditions. Exogenous supplementation of 10 ng/ml each of IL-6 and VEGF165 to insulted PC12 cells conferred neuroprotection, reminiscent of the neuroprotective effect of PLX cells or their conditioned medium. Growth factors as well as co-culture conditioned medium effects were reduced by 70% and 20% upon pretreatment with 240 ng/ml Semaxanib (anti VEGF165) and/or 400 ng/ml neutralizing anti IL-6 antibody, respectively. Therefore, PLX-induced neuroprotection in ischemic PC12 cells may be partially explained by IL-6 and VEGF165 secretion. These findings may also account for the therapeutic effects seen in clinical trials after treatment with these cells.

  2. Plant hemoglobins may be maintained in functional form by reduced flavins in the nuclei, and confer differential tolerance to nitro-oxidative stress.

    PubMed

    Sainz, Martha; Pérez-Rontomé, Carmen; Ramos, Javier; Mulet, Jose Miguel; James, Euan K; Bhattacharjee, Ujjal; Petrich, Jacob W; Becana, Manuel

    2013-12-01

    The heme of bacteria, plant and animal hemoglobins (Hbs) must be in the ferrous state to bind O(2) and other physiological ligands. Here we have characterized the full set of non-symbiotic (class 1 and 2) and 'truncated' (class 3) Hbs of Lotus japonicus. Class 1 Hbs are hexacoordinate, but class 2 and 3 Hbs are pentacoordinate. Three of the globins, Glb1-1, Glb2 and Glb3-1, are nodule-enhanced proteins. The O(2) affinity of Glb1-1 (50 pm) was the highest known for any Hb, and the protein may function as an O(2) scavenger. The five globins were reduced by free flavins, which transfer electrons from NAD(P)H to the heme iron under aerobic and anaerobic conditions. Class 1 Hbs were reduced at very fast rates by FAD, class 2 Hbs at slower rates by both FMN and FAD, and class 3 Hbs at intermediate rates by FMN. The members of the three globin classes were immunolocalized predominantly in the nuclei. Flavins were quantified in legume nodules and nuclei, and their concentrations were sufficient to maintain Hbs in their functional state. All Hbs, except Glb1-1, were expressed in a flavohemoglobin-deficient yeast mutant and found to confer tolerance to oxidative stress induced by methyl viologen, copper or low temperature, indicating an anti-oxidative role for the hemes. However, only Glb1-2 and Glb2 afforded protection against nitrosative stress induced by S-nitrosoglutathione. Because this compound is specifically involved in transnitrosylation reactions with thiol groups, our results suggest a contribution of the single cysteine residues of both proteins in the stress response.

  3. Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

    SciTech Connect

    Sternberg, E.A.; Spizz, G.; Perry, W.M.; Vizard, D.; Weil, T.; Olson, E.N.

    1988-07-01

    Terminal differentiation of skeletal myobalsts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzymte of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers.

  4. Cdc73p and Paf1p are found in a novel RNA polymerase II-containing complex distinct from the Srbp-containing holoenzyme.

    PubMed Central

    Shi, X; Chang, M; Wolf, A J; Chang, C H; Frazer-Abel, A A; Wade, P A; Burton, Z F; Jaehning, J A

    1997-01-01

    The products of the yeast CDC73 and PAF1 genes were originally identified as RNA polymerase II-associated proteins. Paf1p is a nuclear protein important for cell growth and transcriptional regulation of a subset of yeast genes. In this study we demonstrate that the product of CDC73 is a nuclear protein that interacts directly with purified RNA polymerase II in vitro. Deletion of CDC73 confers a temperature-sensitive phenotype. Combination of the cdc73 mutation with the more severe paf1 mutation does not result in an enhanced phenotype, indicating that the two proteins may function in the same cellular processes. To determine the relationship between Cdc73p and Paf1p and the recently described holoenzyme form of RNA polymerase II, we created yeast strains containing glutathione S-transferase (GST)-tagged forms of CDC73, PAF1, and TFG2 functionally replacing the chromosomal copies of the genes. Isolation of GST-tagged Cdc73p and Paf1p complexes has revealed a unique form of RNA polymerase II that contains both Cdc73p and Paf1p but lacks the Srbps found in the holoenzyme. The Cdc73p-Paf1p-RNA polymerase II-containing complex also includes Gal11p, and the general initiation factors TFIIB and TFIIF, but lacks TBP, TFIIH, and transcription elongation factor TFIIS as well as the Srbps. The Srbp-containing holoenzyme does not include either Paf1p or Cdc73p, demonstrating that these two forms of RNA polymerase II are distinct. In confirmation of the hypothesis that the two forms coexist in yeast cells, we found that a TFIIF-containing complex isolated via the GST-tagged Tfg2p construct contains both (i) the Srbps and (ii) Cdc73p and Paf1p. The Srbps and Cdc73p-Paf1p therefore appear to define two complexes with partially redundant, essential functions in the yeast cell. Using the technique of differential display, we have identified several genes whose transcripts require Cdc73p and/or Paf1p for normal levels of expression. Our analysis suggests that there are multiple RNA

  5. Conferences revisited

    NASA Astrophysics Data System (ADS)

    Radcliffe, Jonathan

    2008-08-01

    Way back in the mid-1990s, as a young PhD student, I wrote a Lateral Thoughts article about my first experience of an academic conference (Physics World 1994 October p80). It was a peach of a trip - most of the lab decamped to Grenoble for a week of great weather, beautiful scenery and, of course, the physics. A whole new community was there for me to see in action, and the internationality of it all helped us to forget about England's non-appearance in the 1994 World Cup finals.

  6. Fluctuation and fidelity control of a non-proofreading polymerase

    NASA Astrophysics Data System (ADS)

    Yu, Jin

    2014-03-01

    Polymerases catalyze gene replication and transcription. They modulate activation barriers of nucleotide incorporation and amplify maximal free energy differentiation between the right and wrong nucleotides. It is essential for the polymerases to achieve sufficiently high fidelity at sufficiently high speed. We had noticed a small free energy bias in the translocation of T7 RNA polymerase (RNAP) that aids nucleotide selection. We investigated further how polymerases select against wrong nucleotides efficiently with given kinetics for the right, and with controlled differentiation capacities. We found that early selections on the reaction path outperform the late ones in error reduction. In particular, initial screening seems indispensable for lowering error rates without lowering much the speed. To see how exactly the nucleotide selection proceeds, we studied T7 RNAP also in atomistic simulations. We found that substantial nucleotide selection happens early, prior to full insertion of the nucleotide for complete Watson-Crick base pairing. A highly conserved residue brings up the small translocation energy bias by marginally blocking the active site. The residue senses the nucleotide species upon the nucleotide pre-insertion, and selectively `gates' the nucleotide during insertion. Our studies thus provide a kinetic survey of the nucleotide selection system along with underlying molecular mechanisms.

  7. Rescue of embryonic epithelium reveals that the homozygous deletion of the retinoblastoma gene confers growth factor independence and immortality but does not influence epithelial differentiation or tissue morphogenesis.

    PubMed

    Day, Kathleen C; McCabe, Michael T; Zhao, Xin; Wang, Yuzhuo; Davis, Joanne N; Phillips, John; Von Geldern, Marion; Ried, Thomas; KuKuruga, Mark A; Cunha, Gerald R; Hayward, Simon W; Day, Mark L

    2002-11-15

    The ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb-/-) fetal mice has allowed for the isolation and characterization of the first Rb-/- prostate epithelial cell line. This cell line, designated Rb-/-PrE, was utilized for experiments examining the consequences of Rb loss on an epithelial population. These findings demonstrated that Rb deletion has no discernible effect on prostatic histodifferentiation in Rb-/-PrE cultures. When Rb-/-PrE cells were recombined with embryonic rat urogenital mesenchyme and implanted into athymic male, nude mouse hosts, the recombinants developed into fully differentiated and morphologically normal prostate tissue. The Rb-/-PrE phenotype was characterized by serum independence in culture and immortality in vivo, when compared with wild type controls. Cell cycle analysis revealed elevated S phase DNA content accompanied by increased expression of cyclin E1 and proliferating cell nuclear antigen. Rb-/-PrE cultures also exhibited a diminished ability to growth arrest under high density culture conditions. We believe that the development of Rb-/- prostate tissue and cell lines has provided a unique experimental platform with which to investigate the consequences of Rb deletion in epithelial cells under various physiological conditions. Additionally, the development of this technology will allow similar studies in other tissues and cell populations rescued from Rb-/- fetuses.

  8. Alphavirus polymerase and RNA replication.

    PubMed

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

    2017-01-16

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

  9. Molecular Typing and Differentiation

    EPA Science Inventory

    In this chapter, general background and bench protocols are provided for a number of molecular typing techniques in common use today. Methods for the molecular typing and differentiation of microorganisms began to be widely adopted following the development of the polymerase chai...

  10. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases.

    PubMed

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

    2008-10-04

    Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases.

  11. Mutagenic and Recombinagenic Responses to Defective DNA Polymerase δ Are Facilitated by the Rev1 Protein in pol3-t Mutants of Saccharomyces cerevisiae

    PubMed Central

    Mito, Erica; Mokhnatkin, Janet V.; Steele, Molly C.; Buettner, Victoria L.; Sommer, Steve S.; Manthey, Glenn M.; Bailis, Adam M.

    2008-01-01

    Defective DNA replication can result in substantial increases in the level of genome instability. In the yeast Saccharomyces cerevisiae, the pol3-t allele confers a defect in the catalytic subunit of replicative DNA polymerase δ that results in increased rates of mutagenesis, recombination, and chromosome loss, perhaps by increasing the rate of replicative polymerase failure. The translesion polymerases Pol η, Pol ζ, and Rev1 are part of a suite of factors in yeast that can act at sites of replicative polymerase failure. While mutants defective in the translesion polymerases alone displayed few defects, loss of Rev1 was found to suppress the increased rates of spontaneous mutation, recombination, and chromosome loss observed in pol3-t mutants. These results suggest that Rev1 may be involved in facilitating mutagenic and recombinagenic responses to the failure of Pol δ. Genome stability, therefore, may reflect a dynamic relationship between primary and auxiliary DNA polymerases. PMID:18711219

  12. Tagetitoxin Inhibits RNA Polymerase through Trapping of the Trigger Loop*

    PubMed Central

    Artsimovitch, Irina; Svetlov, Vladimir; Nemetski, Sondra Maureen; Epshtein, Vitaly; Cardozo, Timothy; Nudler, Evgeny

    2011-01-01

    Tagetitoxin (Tgt) inhibits multisubunit chloroplast, bacterial, and some eukaryotic RNA polymerases (RNAPs). A crystallographic structure of Tgt bound to bacterial RNAP apoenzyme shows that Tgt binds near the active site but does not explain why Tgt acts only at certain sites. To understand the Tgt mechanism, we constructed a structural model of Tgt bound to the transcription elongation complex. In this model, Tgt interacts with the β′ subunit trigger loop (TL), stabilizing it in an inactive conformation. We show that (i) substitutions of the Arg residue of TL contacted by Tgt confer resistance to inhibitor; (ii) Tgt inhibits RNAP translocation, which requires TL movements; and (iii) paused complexes and a “slow” enzyme, in which the TL likely folds into an altered conformation, are resistant to Tgt. Our studies highlight the role of TL as a target through which accessory proteins and antibiotics can alter the elongation complex dynamics. PMID:21976682

  13. Conference Abstracts: AEDS '82.

    ERIC Educational Resources Information Center

    Journal of Computers in Mathematics and Science Teaching, 1982

    1982-01-01

    Abstracts from nine selected papers presented at the 1982 Association for Educational Data Systems (AEDS) conference are provided. Copies of conference proceedings may be obtained for fifteen dollars from the Association. (MP)

  14. The General Conference Mennonites.

    ERIC Educational Resources Information Center

    Ediger, Marlow

    General Conference Mennonites and Old Order Amish are compared and contrasted in the areas of physical appearance, religious beliefs, formal education, methods of farming, and home settings. General Conference Mennonites and Amish differ in physical appearance and especially in dress. The General Conference Mennonite men and women dress the same…

  15. EDITORIAL: Conference program

    NASA Astrophysics Data System (ADS)

    2006-04-01

    Some of the papers and talks given at the conference have not been published in this volume of Journal of Physics: Conference Series. The attached PDF file lists the full conference program and indicates (with an asterisk) those papers or talks which are not present in this volume.

  16. Youth Conference Handbook.

    ERIC Educational Resources Information Center

    Brown, Brenda H.

    This handbook is designed to provide practical aid to those who have charge of the planning and organization of a youth conference, Defined as a conference to provide practical information as well as information about possible responsibilities, risks, and consequences of actions, related to the chosen conference topic. Suggestions are given for…

  17. Parent Conferences. Beginnings Workshop.

    ERIC Educational Resources Information Center

    Duffy, Roslyn; And Others

    1997-01-01

    Presents six workshop sessions on parent conferences: (1) "Parents' Perspectives on Conferencing" (R. Duffy); (2) "Three Way Conferences" (G. Zeller); (3) "Conferencing with Parents of Infants" (K. Albrecht); (4) "Conferencing with Parents of School-Agers" (L. G. Miller); (5) "Cross Cultural Conferences" (J. Gonzalez-Mena); and (6) "Working with…

  18. Polymerase Gamma Disease through the Ages

    ERIC Educational Resources Information Center

    Saneto, Russell P.; Naviaux, Robert K.

    2010-01-01

    The most common group of mitochondrial disease is due to mutations within the mitochondrial DNA polymerase, polymerase gamma 1 ("POLG"). This gene product is responsible for replication and repair of the small mitochondrial DNA genome. The structure-function relationship of this gene product produces a wide variety of diseases that at times, seems…

  19. Thermally multiplexed polymerase chain reaction

    PubMed Central

    Phaneuf, Christopher R.; Pak, Nikita; Saunders, D. Curtis; Holst, Gregory L.; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L.; Jerris, Robert; Forest, Craig R.

    2015-01-01

    Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously—each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. PMID:26339317

  20. Transcription by RNA polymerase III: insights into mechanism and regulation

    PubMed Central

    Turowski, Tomasz W.; Tollervey, David

    2016-01-01

    The highly abundant, small stable RNAs that are synthesized by RNA polymerase III (RNAPIII) have key functional roles, particularly in the protein synthesis apparatus. Their expression is metabolically demanding, and is therefore coupled to changing demands for protein synthesis during cell growth and division. Here, we review the regulatory mechanisms that control the levels of RNAPIII transcripts and discuss their potential physiological relevance. Recent analyses have revealed differential regulation of tRNA expression at all steps on its biogenesis, with significant deregulation of mature tRNAs in cancer cells. PMID:27911719

  1. Monitoring DNA polymerase with nanotube-based nanocircuits

    NASA Astrophysics Data System (ADS)

    Li, Yan; Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry; Collins, Philip

    DNA polymerases play an important role in the process of life by accurately and efficiently replicating our genetic information. They use a single-stranded DNA as a template and incorporate nucleotides to create the full, double-stranded DNA. Recent experiments have successfully monitored this process by attaching a Klenow fragment of polymerase I to a carbon nanotube and measuring the current along the tube. Follow-up experiments have shown promise for distinguishing between DNA base pairs when nucleotide analogs are used, thus opening a new avenue for DNA sequencing. In this talk, we present results from computational studies on DNA polymerase I nanocircuits. The enzyme was first equilibrated in molecular dynamics and then density functional theory and Keldysh non-equilibrium Green's function methods were used to calculate the ballistic transmission coefficients and currents for different enzymatic states. Our results show significant change in current when the enzyme alternates between open (idle) and closed (synthesizing) states. We can also differentiate between some template bases when modified nucleotides and gate scanning are used.

  2. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  3. Proteolysis of the proofreading subunit controls the assembly of Escherichia coli DNA polymerase III catalytic core.

    PubMed

    Bressanin, Daniela; Stefan, Alessandra; Piaz, Fabrizio Dal; Cianchetta, Stefano; Reggiani, Luca; Hochkoeppler, Alejandro

    2009-11-01

    The C-terminal region of the proofreading subunit (epsilon) of Escherichia coli DNA polymerase III is shown here to be labile and to contain the residues (identified between F187 and R213) responsible for association with the polymerase subunit (alpha). We also identify two alpha-helices of the polymerase subunit (comprising the residues E311-M335 and G339-D353, respectively) as the determinants of binding to epsilon. The C-terminal region of epsilon is degraded by the ClpP protease assisted by the GroL molecular chaperone, while other factors control the overall concentration in vivo of epsilon. Among these factors, the chaperone DnaK is of primary importance for preserving the integrity of epsilon. Remarkably, inactivation of DnaK confers to Escherichia coli inviable phenotype at 42 degrees C, and viability can be restored over-expressing epsilon. Altogether, our observations indicate that the association between epsilon and alpha subunits of DNA polymerase III depends on small portions of both proteins, the association of which is controlled by proteolysis of epsilon. Accordingly, the factors catalysing (ClpP, GroL) or preventing (DnaK) this proteolysis exert a crucial checkpoint of the assembly of Escherichia coli DNA polymerase III core.

  4. Rifampicin-resistance, rpoB polymorphism and RNA polymerase genetic engineering.

    PubMed

    Alifano, Pietro; Palumbo, Carla; Pasanisi, Daniela; Talà, Adelfia

    2015-05-20

    Following its introduction in 1967, rifampicin has become a mainstay of therapy in the treatment of tuberculosis, leprosy and many other widespread diseases. Its potent antibacterial activity is due to specific inhibition of bacterial RNA polymerase. However, resistance to rifampicin was reported shortly after its introduction in the medical practice. Studies in the model organism Escherichia coli helped to define the molecular mechanism of rifampicin-resistance demonstrating that resistance is mostly due to chromosomal mutations in rpoB gene encoding the RNA polymerase β chain. These studies also revealed the amazing potential of the molecular genetics to elucidate the structure-function relationships in bacterial RNA polymerase. The scope of this paper is to illustrate how rifampicin-resistance has been recently exploited to better understand the regulatory mechanisms that control bacterial cell physiology and virulence, and how this information has been used to maneuver, on a global scale, gene expression in bacteria of industrial interest. In particular, we reviewed recent literature regarding: (i) the effects of rpoB mutations conferring rifampicin-resistance on transcription dynamics, bacterial fitness, physiology, metabolism and virulence; (ii) the occurrence in nature of "mutant-type" or duplicated rifampicin-resistant RNA polymerases; and (iii) the RNA polymerase genetic engineering method for strain improvement and drug discovery.

  5. RNA polymerase gene, microorganism having said gene and the production of RNA polymerase by the use of said microorganism

    DOEpatents

    Kotani, Hirokazu; Hiraoka, Nobutsugu; Obayashi, Akira

    1991-01-01

    SP6 bacteriophage RNA polymerase is produced by cultivating a new microorganism (particularly new strains of Escherichia coli) harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene and recovering SP6 bacteriophage RNA polymerase from the culture broth. SP6 bacteriophage RNA polymerase gene is provided as are new microorganisms harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene.

  6. RNA polymerase II transcription: structure and mechanism.

    PubMed

    Liu, Xin; Bushnell, David A; Kornberg, Roger D

    2013-01-01

    A minimal RNA polymerase II (pol II) transcription system comprises the polymerase and five general transcription factors (GTFs) TFIIB, -D, -E, -F, and -H. The addition of Mediator enables a response to regulatory factors. The GTFs are required for promoter recognition and the initiation of transcription. Following initiation, pol II alone is capable of RNA transcript elongation and of proofreading. Structural studies reviewed here reveal roles of GTFs in the initiation process and shed light on the transcription elongation mechanism. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.

  7. Transcription by RNA polymerases I and III

    PubMed Central

    Paule, Marvin R.; White, Robert J.

    2000-01-01

    The task of transcribing nuclear genes is shared between three RNA polymerases in eukaryotes: RNA polymerase (pol) I synthesises the large rRNA, pol II synthesises mRNA and pol III synthesises tRNA and 5S rRNA. Although pol II has received most attention, pol I and pol III are together responsible for the bulk of transcriptional activity. This survey will summarise what is known about the process of transcription by pol I and pol III, how it happens and the proteins involved. Attention will be drawn to the similarities between the three nuclear RNA polymerase systems and also to their differences. PMID:10684922

  8. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, S.; Richardson, C.

    1997-03-25

    A modified gene encoding a modified DNA polymerase is disclosed. The modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase. 6 figs.

  9. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles

    1997-01-01

    Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.

  10. 10 CFR 501.32 - Conferences (other than prepetition conferences).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Conferences (other than prepetition conferences). 501.32... SANCTIONS Written Comments, Public Hearings and Conferences During Administrative Proceedings § 501.32 Conferences (other than prepetition conferences). (a) At any time following commencement of a...

  11. 47 CFR 1.248 - Prehearing conferences; hearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 1 2010-10-01 2010-10-01 false Prehearing conferences; hearing conferences. 1... Hearing Proceedings Prehearing Procedures § 1.248 Prehearing conferences; hearing conferences. (a) The... to appear at a specified time and place for a conference prior to a hearing, or to submit...

  12. Picornaviral polymerase structure, function, and fidelity modulation.

    PubMed

    Peersen, Olve B

    2017-02-02

    Like all positive strand RNA viruses, the picornaviruses replicate their genomes using a virally encoded RNA-dependent RNA polymerase enzyme known as 3D(pol). Over the past decade we have made tremendous advances in our understanding of 3D(pol) structure and function, including the discovery of a novel mechanism for closing the active site that allows these viruses to easily fine tune replication fidelity and quasispecies distributions. This review summarizes current knowledge of picornaviral polymerase structure and how the enzyme interacts with RNA and other viral proteins to form stable and processive elongation complexes. The picornaviral RdRPs are among the smallest viral polymerases, but their fundamental molecular mechanism for catalysis appears to be generally applicable as a common feature of all positive strand RNA virus polymerases.

  13. Transcription termination by nuclear RNA polymerases

    PubMed Central

    Richard, Patricia; Manley, James L.

    2009-01-01

    Gene transcription in the cell nucleus is a complex and highly regulated process. Transcription in eukaryotes requires three distinct RNA polymerases, each of which employs its own mechanisms for initiation, elongation, and termination. Termination mechanisms vary considerably, ranging from relatively simple to exceptionally complex. In this review, we describe the present state of knowledge on how each of the three RNA polymerases terminates and how mechanisms are conserved, or vary, from yeast to human. PMID:19487567

  14. 76 FR 64083 - Reliability Technical Conference; Notice of Technical Conference

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-17

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Federal Energy Regulatory Commission Reliability Technical Conference; Notice of Technical Conference Take notice that the Federal Energy Regulatory Commission will hold a Technical Conference on Tuesday, November...

  15. The Learning Conference

    ERIC Educational Resources Information Center

    Ravn, Ib

    2007-01-01

    Purpose: The purpose of this paper is to call attention to the fact that conferences for professionals rely on massive one-way communication and hence produce little learning for delegates--and to introduce an alternative, the "learning conference", that involves delegates in fun and productive learning processes.…

  16. ASE Annual Conference 2010

    ERIC Educational Resources Information Center

    McCune, Roger

    2010-01-01

    In this article, the author describes the ASE Annual Conference 2010 which was held at Nottingham after a gap of 22 years. As always, the main conference was preceded by International Day, an important event for science educators from across the world. There were two strands to the programme: (1) "What works for me?"--sharing new ideas…

  17. Lyndon Johnson's Press Conferences.

    ERIC Educational Resources Information Center

    Cooper, Stephen

    Because President Lyndon Johnson understood well the publicity value of the American news media, he sought to exploit them. He saw reporters as "torch bearers" for his programs and policies and used the presidential press conference chiefly for promotional purposes. Although he met with reporters often, his press conferences were usually…

  18. From Conference to Journal

    ERIC Educational Resources Information Center

    McCartney, Robert; Tenenberg, Josh

    2008-01-01

    Revising and extending conference articles for journal publication benefits both authors and readers. The new articles are more complete, and benefit from peer review, feedback from conference presentation, and greater editorial consistency. For those articles that are appropriate, we encourage authors to do this, and present two examples of such…

  19. ICCK Conference Final Report

    SciTech Connect

    Green, William H.

    2013-05-28

    The 7th International Conference on Chemical Kinetics (ICCK) was held July 10-14, 2011, at Massachusetts Institute of Technology (MIT), in Cambridge, MA, hosted by Prof. William H. Green of MIT's Chemical Engineering department. This cross-disciplinary meeting highlighted the importance of fundamental understanding of elementary reactions to the full range of chemical investigations. The specific conference focus was on elementary-step kinetics in both the gas phase and in condensed phase. The meeting provided a unique opportunity to discuss how the same reactive species and reaction motifs manifest under very different reaction conditions (e.g. atmospheric, aqueous, combustion, plasma, in nonaqueous solvents, on surfaces.). The conference featured special sessions on new/improved experimental techniques, improved models and data analysis for interpreting complicated kinetics, computational kinetics (especially rate estimates for large kinetic models), and a panel discussion on how the community should document/archive kinetic data. In the past, this conference had been limited to homogeneous gas-phase and liquid-phase systems. This conference included studies of heterogeneous kinetics which provide rate constants for, or insight into, elementary reaction steps. This Grant from DOE BES covered about half of the subsidies we provided to students and postdocs who attended the conference, by charging them reduced-rate registration fees. The complete list of subsidies provided are listed in Table 1 below. This DOE funding was essential to making the conference affordable to graduate students, and indeed the attendance at this conference was higher than at previous conferences in this series. Donations made by companies provided additional subsidies, leveraging the DOE funding. The conference was very effective in educating graduate students and important in fostering scientific interactions, particularly between scientists studying gas phase and liquid phase kinetics

  20. Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa.

    PubMed

    Crannell, Zachary; Castellanos-Gonzalez, Alejandro; Nair, Gayatri; Mejia, Rojelio; White, A Clinton; Richards-Kortum, Rebecca

    2016-02-02

    This work describes a proof-of-concept multiplex recombinase polymerase amplification (RPA) assay with lateral flow readout that is capable of simultaneously detecting and differentiating DNA from any of the diarrhea-causing protozoa Giardia, Cryptosporidium, and Entamoeba. Together, these parasites contribute significantly to the global burden of diarrheal illness. Differential diagnosis of these parasites is traditionally accomplished via stool microscopy. However, microscopy is insensitive and can miss up to half of all cases. DNA-based diagnostics such as polymerase chain reaction (PCR) are far more sensitive; however, they rely on expensive thermal cycling equipment, limiting their availability to centralized reference laboratories. Isothermal DNA amplification platforms, such as the RPA platform used in this study, alleviate the need for thermal cycling equipment and have the potential to broaden access to more sensitive diagnostics. Until now, multiplex RPA assays have not been developed that are capable of simultaneously detecting and differentiating infections caused by different pathogens. We developed a multiplex RPA assay to detect the presence of DNA from Giardia, Cryptosporidium, and Entamoeba. The multiplex assay was characterized using synthetic DNA, where the limits-of-detection were calculated to be 403, 425, and 368 gene copies per reaction of the synthetic Giardia, Cryptosporidium, and Entamoeba targets, respectively (roughly 1.5 orders of magnitude higher than for the same targets in a singleplex RPA assay). The multiplex assay was also characterized using DNA extracted from live parasites spiked into stool samples where the limits-of-detection were calculated to be 444, 6, and 9 parasites per reaction for Giardia, Cryptosporidium, and Entamoeba parasites, respectively. This proof-of-concept assay may be reconfigured to detect a wide variety of targets by re-designing the primer and probe sequences.

  1. Free RNA polymerase in Escherichia coli.

    PubMed

    Patrick, Michael; Dennis, Patrick P; Ehrenberg, Mans; Bremer, Hans

    2015-12-01

    The frequencies of transcription initiation of regulated and constitutive genes depend on the concentration of free RNA polymerase holoenzyme [Rf] near their promoters. Although RNA polymerase is largely confined to the nucleoid, it is difficult to determine absolute concentrations of [Rf] at particular locations within the nucleoid structure. However, relative concentrations of free RNA polymerase at different growth rates, [Rf]rel, can be estimated from the activities of constitutive promoters. Previous studies indicated that the rrnB P2 promoter is constitutive and that [Rf]rel in the vicinity of rrnB P2 increases with increasing growth rate. Recently it has become possible to directly visualize Rf in growing Escherichia coli cells. Here we examine some of the important issues relating to gene expression based on these new observations. We conclude that: (i) At a growth rate of 2 doublings/h, there are about 1000 free and 2350 non-specifically DNA-bound RNA polymerase molecules per average cell (12 and 28%, respectively, of 8400 total) which are in rapid equilibrium. (ii) The reversibility of the non-specific binding generates more than 1000 free RNA polymerase molecules every second in the immediate vicinity of the DNA. Of these, most rebind non-specifically to the DNA within a few ms; the frequency of non-specific binding is at least two orders of magnitude greater than specific binding and transcript initiation. (iii) At a given amount of RNA polymerase per cell, [Rf] and the density of non-specifically DNA-bound RNA polymerase molecules along the DNA both vary reciprocally with the amount of DNA in the cell. (iv) At 2 doublings/h an E. coli cell contains, on the average, about 1 non-specifically bound RNA polymerase per 9 kbp of DNA and 1 free RNA polymerase per 20 kbp of DNA. However some DNA regions (i.e. near active rRNA operons) may have significantly higher than average [Rf].

  2. A genetic system to identify DNA polymerase β mutator mutants

    PubMed Central

    Washington, Stacy L.; Yoon, Margaret S.; Chagovetz, Alexander M.; Li, Shu-Xia; Clairmont, Caroline A.; Preston, Bradley D.; Eckert, Kristin A.; Sweasy, Joann B.

    1997-01-01

    DNA polymerase β (pol β) is a 39-kDa protein that functions in DNA repair processes in mammalian cells. As a first step toward understanding mechanisms of polymerase fidelity, we developed a genetic method to identify mammalian pol β mutator mutants. This screen takes advantage of a microbial genetics assay and the ability of rat pol β to substitute for Escherichia coli DNA polymerase I in DNA replication in vivo. Using this screen, we identified 13 candidate pol β mutator mutants. Three of the candidate mutator mutants were further characterized in vivo and shown to confer an increased spontaneous mutation frequency over that of wild-type pol β to our bacterial strain. Purification and subsequent analysis of one of our putative mutator proteins, the pol β-14 protein, showed that it possesses intrinsic mutator activity in four different assays that measure the fidelity of DNA synthesis. Therefore, residue 265, which is altered in pol β-14 and another of our mutant proteins, pol β-166, is probably critical for accurate DNA synthesis by pol β. Thus, our genetic method of screening for pol β mutator mutants is useful in identifying active mammalian DNA polymerase mutants that encode enzymes that catalyze DNA synthesis with altered fidelity compared with the wild-type pol β enzyme. PMID:9037051

  3. Escherichia coli RNA polymerase is the target of the cyclopeptide antibiotic microcin J25.

    PubMed

    Delgado, M A; Rintoul, M R; Farías, R N; Salomón, R A

    2001-08-01

    Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded, cyclic peptide antibiotic consisting of 21 unmodified amino acid residues. It is primarily active on gram-negative bacteria related to the producer strain, inducing cell filamentation in an SOS-independent way. A mutation causing resistance to MccJ25 was isolated. Genetic analysis indicated that it resided in the rpoC gene, encoding the beta' subunit of RNA polymerase, at 90 min on the E. coli genetic map. The mutation was genetically crossed on to a plasmid containing the wild-type rpoC gene. The presence of the recombinant plasmid conferred complete resistance to otherwise sensitive strains. Nucleotide sequencing of the plasmid-borne, mutant rpoC gene revealed a ACC (Thr)-to-ATC (Ile) change at codon 931, within homology block G, an evolutionarily conserved region in the large subunits of all RNA polymerases. MccJ25 decreased RNA synthesis both in vivo and in vitro. These results point to the RNA polymerase as the target of microcin action. We favor the possibility that the filamentous phenotype induced by MccJ25 results from impaired transcription of genes coding for cell division proteins. As far as we know, MccJ25 is the first peptide antibiotic shown to affect RNA polymerase.

  4. High-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop

    PubMed Central

    Erinne, Olivia C.; Dave, Jui M.; Cui, Ping; Jin, Huiyan; Muthukrishnan, Nandhini; Tang, Leung K.; Lam, Kenny C.; Strohner, Ralf; Van den Brulle, Jan; Sze, Sing-Hoi; Kaplan, Craig D.

    2016-01-01

    The active sites of multisubunit RNA polymerases have a “trigger loop” (TL) that multitasks in substrate selection, catalysis, and translocation. To dissect the Saccharomyces cerevisiae RNA polymerase II TL at individual-residue resolution, we quantitatively phenotyped nearly all TL single variants en masse. Three mutant classes, revealed by phenotypes linked to transcription defects or various stresses, have distinct distributions among TL residues. We find that mutations disrupting an intra-TL hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding through destabilization of a catalytically inactive TL state, confer phenotypes consistent with pocket disruption and increased catalysis. Furthermore, allele-specific genetic interactions among TL and TL-proximal domain residues support the contribution of the funnel and bridge helices (BH) to TL dynamics. Our structural genetics approach incorporates structural and phenotypic data for high-resolution dissection of transcription mechanisms and their evolution, and is readily applicable to other essential yeast proteins. PMID:27898685

  5. DNA polymerase activity of tomato fruit chromoplasts.

    PubMed

    Serra, E C; Carrillo, N

    1990-11-26

    DNA polymerase activity was measured in chromoplasts of ripening tomato fruits. Plastids isolated from young leaves or mature red fruits showed similar DNA polymerase activities. The same enzyme species was present in either chloroplasts or chromoplasts as judged by pH and temperature profiles, sensitivities towards different inhibitors and relative molecular mass (Mr 88 kDa). The activities analyzed showed the typical behaviour of plastid-type polymerases. The results presented here suggest that chromoplast maintain their DNA synthesis potential in fruit tissue at chloroplast levels. Consequently, the sharp decrease of the plastid chromosome transcription observed at the onset of fruit ripening could not be due to limitations in the availability of template molecules. Other mechanisms must be involved in the inhibition of chromoplast RNA synthesis.

  6. 48 CFR 6101.11 - Conferences; conference memorandum [Rule 11].

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Conferences; conference memorandum . 6101.11 Section 6101.11 Federal Acquisition Regulations System CIVILIAN BOARD OF CONTRACT APPEALS, GENERAL SERVICES ADMINISTRATION CONTRACT DISPUTE CASES 6101.11 Conferences; conference...

  7. In Vitro Resistance Study of AG-021541, a Novel Nonnucleoside Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

    SciTech Connect

    Shi, S.T.; Herlihy, K.J.; Graham, J.P.; Fuhrman, S.A.; Doan, C.; Parge, H.; Hickey, M.; Gao, J.; Yu, X.; Chau, F.; Gonzalez, J.; Li, H.; Lewis, C.; Patrick, A.K.; Duggal, R.

    2009-05-27

    A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 A away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 muM. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.

  8. DNA sequencing using polymerase substrate-binding kinetics

    PubMed Central

    Previte, Michael John Robert; Zhou, Chunhong; Kellinger, Matthew; Pantoja, Rigo; Chen, Cheng-Yao; Shi, Jin; Wang, BeiBei; Kia, Amirali; Etchin, Sergey; Vieceli, John; Nikoomanzar, Ali; Bomati, Erin; Gloeckner, Christian; Ronaghi, Mostafa; He, Molly Min

    2015-01-01

    Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications. PMID:25612848

  9. Insider conference tips

    NASA Astrophysics Data System (ADS)

    Tennant, Jill

    2012-01-01

    Attending an educator conference and its associated exhibit hall can be a rewarding experience for your brain. But if you keep in mind these insider's tips, your feet, arms, stomach, and wallet will also thank you.

  10. Lunar & Planetary Science Conference.

    ERIC Educational Resources Information Center

    Warner, Jeffrey L.; And Others

    1982-01-01

    Summaries of different topics discussed at the Lunar and Planetary Science Conference are presented to provide updated information to nonplanetologists. Some topics include Venus, isotopes, chondrites, creation science, cosmic dust, cratering, moons and rings, igneous rocks, and lunar soil. (DC)

  11. Aircraft Engine Emissions. [conference

    NASA Technical Reports Server (NTRS)

    1977-01-01

    A conference on a aircraft engine emissions was held to present the results of recent and current work. Such diverse areas as components, controls, energy efficient engine designs, and noise and pollution reduction are discussed.

  12. Tackling conference carbon footprints

    NASA Astrophysics Data System (ADS)

    Grozier, Jim

    2016-12-01

    In reply to Margaret Harris's Lateral Thoughts article "Putting my foot down", which discussed the challenges of attending a conference with a physical disability (October p76) and a subsequent letter by Anna Wood (November p18).

  13. DNA sequencing conference, 2

    SciTech Connect

    Cook-Deegan, R.M.; Venter, J.C.; Gilbert, W.; Mulligan, J.; Mansfield, B.K.

    1991-06-19

    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  14. A movie of RNA polymerase II transcription.

    PubMed

    Cheung, Alan C M; Cramer, Patrick

    2012-06-22

    We provide here a molecular movie that captures key aspects of RNA polymerase II initiation and elongation. To create the movie, we combined structural snapshots of the initiation-elongation transition and of elongation, including nucleotide addition, translocation, pausing, proofreading, backtracking, arrest, reactivation, and inhibition. The movie reveals open questions about the mechanism of transcription and provides a useful teaching tool.

  15. Catalytic editing properties of DNA polymerases.

    PubMed Central

    Canard, B; Cardona, B; Sarfati, R S

    1995-01-01

    Enzymatic incorporation of 2',3'-dideoxynucleotides into DNA results in chain termination. We report that 3'-esterified 2'-deoxynucleoside 5'-triphosphates (dNTPs) are false chain-terminator substrates since DNA polymerases, including human immunodeficiency virus reverse transcriptase, can incorporate them into DNA and, subsequently, use this new 3' end to insert the next correctly paired dNTP. Likewise, a DNA substrate with a primer chemically esterified at the 3' position can be extended efficiently upon incubation with dNTPs and T7 DNA polymerase lacking 3'-to-5' exonuclease activity. This enzyme is also able to use dTTP-bearing reporter groups in the 3' position conjugated through amide or thiourea bonds and cleave them to restore a DNA chain terminated by an amino group at the 3' end. Hence, a number of DNA polymerases exhibit wide catalytic versatility at the 3' end of the nascent DNA strand. As part of the polymerization mechanism, these capabilities extend the number of enzymatic activities associated with these enzymes and also the study of interactions between DNA polymerases and nucleotide analogues. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7479898

  16. The bacterial enhancer-dependent RNA polymerase

    PubMed Central

    Zhang, Nan; Darbari, Vidya C.; Glyde, Robert; Zhang, Xiaodong; Buck, Martin

    2016-01-01

    Transcription initiation is highly regulated in bacterial cells, allowing adaptive gene regulation in response to environment cues. One class of promoter specificity factor called sigma54 enables such adaptive gene expression through its ability to lock the RNA polymerase down into a state unable to melt out promoter DNA for transcription initiation. Promoter DNA opening then occurs through the action of specialized transcription control proteins called bacterial enhancer-binding proteins (bEBPs) that remodel the sigma54 factor within the closed promoter complexes. The remodelling of sigma54 occurs through an ATP-binding and hydrolysis reaction carried out by the bEBPs. The regulation of bEBP self-assembly into typically homomeric hexamers allows regulated gene expression since the self-assembly is required for bEBP ATPase activity and its direct engagement with the sigma54 factor during the remodelling reaction. Crystallographic studies have now established that in the closed promoter complex, the sigma54 factor occupies the bacterial RNA polymerase in ways that will physically impede promoter DNA opening and the loading of melted out promoter DNA into the DNA-binding clefts of the RNA polymerase. Large-scale structural re-organizations of sigma54 require contact of the bEBP with an amino-terminal glutamine and leucine-rich sequence of sigma54, and lead to domain movements within the core RNA polymerase necessary for making open promoter complexes and synthesizing the nascent RNA transcript. PMID:27789741

  17. Determining Annealing Temperatures for Polymerase Chain Reaction

    ERIC Educational Resources Information Center

    Porta, Angela R.; Enners, Edward

    2012-01-01

    The polymerase chain reaction (PCR) is a common technique used in high school and undergraduate science teaching. Students often do not fully comprehend the underlying principles of the technique and how optimization of the protocol affects the outcome and analysis. In this molecular biology laboratory, students learn the steps of PCR with an…

  18. Polymerase Chain Reaction for Educational Settings.

    ERIC Educational Resources Information Center

    Garrison, Stephen J.; dePamphillis, Claude

    1994-01-01

    Suggests the incorporation of the Polymerase Chain Reaction (PCR) technique into high school and college biology laboratories. Discusses the following sections: (1) current PCR applications; (2) PCR technique; (3) Manual and Machine PCR; (4) Manual PCR Preparations and Procedure; (5) Materials, Supplies, and Recipes; (6) Primer Selection; and (7)…

  19. Circulating polymerase chain reaction chips utilizing multiple-membrane activation

    NASA Astrophysics Data System (ADS)

    Wang, Chih-Hao; Chen, Yi-Yu; Liao, Chia-Sheng; Hsieh, Tsung-Min; Luo, Ching-Hsing; Wu, Jiunn-Jong; Lee, Huei-Huang; Lee, Gwo-Bin

    2007-02-01

    This paper reports a new micromachined, circulating, polymerase chain reaction (PCR) chip for nucleic acid amplification. The PCR chip is comprised of a microthermal control module and a polydimethylsiloxane (PDMS)-based microfluidic control module. The microthermal control modules are formed with three individual heating and temperature-sensing sections, each modulating a specific set temperature for denaturation, annealing and extension processes, respectively. Micro-pneumatic valves and multiple-membrane activations are used to form the microfluidic control module to transport sample fluids through three reaction regions. Compared with other PCR chips, the new chip is more compact in size, requires less time for heating and cooling processes, and has the capability to randomly adjust time ratios and cycle numbers depending on the PCR process. Experimental results showed that detection genes for two pathogens, Streptococcus pyogenes (S. pyogenes, 777 bps) and Streptococcus pneumoniae (S. pneumoniae, 273 bps), can be successfully amplified using the new circulating PCR chip. The minimum number of thermal cycles to amplify the DNA-based S. pyogenes for slab gel electrophoresis is 20 cycles with an initial concentration of 42.5 pg µl-1. Experimental data also revealed that a high reproducibility up to 98% could be achieved if the initial template concentration of the S. pyogenes was higher than 4 pg µl-1. The preliminary results of the current paper were presented at the 19th IEEE International Conference on Micro Electro Mechanical Systems (IEEE MEMS 2006), Istanbul, Turkey, 22-26 January, 2006.

  20. Multiphoton processes: conference proceedings

    SciTech Connect

    Lambropoulos, P.; Smith, S.J.

    1984-01-01

    The chapters of this volume represent the invited papers delivered at the conference. They are arranged according to thermatic proximity beginning with atoms and continuing with molecules and surfaces. Section headings include multiphoton processes in atoms, field fluctuations and collisions in multiphoton process, and multiphoton processes in molecules and surfaces. Abstracts of individual items from the conference were prepared separately for the data base. (GHT)

  1. Conference scene: DGVS spring conference 2009.

    PubMed

    Kolligs, Frank Thomas

    2009-10-01

    The 3rd annual DGVS Spring Conference of the German Society for Gastroenterology (Deutsche Gesellschaft für Verdauungs- und Stoffwechselkrankheiten) was held at the Seminaris Campus Hotel in Berlin, Germany, on 8-9 May, 2009. The conference was organized by Roland Schmid and Matthias Ebert from the Technical University of Munich, Germany. The central theme of the meeting was 'translational gastrointestinal oncology: towards personalized medicine and individualized therapy'. The conference covered talks on markers for diagnosis, screening and surveillance of colorectal cancer, targets for molecular therapy, response prediction in clinical oncology, development and integration of molecular imaging in gastrointestinal oncology and translational research in clinical trial design. Owing to the broad array of topics and limitations of space, this article will focus on biomarkers, response prediction and the integration of biomarkers into clinical trials. Presentations mentioned in this summary were given by Matthias Ebert (Technical University of Munich, Germany), Esmeralda Heiden (Epigenomics, Berlin, Germany), Frank Kolligs (University of Munich, Germany), Florian Lordick (University of Heidelberg, Germany), Hans Jorgen Nielsen (University of Copenhagen, Denmark), Anke Reinacher-Schick (University of Bochum, Germany), Christoph Röcken (University of Berlin, Germany), Wolff Schmiegel (University of Bochum, Germany) and Thomas Seufferlein (University of Halle, Germany).

  2. Independent Structural Domains in Paramyxovirus Polymerase Protein*

    PubMed Central

    Dochow, Melanie; Krumm, Stefanie A.; Crowe, James E.; Moore, Martin L.; Plemper, Richard K.

    2012-01-01

    All enzymatic activities required for genomic replication and transcription of nonsegmented negative strand RNA viruses (or Mononegavirales) are believed to be concentrated in the viral polymerase (L) protein. However, our insight into the organization of these different enzymatic activities into a bioactive tertiary structure remains rudimentary. Fragments of Mononegavirales polymerases analyzed to date cannot restore bioactivity through trans-complementation, unlike the related L proteins of segmented NSVs. We investigated the domain organization of phylogenetically diverse Paramyxovirus L proteins derived from measles virus (MeV), Nipah virus (NiV), and respiratory syncytial virus (RSV). Through a comprehensive in silico and experimental analysis of domain intersections, we defined MeV L position 615 as an interdomain candidate in addition to the previously reported residue 1708. Only position 1708 of MeV and the homologous positions in NiV and RSV L also tolerated the insertion of epitope tags. Splitting of MeV L at residue 1708 created fragments that were unable to physically interact and trans-complement, but strikingly, these activities were reconstituted by the addition of dimerization tags to the fragments. Equivalently split fragments of NiV, RSV, and MeV L oligomerized with comparable efficiency in all homo- and heterotypic combinations, but only the homotypic pairs were able to trans-complement. These results demonstrate that synthesis as a single polypeptide is not required for the Mononegavirales polymerases to adopt a proper tertiary conformation. Paramyxovirus polymerases are composed of at least two truly independent folding domains that lack a traditional interface but require molecular compatibility for bioactivity. The functional probing of the L domain architecture through trans-complementation is anticipated to be applicable to all Mononegavirales polymerases. PMID:22215662

  3. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells.

    PubMed

    Min, Irene M; Waterfall, Joshua J; Core, Leighton J; Munroe, Robert J; Schimenti, John; Lis, John T

    2011-04-01

    Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ∼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, "bivalent" ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5' proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation.

  4. Role of DNA polymerase gamma in adenovirus DNA replication. Mechanism of inhibition by 2',3'-dideoxynucleoside 5'-triphosphates.

    PubMed

    van der Vliet, P C; Kwant, M M

    1981-04-28

    In contrast to cellular or SV40 DNA replication, adenovirus type 5 (Ad5) or type 2 (Ad2) DNA synthesis in isolated nuclei is strongly inhibited by low concentrations of 2',3'-dideoxythymidine 5'-triphosphate (ddTTP). On the basis of differential sensitivity of cellular DNA polymerases, a role of DNA polymerase gamma in adenovirus DNA replication has been proposed. We have investigated the mechanism of inhibition of adenovirus DNA synthesis, using [alpha-32P]ddTTP and other dNTP analogues. Both ddATP and ddGTP were as inhibitory as ddTTP, while ddCTP had an even stronger effect on adenovirus DNA replication. DNA polymerase alpha was resistant to all four ddNTP's, while DNA polymerase gamma was very sensitive. The inhibition by ddTTP in isolated infected nuclei was slowly reversible. [alpha-32P]ddTTP was incorporated into Ad5 DNA as a chain-terminating nucleotide, and the analogue could be used as a substrate by DNA polymerase gamma. Under similar conditions, incorporation in cellular DNA or using DNA polymerase alpha was not observed. The nucleoside analogues ddA and ddC suppressed adenovirus. DNA replication in intact cells and reduced plaque formation. These results provide further evidence for a function of DNA polymerase gamma in adenovirus DNA synthesis.

  5. Microbial metagenome profiling using amplicon length heterogeneity-polymerase chain reaction proves more effective than elemental analysis in discriminating soil specimens.

    PubMed

    Moreno, Lilliana I; Mills, DeEtta K; Entry, James; Sautter, Robert T; Mathee, Kalai

    2006-11-01

    The combination of soil's ubiquity and its intrinsic abiotic and biotic information can contribute greatly to the forensic field. Although there are physical and chemical characterization methods of soil comparison for forensic purposes, these require a level of expertise not always encountered in crime laboratories. We hypothesized that soil microbial community profiling could be used to discriminate between soil types by providing biological fingerprints that confer uniqueness. Three of the six Miami-Dade soil types were randomly selected and sampled. We compared the microbial metagenome profiles generated using amplicon length heterogeneity-polymerase chain reaction analysis of the 16S rRNA genes with inductively coupled plasma optical emission spectroscopy analysis of 13 elements (Al, B, Ca, Cu, Fe, K, Mg, Mn, Na, P, S, Si, and Zn) that are commonly encountered in soils. Bray-Curtis similarity index and analysis of similarity were performed on all data to establish differences within sites, among sites, and across two seasons. These data matrices were used to group samples that shared similar community patterns using nonmetric multidimensional scaling analysis. We concluded that while chemical characterization could provide some differentiation between soils, microbial metagenome profiling was better able to discriminate between the soil types and had a high degree of reproducibility, therefore proving to be a potential tool for forensic soil comparisons.

  6. Nuclear Rocket Technology Conference

    NASA Technical Reports Server (NTRS)

    1966-01-01

    The Lewis Research Center has a strong interest in nuclear rocket propulsion and provides active support of the graphite reactor program in such nonnuclear areas as cryogenics, two-phase flow, propellant heating, fluid systems, heat transfer, nozzle cooling, nozzle design, pumps, turbines, and startup and control problems. A parallel effort has also been expended to evaluate the engineering feasibility of a nuclear rocket reactor using tungsten-matrix fuel elements and water as the moderator. Both of these efforts have resulted in significant contributions to nuclear rocket technology. Many successful static firings of nuclear rockets have been made with graphite-core reactors. Sufficient information has also been accumulated to permit a reasonable Judgment as to the feasibility of the tungsten water-moderated reactor concept. We therefore consider that this technoIogy conference on the nuclear rocket work that has been sponsored by the Lewis Research Center is timely. The conference has been prepared by NASA personnel, but the information presented includes substantial contributions from both NASA and AEC contractors. The conference excludes from consideration the many possible mission requirements for nuclear rockets. Also excluded is the direct comparison of nuclear rocket types with each other or with other modes of propulsion. The graphite reactor support work presented on the first day of the conference was partly inspired through a close cooperative effort between the Cleveland extension of the Space Nuclear Propulsion Office (headed by Robert W. Schroeder) and the Lewis Research Center. Much of this effort was supervised by Mr. John C. Sanders, chairman for the first day of the conference, and by Mr. Hugh M. Henneberry. The tungsten water-moderated reactor concept was initiated at Lewis by Mr. Frank E. Rom and his coworkers. The supervision of the recent engineering studies has been shared by Mr. Samuel J. Kaufman, chairman for the second day of the

  7. Human DNA polymerase alpha gene: sequences controlling expression in cycling and serum-stimulated cells.

    PubMed Central

    Pearson, B E; Nasheuer, H P; Wang, T S

    1991-01-01

    We have investigated the DNA polymerase alpha promoter sequence requirements for the expression of a heterologous gene in actively cycling cells and following serum addition to serum-deprived cells. An 11.4-kb genomic clone that spans the 5' end of this gene and includes 1.62 kb of sequence upstream from the translation start site was isolated. The transcription start site was mapped at 46 +/- 1 nucleotides upstream from the translation start site. The upstream sequence is GC rich and lacks a TATA sequence but has a CCAAT sequence on the opposite strand. Analysis of a set of deletion constructs in transient transfection assays demonstrated that efficient expression of the reporter in cycling cells requires 248 bp of sequence upstream from the cap site. Clustered within these 248 nucleotides are sequences similar to consensus sequences for Sp1-, Ap1-, Ap2-, and E2F-binding sites. The CCAAT sequence and the potential E2F- and Ap1-binding sites are shown to be protected from DNase I digestion by partially purified nuclear proteins. The DNA polymerase alpha promoter can confer upon the reporter an appropriate, late response to serum addition. No single sequence element could be shown to confer serum inducibility. Rather, multiple sequence elements appear to mediate the full serum response. Images PMID:2005899

  8. Involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism.

    PubMed

    Agudo, Rubén; de la Higuera, Ignacio; Arias, Armando; Grande-Pérez, Ana; Domingo, Esteban

    2016-07-01

    We previously characterized a foot-and-mouth disease virus (FMDV) with three amino acid replacements in its polymerase (3D) that conferred resistance to the mutagenic nucleoside analogue ribavirin. Here we show that passage of this mutant in the presence of high ribavirin concentrations resulted in selection of viruses with the additional replacement I248T in 2C. This 2C substitution alone (even in the absence of replacements in 3D) increased FMDV fitness mainly in the presence of ribavirin, prevented an incorporation bias in favor of A and U associated with ribavirin mutagenesis, and conferred the ATPase activity of 2C decreased sensitivity to ribavirin-triphosphate. Since in previous studies we described that 2C with I248T was selected under different selective pressures, this replacement qualifies as a joker substitution in FMDV evolution. The results have identified a role of 2C in nucleotide incorporation, and have unveiled a new polymerase-independent mechanism of virus escape to lethal mutagenesis.

  9. Structural biology of bacterial RNA polymerase.

    PubMed

    Murakami, Katsuhiko S

    2015-05-11

    Since its discovery and characterization in the early 1960s (Hurwitz, J. The discovery of RNA polymerase. J. Biol. Chem. 2005, 280, 42477-42485), an enormous amount of biochemical, biophysical and genetic data has been collected on bacterial RNA polymerase (RNAP). In the late 1990s, structural information pertaining to bacterial RNAP has emerged that provided unprecedented insights into the function and mechanism of RNA transcription. In this review, I list all structures related to bacterial RNAP (as determined by X-ray crystallography and NMR methods available from the Protein Data Bank), describe their contributions to bacterial transcription research and discuss the role that small molecules play in inhibiting bacterial RNA transcription.

  10. Enzymatic initiation of DNA synthesis by yeast DNA polymerases.

    PubMed Central

    Plevani, P; Chang, L M

    1977-01-01

    Partially purified yeast RNA polymerases (RNA nucleotidyltransferases) initiate DNA synthesis by yeast DNA polymerase (DNA nucleotidyltransferase) I and to a lesser extent yeast DNA polymerase II in the replication of single-stranded DNA. The enzymatic initiation of DNA synthesis on phage fd DNA template occurs with dNTPs alone and is further stimulated by the presence of rNTPs in DNA polymerase I reactions. The presence of rNTPs has no effect on the RNA polymerase initiation of the DNA polymerase II reaction. RNA polymerases I and III are more efficient in initiation of DNA synthesis than RNA polymerase II. Analyses of the products of fd DNA replication show noncovalent linkage between the newly synthesized DNA and the template DNA, and covalent linkage between the newly synthesized RNA and DNA. PMID:325562

  11. The architecture of RNA polymerase fidelity.

    PubMed

    Kaplan, Craig D

    2010-06-22

    The basis for transcriptional fidelity by RNA polymerase is not understood, but the 'trigger loop', a conserved structural element that is rearranged in the presence of correct substrate nucleotides, is thought to be critical. A study just published in BMC Biology sheds new light on the ways in which the trigger loop may promote selection of correct nucleotide triphosphate substrates. See research article http://www.biomedcentral.com/1741-7007/8/54.

  12. DNA-polymerase inhibitors. Rifamycin derivatives.

    PubMed Central

    Frolova, L Y; Meldrays, Y A; Kochkina, L L; Giller, S A; Eremeyev, A V; Grayevskaya, N A; Kisselev, L L

    1977-01-01

    Ten new derivatives of the antibiotic rifamycin with variable side chains at position 3 were synthesized. The inhibitory activity of these derivatives against DNA-polymerases isolated from avian myeloblastosis virus, E. coli and calf thymus were studied at various conditions. 3-(2,4,6-trinitrophenylhydrazone-(methyl) rifamycin SV is a strong inhibitor for all the polymerases tested and belongs to the C class inhibitors of reverse transcriptase. 3-(monoallylhydrazone-(methyl) rifamycin SV possesses a selective action on polymerases: at 0.1 mg/ml concentration it almost completely inhibits the reverse transcriptase and less than half of the bacterial and eukaryotic enzymes. A drug is found which strongly inhibits the DNA-polymerases from E. coli and calf thymus and weakly the viral enzyme. The inhibitory effect on reverse transcriptase is independent of the choice of template-primer; it could be overcome by the addition of excess enzyme but not of excess template-primer; the inhibition could be completely reversed by dilution of the drug-enzyme mixture. From Lineweaver-Burk analysis, the inhibition is noncompetitive with respect to the template-primer and, thus the drugs bind to the site different from the active site for the template-primer. From protective action of the template-primer and other data it might be suggested that the rifamycin derivatives act at an early step(s) in DNA synthesis catalyzed by reverse transcriptase. The obtained data are in agreement with the results for other derivatives of rifamycin SV described in literature. PMID:68462

  13. DNA polymerase mu, a candidate hypermutase?

    PubMed Central

    Ruiz, J F; Domínguez, O; Laín de Lera, T; Garcia-Díaz, M; Bernad, A; Blanco, L

    2001-01-01

    A novel DNA polymerase (Pol mu) has been recently identified in human cells. The amino-acid sequence of Pol mu is 42% identical to that of terminal deoxynucleotidyl transferase (TdT), a DNA-independent DNA polymerase that contributes to antigen-receptor diversity. In this paper we review the evidence supporting the role of Pol mu in somatic hypermutation of immunoglobulin genes, a T-dependent process that selectively occurs at germinal centres: (i) preferential expression in secondary lymphoid organs; (ii) expression associated to developing germinal centres; and (iii) very low base discrimination during DNA-dependent DNA polymerization by Pol mu, a mutator phenotype enormously accentuated by the presence of activating Mn2+ ions. Moreover, its similarity to TdT, together with extrapolation to the crystal structure of DNA polymerase beta complexed (Pol beta) with DNA, allows us to discuss the structural basis for the unprecedented error proneness of Pol mu, and to predict that Pol mu is structurally well suited to participate also in DNA end-filling steps occurring both during V(D)J recombination and repair of DNA double-strand breaks that are processed by non-homologous end-joining. PMID:11205337

  14. 1,5-Benzodiazepines, a Novel Class of Hepatitis C Virus Polymerase Nonnucleoside Inhibitors ▿ †

    PubMed Central

    Nyanguile, Origène; Pauwels, Frederik; Van den Broeck, Walter; Boutton, Carlo W.; Quirynen, Ludo; Ivens, Tania; van der Helm, Liesbet; Vandercruyssen, Geneviève; Mostmans, Wendy; Delouvroy, Frédéric; Dehertogh, Pascale; Cummings, Maxwell D.; Bonfanti, Jean-Francois; Simmen, Kenneth A.; Raboisson, Pierre

    2008-01-01

    The exogenous control of hepatitis C virus (HCV) replication can be mediated through the inhibition of the RNA-dependent RNA polymerase (RdRp) activity of NS5B. Small-molecule inhibitors of NS5B include nucleoside and nonnucleoside analogs. Here, we report the discovery of a novel class of HCV polymerase nonnucleoside inhibitors, 1,5-benzodiazepines (1,5-BZDs), identified by high-throughput screening of a library of small molecules. A fluorescence-quenching assay and X-ray crystallography revealed that 1,5-BZD 4a bound stereospecifically to NS5B next to the catalytic site. When introduced into replicons, mutations known to confer resistance against chemotypes that bind at this site were detrimental to inhibition by 1,5-BZD 7a. Using a panel of enzyme isolates that covered genotypes 1 to 6, we showed that compound 4a inhibited genotype 1 only. In mechanistic studies, 4a was found to inhibit the RdRp activity of NS5B noncompetitively with GTP and to inhibit the formation of the first phosphodiester bond during the polymerization cycle. The specificity for the HCV target was evaluated by profiling the 1,5-BZDs against other viral and human polymerases, as well as BZD receptors. PMID:18852280

  15. Structure of the SSB-DNA polymerase III interface and its role in DNA replication

    SciTech Connect

    Marceau, Aimee H; Bahng, Soon; Massoni, Shawn C; George, Nicholas P; Sandler, Steven J; Marians, Kenneth J; Keck, James L

    2012-05-22

    Interactions between single-stranded DNA-binding proteins (SSBs) and the DNA replication machinery are found in all organisms, but the roles of these contacts remain poorly defined. In Escherichia coli, SSB's association with the χ subunit of the DNA polymerase III holoenzyme has been proposed to confer stability to the replisome and to aid delivery of primers to the lagging-strand DNA polymerase. Here, the SSB-binding site on χ is identified crystallographically and biochemical and cellular studies delineate the consequences of destabilizing the χ/SSB interface. An essential role for the χ/SSB interaction in lagging-strand primer utilization is not supported. However, sequence changes in χ that block complex formation with SSB lead to salt-dependent uncoupling of leading- and lagging-strand DNA synthesis and to a surprising obstruction of the leading-strand DNA polymerase in vitro, pointing to roles for the χ/SSB complex in replisome establishment and maintenance. Destabilization of the χ/SSB complex in vivo produces cells with temperature-dependent cell cycle defects that appear to arise from replisome instability.

  16. 78 FR 27963 - Reliability Technical Conference; Notice of Technical Conference

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-13

    ... Energy Regulatory Commission Reliability Technical Conference; Notice of Technical Conference Take notice... the conference is to discuss policy issues related to the reliability of the Bulk-Power System. A more... Webcast. Anyone with Internet access who desires to listen to this event can do so by navigating to...

  17. Turbomachinery controls conference (TCC) 1995

    SciTech Connect

    1995-09-01

    The proceedings of the Turbomachinery Controls Conference 1995 are presented. Eleven papers were presented at the conference. A separate abstract was prepared for each of the nine papers for inclusion in the Energy Science and Technology Database.

  18. ARC Conference Showcases Telecommunications Services.

    ERIC Educational Resources Information Center

    Baldwin, Fred D.

    1996-01-01

    The Appalachian Regional Commission's 1996 Conference, "Building Blocks for Using Telecommunications and Information Technology," held in Binghamton, New York, focused on the role of telecommunications in Appalachia in education and training, telemedicine, business, and government. Highlights conference presentations on special…

  19. Twitter use in physics conferences.

    PubMed

    Webb, Stephen

    An analysis of Twitter use in 116 conferences suggests that the service is used more extensively at PACS10 conferences (those devoted to the physics of elementary particles and fields) and PACS90 conferences (those devoted to geophysics, astronomy, and astrophysics) than at conferences in other fields of physics. Furthermore, Twitter is used in a qualitatively different manner. A possible reason for these differences is discussed.

  20. Conference on Navajo Orthography.

    ERIC Educational Resources Information Center

    Ohannessian, Sirarpi; And Others

    This report on the Conference on Navajo Orthography, held in Albuquerque, New Mexico on May 2-3, 1969 constitutes a summary of the discussion and decisions of a meeting which was convened by the Center for Applied Linguistics under contract with the Bureau of Indian Affairs to agree on an orthography for the Navajo language. The immediate purpose…

  1. DEVELOP students attend conference

    NASA Technical Reports Server (NTRS)

    2009-01-01

    Last month, Madeline Brozen and Jason Jones of the DEVELOP Program at John C. Stennis Space Center joined members from the program's national office at Langley Research Center to attend the Southern Growth Policies Board annual conference in Biloxi. Pictured are (l to r): Karen Allsbrook, Jonathan Gleason, Gov. Haley Barbour, Madeline Brozen, Lindsay Rogers and Tracey Silcox.

  2. Hydrogen Conference: Workshop Proceedings

    SciTech Connect

    1989-10-01

    Hydrogen is currently a major chemical/fuel with long-term energy system benefits that may impact the industry's physical and economic well-being. EPRI's recent hydrogen conference concluded that to be competitive, the production cost must take into account environmental and end-use efficiency benefits.

  3. The interparliamentary conference

    SciTech Connect

    Not Available

    1990-01-01

    The purpose of this conference was to provide a forum for exchange of information on environmental problems with global origins and consequences. The areas of major concern included the following: global climate change; deforestation and desertification; preservation of biological diversity; safeguarding oceans and water resources; population growth; destruction of the stratospheric ozone layer; and sustainable development.

  4. Conference summary - Personal views

    NASA Astrophysics Data System (ADS)

    Lub, J.

    2016-05-01

    This is a collection of remarks on the three and a half days of the RR Lyrae 2015 Conference, limited only by my own lack of attention and understanding. I end with some personal recollections on my complete failure, even though doing the necessary calculations, to spot the importance and the possible application of Fourier amplitudes and phases of the RR Lyrae light curves.

  5. Government Quality Conference Proceedings

    NASA Technical Reports Server (NTRS)

    1992-01-01

    The Government Quality Conference was an attempt to bring together executive organizations and senior individuals in the Federal Government that have a desire to improve productivity. It was designed to provide an exchange of ideas based on experience, and to encourage individual management initiatives to tap the capabilities of Federal employees.

  6. 2002 NASPSA Conference Abstracts.

    ERIC Educational Resources Information Center

    Journal of Sport & Exercise Psychology, 2002

    2002-01-01

    Contains abstracts from the 2002 conference of the North American Society for the Psychology of Sport and Physical Activity. The publication is divided into three sections: the preconference workshop, "Effective Teaching Methods in the Classroom;" symposia (motor development, motor learning and control, and sport psychology); and free…

  7. Effective Parent Conferences

    ERIC Educational Resources Information Center

    Elmore, Michael

    2008-01-01

    Conferences with an upset parent, teacher, or student should be opportunities for schools to build relationships with the community and to foster a positive school culture. But in reality, they are often held because a problem has arisen and often end with stakeholders having a less-than-positive image of the school. Understanding the steps that…

  8. Conference Abstracts: AEDS '84.

    ERIC Educational Resources Information Center

    Baird, William E.

    1985-01-01

    The Association of Educational Data Systems (AEDS) conference included 102 presentations. Abstracts of seven of these presentations are provided. Topic areas considered include LOGO, teaching probability through a computer game, writing effective computer assisted instructional materials, computer literacy, research on instructional…

  9. Annual Conference Abstracts

    ERIC Educational Resources Information Center

    Journal of Engineering Education, 1972

    1972-01-01

    Includes abstracts of papers presented at the 80th Annual Conference of the American Society for Engineering Education. The broad areas include aerospace, affiliate and associate member council, agricultural engineering, biomedical engineering, continuing engineering studies, chemical engineering, civil engineering, computers, cooperative…

  10. Microbicides 2006 conference

    PubMed Central

    Ramjee, Gita; Shattock, Robin; Delany, Sinead; McGowan, Ian; Morar, Neetha; Gottemoeller, Megan

    2006-01-01

    Current HIV/AIDS statistics show that women account for almost 60% of HIV infections in Sub-Saharan Africa. HIV prevention tools such as male and female condoms, abstinence and monogamy are not always feasible options for women due to various socio-economic and cultural factors. Microbicides are products designed to be inserted in the vagina or rectum prior to sex to prevent HIV acquisition. The biannual Microbicides conference took place in Cape Town, South Africa from 23–26 April 2006. The conference was held for the first time on the African continent, the region worst affected by the HIV/AIDS pandemic. The conference brought together a record number of 1,300 scientists, researchers, policy makers, healthcare workers, communities and advocates. The conference provided an opportunity for an update on microbicide research and development as well as discussions around key issues such as ethics, acceptability, access and community involvement. This report discusses the current status of microbicide research and development, encompassing basic and clinical science, social and behavioural science, and community mobilisation and advocacy activities. PMID:17038196

  11. APPA 2011 Conference Highlights

    ERIC Educational Resources Information Center

    Facilities Manager, 2011

    2011-01-01

    This article presents highlights of APPA conference that was held on July 16-18, 2011. The highlights feature photos of 2011-2012 board of directors, outgoing senior regional representatives to the board, meritorious service award, APPA fellow, president's recognition and gavel exchange, and diamond business partner award.

  12. Annual Conference Abstracts

    ERIC Educational Resources Information Center

    Engineering Education, 1976

    1976-01-01

    Presents the abstracts of 158 papers presented at the American Society for Engineering Education's annual conference at Knoxville, Tennessee, June 14-17, 1976. Included are engineering topics covering education, aerospace, agriculture, biomedicine, chemistry, computers, electricity, acoustics, environment, mechanics, and women. (SL)

  13. REGIONAL CONFERENCE SUMMARIES, 1966.

    ERIC Educational Resources Information Center

    Bureau of Adult, Vocational, and Technical Education (DHEW/OE), Washington, DC. Div. of Vocational and Technical Education.

    AN AVERAGE OF 200 TEACHER EDUCATORS, STATE DIRECTORS, LAYMEN, AND REPRESENTATIVES OF VARIOUS AGENCIES ATTENDED EACH OF NINE REGIONAL CONFERENCES CONDUCTED THROUGHOUT THE UNITED STATES TO DISCUSS THE INFLUENCE OF SOCIAL AND ECONOMIC CHANGES AND PROBLEMS IN PLANNING AND CONDUCTING VOCATIONAL AND TECHNICAL EDUCATION PROGRAMS. MAJOR SPEECHES PRESENTED…

  14. Metabolic Engineering X Conference

    SciTech Connect

    Flach, Evan

    2015-05-07

    The International Metabolic Engineering Society (IMES) and the Society for Biological Engineering (SBE), both technological communities of the American Institute of Chemical Engineers (AIChE), hosted the Metabolic Engineering X Conference (ME-X) on June 15-19, 2014 at the Westin Bayshore in Vancouver, British Columbia. It attracted 395 metabolic engineers from academia, industry and government from around the globe.

  15. Conference Rules, Part 1

    ERIC Educational Resources Information Center

    Kerber, Linda K.

    2008-01-01

    Most academic conferences are preceded by some effort to make the sessions different from the usual format, but the usual format overwhelmingly prevails. That is: Each panel discussion runs no longer than two hours, during which two, three, or four specialists stand at a lectern and talk. Sometimes they will read a prepared paper; sometimes they…

  16. Open Mind Conference

    NASA Technical Reports Server (NTRS)

    King, Alexander H.

    1995-01-01

    Open Mind, The Association for the achievement of diversity in higher education, met in conference in Albuquerque, New Mexico, between October 16 and 18, 1992. A number of workgroups met to discuss the goals, structure, and generally evaluate the Association and its achievements. A summary of the workgroup sessions and their minutes are included.

  17. A Conference of Hope.

    ERIC Educational Resources Information Center

    American Printing House for the Blind, Louisville, KY. Dept. of Educational Research.

    Presented are the proceedings of the First Historic Helen Keller World Conference on Services to Deaf-Blind Youths and Adults, held in New York City in September, 1977 on the theme "The Deaf-Blind Person in the Community." Reports have the following titles and authors: "Definition, Demography, Causes and Prevention of Deaf-Blindness; Finding and…

  18. ALA Conference 2009: Chicago Hope

    ERIC Educational Resources Information Center

    Berry, John N., III

    2009-01-01

    There is joy among those who have the funds to go to Chicago for the American Library Association (ALA) annual conference, July 9-15. Every librarian knows there is nothing better than a Chicago gathering, with the city's wonderful haunts, museums, restaurants, and fine memories of past conferences. The conference program covers nearly every…

  19. Summary: A Very Timely Conference

    NASA Astrophysics Data System (ADS)

    Wyse, Rosemary F. G.

    2012-04-01

    The conference poster includes a very apt phrase that describes a primary motivation for this conference: Time discovers truth. This aphorism, attributed to Seneca, was certainly affirmed by the many exciting talks and discussions at this conference, in both formal and informal settings.

  20. Making Connections: Attending Professional Conferences

    ERIC Educational Resources Information Center

    Cherrstrom, Catherine A.

    2012-01-01

    Attending a professional conference is an effective way to explore and advance knowledge, skills, and careers. For graduate students, attending a conference is an effective way to explore academic fields and new professions. However, attending a professional conference requires precious resources--time and money--so the decision to attend, or not,…

  1. Evidence that sigma factors are components of chloroplast RNA polymerase.

    PubMed Central

    Troxler, R F; Zhang, F; Hu, J; Bogorad, L

    1994-01-01

    Plastid genes are transcribed by DNA-dependent RNA polymerase(s), which have been incompletely characterized and have been examined in a limited number of species. Plastid genomes contain rpoA, rpoB, rpoC1, and rpoC2 coding for alpha, beta, beta', and beta" RNA polymerase subunits that are homologous to the alpha, beta, and beta' subunits that constitute the core moiety of RNA polymerase in bacteria. However, genes with homology to sigma subunits in bacteria have not been found in plastid genomes. An antibody directed against the principal sigma subunit of RNA polymerase from the cyanobacterium Anabaena sp. PCC 7120 was used to probe western blots of purified chloroplast RNA polymerase from maize, rice, Chlamydomonas reinhardtii, and Cyanidium caldarium. Chloroplast RNA polymerase from maize and rice contained an immunoreactive 64-kD protein. Chloroplast RNA polymerase from C. reinhardtii contained immunoreactive 100- and 82-kD proteins, and chloroplast RNA polymerase from C. caldarium contained an immunoreactive 32-kD protein. The elution profile of enzyme activity of both algal chloroplast RNA polymerases coeluted from DEAE with the respective immunoreactive proteins, indicating that they are components of the enzyme. These results provide immunological evidence for sigma-like factors in chloroplast RNA polymerase in higher plants and algae. PMID:8159791

  2. Identification of mammalian-adapting mutations in the polymerase complex of an avian H5N1 influenza virus.

    PubMed

    Taft, Andrew S; Ozawa, Makoto; Fitch, Adam; Depasse, Jay V; Halfmann, Peter J; Hill-Batorski, Lindsay; Hatta, Masato; Friedrich, Thomas C; Lopes, Tiago J S; Maher, Eileen A; Ghedin, Elodie; Macken, Catherine A; Neumann, Gabriele; Kawaoka, Yoshihiro

    2015-06-17

    Avian influenza viruses of the H5N1 subtype pose a serious global health threat due to the high mortality (>60%) associated with the disease caused by these viruses and the lack of protective antibodies to these viruses in the general population. The factors that enable avian H5N1 influenza viruses to replicate in humans are not completely understood. Here we use a high-throughput screening approach to identify novel mutations in the polymerase genes of an avian H5N1 virus that confer efficient polymerase activity in mammalian cells. Several of the identified mutations (which have previously been found in natural isolates) increase viral replication in mammalian cells and virulence in infected mice compared with the wild-type virus. The identification of amino-acid mutations in avian H5N1 influenza virus polymerase complexes that confer increased replication and virulence in mammals is important for the identification of circulating H5N1 viruses with an increased potential to infect humans.

  3. Identification of mammalian-adapting mutations in the polymerase complex of an avian H5N1 influenza virus

    PubMed Central

    Taft, Andrew S.; Ozawa, Makoto; Fitch, Adam; Depasse, Jay V.; Halfmann, Peter J.; Hill-Batorski, Lindsay; Hatta, Masato; Friedrich, Thomas C.; Lopes, Tiago J. S.; Maher, Eileen A.; Ghedin, Elodie; Macken, Catherine A.; Neumann, Gabriele; Kawaoka, Yoshihiro

    2015-01-01

    Avian influenza viruses of the H5N1 subtype pose a serious global health threat due to the high mortality (>60%) associated with the disease caused by these viruses and the lack of protective antibodies to these viruses in the general population. The factors that enable avian H5N1 influenza viruses to replicate in humans are not completely understood. Here we use a high-throughput screening approach to identify novel mutations in the polymerase genes of an avian H5N1 virus that confer efficient polymerase activity in mammalian cells. Several of the identified mutations (which have previously been found in natural isolates) increase viral replication in mammalian cells and virulence in infected mice compared with the wild-type virus. The identification of amino-acid mutations in avian H5N1 influenza virus polymerase complexes that confer increased replication and virulence in mammals is important for the identification of circulating H5N1 viruses with an increased potential to infect humans. PMID:26082035

  4. Conditions for Using DNA Polymerase I as an RNA-Dependent DNA Polymerase

    PubMed Central

    Gulati, S. C.; Kacian, D. L.; Spiegelman, S.

    1974-01-01

    Conditions are described for using Escherichia coli DNA polymerase I for synthesizing complementary DNA copies of natural RNA molecules, which are suitable for use in hybridization experiments. The molar ratio of enzyme to template is critical; below a certain level, synthesis is not observed. Hybrids formed with the complementary DNA are of comparable specificity and stability to those formed with complementary DNAs synthesized by viral RNA-directed DNA polymerase. Synthesis of dA-dT polymers, a common occurrence with this enzyme, can be eliminated by including distamycin in the reaction mixture. PMID:4133845

  5. Interplay between polymerase II- and polymerase III-assisted expression of overlapping genes.

    PubMed

    Lukoszek, Radoslaw; Mueller-Roeber, Bernd; Ignatova, Zoya

    2013-11-15

    Up to 15% of the genes in different genomes overlap. This architecture, although beneficial for the genome size, represents an obstacle for simultaneous transcription of both genes. Here we analyze the interference between RNA-polymerase II (Pol II) and RNA-polymerase III (Pol III) when transcribing their target genes encoded on opposing strands within the same DNA fragment in Arabidopsis thaliana. The expression of a Pol II-dependent protein-coding gene negatively correlated with the transcription of a Pol III-dependent, tRNA-coding gene set. We suggest that the architecture of the overlapping genes introduces an additional layer of control of gene expression.

  6. Low-Fidelity Polymerases of Alphaviruses Recombine at Higher Rates To Overproduce Defective Interfering Particles

    PubMed Central

    Poirier, Enzo Z.; Mounce, Bryan C.; Rozen-Gagnon, Kathryn; Hooikaas, Peter Jan; Stapleford, Kenneth A.; Moratorio, Gonzalo

    2015-01-01

    ABSTRACT Low-fidelity RNA-dependent RNA polymerases for many RNA virus mutators have been shown to confer attenuated phenotypes, presumably due to increased mutation rates. Additionally, for many RNA viruses, replication to high titers results in the production of defective interfering particles (DIs) that also attenuate infection. We hypothesized that fidelity, recombination, and DI production are tightly linked. We show that a Sindbis virus mutator replicating at a high multiplicity of infection manifests an earlier and greater accumulation of DIs than its wild-type counterpart. The isolated DIs interfere with the replication of full-length virus in a dose-dependent manner. Importantly, the ability of the mutator virus to overproduce DIs could be linked to an increased recombination frequency. These data confirm that RNA-dependent RNA polymerase fidelity and recombination are inversely correlated for this mutator. Our findings suggest that defective interference resulting from higher recombination rates may be more detrimental to RNA virus mutators than the increase in mutational burden. IMPORTANCE Replication, adaptation, and evolution of RNA viruses rely in large part on their low-fidelity RNA-dependent RNA polymerase. Viruses artificially modified in their polymerases to decrease fidelity (mutator viruses) are attenuated in vivo, demonstrating the important role of fidelity in viral fitness. However, attenuation was attributed solely to the modification of the viral mutation rate and the accumulation of detrimental point mutations. In this work, we described an additional phenotype of mutator viruses: an increased recombination rate leading to defective interfering particle (DI) overproduction. Because DIs are known for their inhibitory effect on viral replication, our work suggests that fidelity variants may be attenuated in vivo via several mechanisms. This has important implications in the development of fidelity variants as live attenuated vaccine strains

  7. Transcriptional analysis of the DNA polymerase gene of Bombyx mori parvo-like virus (China isolate).

    PubMed

    Wang, Yong-Jie; Chen, Ke-Ping; Yao, Qin; Han, Xu

    2007-04-01

    The Bombyx mori parvo-like virus (China isolate) DNA polymerase (BmDNV-3 dnapol) gene has been tentatively identified based on the presence of conserved motifs. In the present study, we perform a transcriptional analysis of the BmDNV-3 dnapol gene using the total RNA isolated from BmDNV-3 infected silkworm at different times. Northern blot analysis with a BmDNV-3 dnapol-specific riboprobe showed a major transcript of 3.3 kb. 5'-RACE revealed that the major transcription start point was located 20 nucleotides downstream of the TATA box. In a temporal expression analysis using differential RT-PCR, BmDNV-3 dnapol transcript was detected at low levels at 6 h.p.i., increased from 6 to 36 h.p.i., and remained fairly constant thereafter. Analysis of the predicted DNA polymerase sequence using neighborjoining and protein parsimony algorithms indicated that the predicted 1115-residue polypeptide contained five motifs associated with DNA polymerases synthetic activities and three additional motifs associated with polymerases possessing 3' to 5' exonuclease activity. The molecular phylogenetic analysis of this gene supported the placement of Bombyx mori parvo-like virus in a separate virus family.

  8. Co-evolution of RNA polymerase with RbpA in the phylum Actinobacteria

    PubMed Central

    Dey, Abhinav; Adithi, V.R.; Chatterji, Dipankar

    2012-01-01

    The role of RbpA in the backdrop of M. smegmatis showed that it rescues mycobacterial RNA polymerase from rifampicin-mediated inhibition (Dey et al., 2010; Dey et al., 2011). Paget and co-workers (Paget et al., 2001; Newell et al., 2006) have revealed that RbpA homologs occur exclusively in actinobacteria. Newell et al. (2006) showed that MtbRbpA, when complemented in a ∆rbpA mutant of S. coelicolor, showed a low recovery of MIC (from 0.75 to 2 μg/ml) as compared to complementation by native RbpA of S. coelicolor (MIC increases from 0.75 to 11 μg/ml). Our studies on MsRbpA show that it is a differential marker for M. smegmatis RNA polymerase as compared to E. coli RNA polymerase at IC50 levels of rifampicin. A recent sequence-based analysis by Lane and Darst (2010) has shown that RNA polymerases from Proteobacteria and Actinobacteria have had a divergent evolution. E. coli is a representative of Proteobacteria and M. smegmatis is an Actinobacterium. RbpA has an exclusive occurrence in Actinobacteria. Since protein–protein interactions might not be conserved across different species, therefore, the probable reason for the indifference of MsRbpA toward E. coli RNA polymerase could be the lineage-specific differences between actinobacterial and proteobacterial RNA polymerases. These observations led us to ask the question as to whether the evolution of RbpA in Actinobacteria followed the same route as that of RNA polymerase subunits from actinobacterial species. We show that the exclusivity of RbpA in Actinobacteria and the unique evolution of RNA polymerase in this phylum share a co-evolutionary link. We have addressed this issue by a blending of experimental and bioinformatics based approaches. They comprise of induction of bacterial cultures coupled to rifampicin-tolerance, transcription assays and statistical comparison of phylogenetic trees for different pairs of proteins in actinobacteria. PMID:27896048

  9. Evolution of DNA polymerases: an inactivated polymerase-exonuclease module in Pol ε and a chimeric origin of eukaryotic polymerases from two classes of archaeal ancestors

    PubMed Central

    Tahirov, Tahir H; Makarova, Kira S; Rogozin, Igor B; Pavlov, Youri I; Koonin, Eugene V

    2009-01-01

    Background Evolution of DNA polymerases, the key enzymes of DNA replication and repair, is central to any reconstruction of the history of cellular life. However, the details of the evolutionary relationships between DNA polymerases of archaea and eukaryotes remain unresolved. Results We performed a comparative analysis of archaeal, eukaryotic, and bacterial B-family DNA polymerases, which are the main replicative polymerases in archaea and eukaryotes, combined with an analysis of domain architectures. Surprisingly, we found that eukaryotic Polymerase ε consists of two tandem exonuclease-polymerase modules, the active N-terminal module and a C-terminal module in which both enzymatic domains are inactivated. The two modules are only distantly related to each other, an observation that suggests the possibility that Pol ε evolved as a result of insertion and subsequent inactivation of a distinct polymerase, possibly, of bacterial descent, upstream of the C-terminal Zn-fingers, rather than by tandem duplication. The presence of an inactivated exonuclease-polymerase module in Pol ε parallels a similar inactivation of both enzymatic domains in a distinct family of archaeal B-family polymerases. The results of phylogenetic analysis indicate that eukaryotic B-family polymerases, most likely, originate from two distantly related archaeal B-family polymerases, one form giving rise to Pol ε, and the other one to the common ancestor of Pol α, Pol δ, and Pol ζ. The C-terminal Zn-fingers that are present in all eukaryotic B-family polymerases, unexpectedly, are homologous to the Zn-finger of archaeal D-family DNA polymerases that are otherwise unrelated to the B family. The Zn-finger of Polε shows a markedly greater similarity to the counterpart in archaeal PolD than the Zn-fingers of other eukaryotic B-family polymerases. Conclusion Evolution of eukaryotic DNA polymerases seems to have involved previously unnoticed complex events. We hypothesize that the archaeal

  10. Polymerase chain reaction assay for avian polyomavirus.

    PubMed

    Phalen, D N; Wilson, V G; Graham, D L

    1991-05-01

    A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By using the polymerase chain reaction, BFDV was detected in adult, nestling, and embryo budgerigar (Melopsitticus undulatus) tissue DNAs and in sera from adult and nestling budgerigars. These results suggest the possibility of persistent infections in adult birds and lend further support to previously described evidence of possible in ovo transmission. BFDV was also detected in chicken embryo fibroblast cell cultures and chicken eggs inoculated with the virus. A 550-bp product with identical restriction enzyme sites was amplified from a suspected polyomavirus isolated from a peach-faced lovebird (Agapornis pesonata) and from tissue DNA from a Hahn's macaw (Ara nobilis) and a sun conure (Aratinga solstitialis) with histological lesions suggestive of polyomavirus infection. These fragments also hybridized with a BFDV-derived probe, proving that they were derived from a polyomavirus very similar, if not identical, to BFDV.

  11. Polymerase chain reaction assay for avian polyomavirus.

    PubMed Central

    Phalen, D N; Wilson, V G; Graham, D L

    1991-01-01

    A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By using the polymerase chain reaction, BFDV was detected in adult, nestling, and embryo budgerigar (Melopsitticus undulatus) tissue DNAs and in sera from adult and nestling budgerigars. These results suggest the possibility of persistent infections in adult birds and lend further support to previously described evidence of possible in ovo transmission. BFDV was also detected in chicken embryo fibroblast cell cultures and chicken eggs inoculated with the virus. A 550-bp product with identical restriction enzyme sites was amplified from a suspected polyomavirus isolated from a peach-faced lovebird (Agapornis pesonata) and from tissue DNA from a Hahn's macaw (Ara nobilis) and a sun conure (Aratinga solstitialis) with histological lesions suggestive of polyomavirus infection. These fragments also hybridized with a BFDV-derived probe, proving that they were derived from a polyomavirus very similar, if not identical, to BFDV. Images PMID:1647403

  12. Solving the RNA polymerase I structural puzzle

    SciTech Connect

    Moreno-Morcillo, María; Taylor, Nicholas M. I.; Gruene, Tim; Legrand, Pierre; Rashid, Umar J.; Ruiz, Federico M.; Steuerwald, Ulrich; Müller, Christoph W.; Fernández-Tornero, Carlos

    2014-10-01

    Details of the RNA polymerase I crystal structure determination provide a framework for solution of the structures of other multi-subunit complexes. Simple crystallographic experiments are described to extract relevant biological information such as the location of the enzyme active site. Knowing the structure of multi-subunit complexes is critical to understand basic cellular functions. However, when crystals of these complexes can be obtained they rarely diffract beyond 3 Å resolution, which complicates X-ray structure determination and refinement. The crystal structure of RNA polymerase I, an essential cellular machine that synthesizes the precursor of ribosomal RNA in the nucleolus of eukaryotic cells, has recently been solved. Here, the crucial steps that were undertaken to build the atomic model of this multi-subunit enzyme are reported, emphasizing how simple crystallographic experiments can be used to extract relevant biological information. In particular, this report discusses the combination of poor molecular replacement and experimental phases, the application of multi-crystal averaging and the use of anomalous scatterers as sequence markers to guide tracing and to locate the active site. The methods outlined here will likely serve as a reference for future structural determination of large complexes at low resolution.

  13. Energy Conferences and Symposia; (USA)

    SciTech Connect

    Osborne, J.H.; Simpson, W.F. Jr.

    1991-01-01

    Energy Conferences and Symposia, a monthly publication, was instituted to keep scientists, engineers, managers, and related energy professionals abreast of meetings sponsored by the Department of Energy (DOE) and by other technical associations. Announcements cover conference, symposia, workshops, congresses, and other formal meetings pertaining to DOE programmatic interests. Complete meeting information, including title, sponsor, and contact, is presented in the main section, which is arranged alphabetically by subject area. Within a subject, citations are sorted by beginning data of the meeting. New listings are indicated by a bullet after the conference number and DOE-sponsored conferences are indicated by a star. Two indexes are provided for cross referencing conference information. The Chronological Index lists conference titles by dates and gives the subject area where complete information they may be found. The Location Index is alphabetically sorted by the city where the conference will be held.

  14. Unusual organization of a developmentally regulated mitochondrial RNA polymerase (TBMTRNAP) gene in Trypanosoma brucei

    PubMed Central

    Clement, Sandra L.; Koslowsky, Donna J.

    2009-01-01

    We report here the characterization of a developmentally regulated mitochondrial RNA polymerase transcript in the parasitic protozoan, Trypanosoma brucei. The 3822 bp protein-coding region of the T. brucei mitochondrial RNA polymerase (TBMTRNAP) gene is predicted to encode a 1274 amino acid polypeptide, the carboxyl-terminal domain of which exhibits 29–37% identity with the mitochondrial RNA polymerases from other organisms in the molecular databases. Interestingly, the TBMTRNAP mRNA is one of several mature mRNA species post-transcriptionally processed from a stable, polycistronic precursor. Alternative polyadenylation of the TBMTRNAP mRNA produces two mature transcripts that differ by 500 nt and that show stage-specific differences in abundance during the T. brucei life cycle. This alternative polyadenylation event appears to be accompanied by the alternative splicing of a high abundance, non-coding downstream transcript of unknown function. Our finding that the TBMTRNAP gene is transcribed into two distinct mRNAs subject to differential regulation during the T. brucei life cycle suggests that mitochondrial differentiation might be achieved in part through the regulated expression of this gene. PMID:11470527

  15. Mississippi Climate & Hydrology Conference

    SciTech Connect

    Lawford, R.; Huang, J.

    2002-05-01

    The GEWEX Continental International Project (GCIP), which started in 1995 and completed in 2001, held its grand finale conference in New Orleans, LA in May 2002. Participants at this conference along with the scientists funded through the GCIP program are invited to contribute a paper to a special issue of Journal of Geophysical Research (JGR). This special JGR issue (called GCIP3) will serve as the final report on scientific research conducted by GCIP investigators. Papers are solicited on the following topical areas, but are not limited to, (1) water energy budget studies; (2) warm season precipitation; (3) predictability and prediction system; (4) coupled land-atmosphere models; (5) climate and water resources applications. The research areas cover observations, modeling, process studies and water resources applications.

  16. Metabolic Engineering VII Conference

    SciTech Connect

    Kevin Korpics

    2012-12-04

    The aims of this Metabolic Engineering conference are to provide a forum for academic and industrial researchers in the field; to bring together the different scientific disciplines that contribute to the design, analysis and optimization of metabolic pathways; and to explore the role of Metabolic Engineering in the areas of health and sustainability. Presentations, both written and oral, panel discussions, and workshops will focus on both applications and techniques used for pathway engineering. Various applications including bioenergy, industrial chemicals and materials, drug targets, health, agriculture, and nutrition will be discussed. Workshops focused on technology development for mathematical and experimental techniques important for metabolic engineering applications will be held for more in depth discussion. This 2008 meeting will celebrate our conference tradition of high quality and relevance to both industrial and academic participants, with topics ranging from the frontiers of fundamental science to the practical aspects of metabolic engineering.

  17. The DnaE polymerase from Deinococcus radiodurans features RecA-dependent DNA polymerase activity

    PubMed Central

    Randi, Lorenzo; Perrone, Alessandro; Maturi, Mirko; Dal Piaz, Fabrizio; Camerani, Michela; Hochkoeppler, Alejandro

    2016-01-01

    We report in the present study on the catalytic properties of the Deinococcus radiodurans DNA polymerase III α subunit (αDr). The αDr enzyme was overexpressed in Escherichia coli, both in soluble form and as inclusion bodies. When purified from soluble protein extracts, αDr was found to be tightly associated with E. coli RNA polymerase, from which αDr could not be dissociated. On the contrary, when refolded from inclusion bodies, αDr was devoid of E. coli RNA polymerase and was purified to homogeneity. When assayed with different DNA substrates, αDr featured slower DNA extension rates when compared with the corresponding enzyme from E. coli (E. coli DNA Pol III, αEc), unless under high ionic strength conditions or in the presence of manganese. Further assays were performed using a ssDNA and a dsDNA, whose recombination yields a DNA substrate. Surprisingly, αDr was found to be incapable of recombination-dependent DNA polymerase activity, whereas αEc was competent in this action. However, in the presence of the RecA recombinase, αDr was able to efficiently extend the DNA substrate produced by recombination. Upon comparing the rates of RecA-dependent and RecA-independent DNA polymerase activities, we detected a significant activation of αDr by the recombinase. Conversely, the activity of αEc was found maximal under non-recombination conditions. Overall, our observations indicate a sharp contrast between the catalytic actions of αDr and αEc, with αDr more performing under recombination conditions, and αEc preferring DNA substrates whose extension does not require recombination events. PMID:27789781

  18. SALT Science Conference 2015

    NASA Astrophysics Data System (ADS)

    Buckley, David; Schroeder, Anja

    2015-06-01

    The Southern African Large Telescope (SALT) has seen great changes in the last years following the beginning of full time science operations in 2011. The three first generation instruments, namely the SALTICAM imager, the Robert Stobie Spectrograph (RSS) and its multiple modes and finally in 2014, the new High Resolution Spectrograph (HRS), have commissioned it. The SALT community now eagerly anticipate the installation and commissioning of the near-infrared arm of RSS, likely to commence in 2016. The the third "Science with SALT" conference was held at the Stellenbosch Institute of Advanced Study from 1-5 June 2015. The goals of this conference were to: -Present and discuss recent results from SALT observations; -Anticipate scientific programs that will be carried out with new SALT instrumentation such as RSS-NIR; -Provide a scientific environment in which to foster inter-institutional and inter-facility collaborations between scientists at the different SALT partners; -Provide an opportunity for students and postdocs to become more engaged in SALT science and operations; -Encourage the scientific strategic planning that will be necessary to insure an important role for SALT in an era of large astronomical facilities in the southern hemisphere such as MeerKAT, the SKA, LSST, and ALMA; -Consider options for future instrumentation and technical development of SALT; and, -Present, discuss, and engage in the SALT Collateral Benefits program led by SAAO. Conference proceedings editors: David Buckley and Anja Schroeder

  19. 2004 Mutagenesis Gordon Conference

    SciTech Connect

    Dr. Sue Jinks-Robertson

    2005-09-16

    Mutations are genetic alterations that drive biological evolution and cause many, if not all, human diseases. Mutation originates via two distinct mechanisms: ''vertical'' variation is de novo change of one or few bases, whereas ''horizontal'' variation occurs by genetic recombination, which creates new mosaics of pre-existing sequences. The Mutagenesis Conference has traditionally focused on the generation of mutagenic intermediates during normal DNA synthesis or in response to environmental insults, as well as the diverse repair mechanisms that prevent the fixation of such intermediates as permanent mutations. While the 2004 Conference will continue to focus on the molecular mechanisms of mutagenesis, there will be increased emphasis on the biological consequences of mutations, both in terms of evolutionary processes and in terms of human disease. The meeting will open with two historical accounts of mutation research that recapitulate the intellectual framework of this field and thereby place the current research paradigms into perspective. The two introductory keynote lectures will be followed by sessions on: (1) mutagenic systems, (2) hypermutable sequences, (3) mechanisms of mutation, (4) mutation avoidance systems, (5) mutation in human hereditary and infectious diseases, (6) mutation rates in evolution and genotype-phenotype relationships, (7) ecology, mutagenesis and the modeling of evolution and (8) genetic diversity of the human population and models for human mutagenesis. The Conference will end with a synthesis of the meeting as the keynote closing lecture.

  20. CONFERENCE NOTE: Forthcoming Conference on Frequency Metrology

    NASA Astrophysics Data System (ADS)

    1981-04-01

    The Third Symposium on Frequency Standards and Metrology will be held 5 7 October 1981 at the Centre Paul Langevin, Aussois, Savoie, France. This Conference will follow the lines of its predecessors at Forêt Montmorency, Quebec, Canada in September 1971 and at Copper Mountain, Colorado, USA in July 1976. It is intended to serve as a discussion forum on recent progress and ideas relating to precision frequency standards, the associated metrology and the specific fields of applications. A tentative list of the topics to be covered is the following: Progress in the field of atomic/molecular frequency standards throughout the electromagnetic spectrum Current trends and discussion of the precision capabilities of new techniques (Ramsey fringes in optics, cooling of atoms and ions . . . ) System application (VLBI, navigation . . .) and scientific applications (relativity, geodesy . . .) of atomic/molecular frequency standards and needs in these fields Modern distant time and frequency comparisons Progress in frequency synthesis of microwave to visible frequencies, etc. Most of the talks will be by invitation. Time will be provided for discussion, as well as for presentation of selected late ideas and results. Those interested in the Symposium should communicate with: Dr C Audoin, Laboratoire de l'Horloge Atomique, Bât. 221, Université Paris-Sud, 91405 Orsay Cedex, France.

  1. A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription

    PubMed Central

    1991-01-01

    An antibody was identified previously that recognizes sites of polymerase II transcription on lampbrush chromosomes, puffs on polytene chromosomes, and many small granules in the nucleoplasm of all cells tested. This antibody binds a conserved family of phosphorylated polypeptides in vertebrate and invertebrate cells. We developed a method for purifying these proteins that involves differential solubility in MgCl2. We isolated a Drosophila cDNA encoding one of the proteins using information obtained from microsequencing. In vivo expression studies show that this protein is concentrated on sites of polymerase II transcription and that it is highly phosphorylated. The protein shares a high degree of homology with proteins involved in alternative splicing of pre-mRNA suggesting the possibility that this protein plays a role in pre-mRNA splicing. PMID:1717489

  2. PREFACE: Wake Conference 2015

    NASA Astrophysics Data System (ADS)

    Barney, Andrew; Nørkær Sørensen, Jens; Ivanell, Stefan

    2015-06-01

    The 44 papers in this volume constitute the proceedings of the 2015 Wake Conference, held in Visby on the island of Gotland in Sweden. It is the fourth time this conference has been held. The Wake Conference series started in Visby, where it was held in 2009 and 2011. In 2013 it took place in Copenhagen where it was combined with the International Conference on Offshore Wind Energy and Ocean Energy. In 2015 it is back where it started in Visby, where it takes place at Uppsala University Campus Gotland, June 9th-11th. The global yearly production of electrical energy by wind turbines has grown tremendously in the past decade and it now comprises more than 3% of the global electrical power consumption. Today the wind power industry has a global annual turnover of more than 50 billion USD and an annual average growth rate of more than 20%. State-of-the-art wind turbines have rotor diameters of up to 150 m and 8 MW installed capacity. These turbines are often placed in large wind farms that have a total production capacity corresponding to that of a nuclear power plant. In order to make a substantial impact on one of the most significant challenges of our time, global warming, the industry's growth has to continue for a decade or two yet. This in turn requires research into the physics of wind turbine wakes and wind farms. Modern wind turbines are today clustered in wind farms in which the turbines are fully or partially influenced by the wake of upstream turbines. As a consequence, the wake behind the wind turbines has a lower mean wind speed and an increased turbulence level, as compared to the undisturbed flow outside the farm. Hence, wake interaction results in decreased total production of power, caused by lower kinetic energy in the wind, and an increase in the turbulence intensity. Therefore, understanding the physical nature of the vortices and their dynamics in the wake of a turbine is important for the optimal design of a wind farm. This conference is aimed

  3. Chimeric Bivalent Virus-Like Particle Vaccine for H5N1 HPAI and ND Confers Protection against a Lethal Challenge in Chickens and Allows a Strategy of Differentiating Infected from Vaccinated Animals (DIVA)

    PubMed Central

    Noh, Jin-Yong; Park, Jae-Keun; Lee, Dong-Hun; Yuk, Seong-Su; Kwon, Jung-Hoon; Lee, Sang-Won; Lee, Joong-Bok; Park, Seung-Yong; Choi, In-Soo; Song, Chang-Seon

    2016-01-01

    Highly pathogenic avian influenza (HPAI) and Newcastle disease (ND) are considered as the most devastating poultry infections, owing to their worldwide distribution and economical threat. Vaccines have been widely used to control these diseases in the poultry industry in endemic countries. However, vaccination policy without differentiating infected animals from vaccinated animals (DIVA) makes the virus surveillance difficult. In this study, we developed a bivalent virus-like particle (VLP) vaccine that is composed of the hemagglutinin (HA) and matrix 1 (M1) proteins of the H5N1 HPAI virus (HPAIV) and a chimeric protein containing the ectodomain of the ND virus (NDV) fusion (F) protein fused with the cytoplasmic and transmembrane domains of the HPAIV HA protein. A single immunization of chickens with the chimeric VLP vaccine induced high levels of hemagglutination inhibition (HI) antibody titers against H5N1 HPAI virus and anti-NDV antibody detected in ELISA and protected chickens against subsequent lethal HPAIV and NDV infections. Furthermore, we could easily perform DIVA test using the commercial NP-cELISA tests against HPAIV and HI assay against NDV. These results strongly suggest that utilization of chimeric VLP vaccine in poultry species would be a promising strategy for the better control of HPAI and ND simultaneously. PMID:27626934

  4. Chimeric Bivalent Virus-Like Particle Vaccine for H5N1 HPAI and ND Confers Protection against a Lethal Challenge in Chickens and Allows a Strategy of Differentiating Infected from Vaccinated Animals (DIVA).

    PubMed

    Noh, Jin-Yong; Park, Jae-Keun; Lee, Dong-Hun; Yuk, Seong-Su; Kwon, Jung-Hoon; Lee, Sang-Won; Lee, Joong-Bok; Park, Seung-Yong; Choi, In-Soo; Song, Chang-Seon

    2016-01-01

    Highly pathogenic avian influenza (HPAI) and Newcastle disease (ND) are considered as the most devastating poultry infections, owing to their worldwide distribution and economical threat. Vaccines have been widely used to control these diseases in the poultry industry in endemic countries. However, vaccination policy without differentiating infected animals from vaccinated animals (DIVA) makes the virus surveillance difficult. In this study, we developed a bivalent virus-like particle (VLP) vaccine that is composed of the hemagglutinin (HA) and matrix 1 (M1) proteins of the H5N1 HPAI virus (HPAIV) and a chimeric protein containing the ectodomain of the ND virus (NDV) fusion (F) protein fused with the cytoplasmic and transmembrane domains of the HPAIV HA protein. A single immunization of chickens with the chimeric VLP vaccine induced high levels of hemagglutination inhibition (HI) antibody titers against H5N1 HPAI virus and anti-NDV antibody detected in ELISA and protected chickens against subsequent lethal HPAIV and NDV infections. Furthermore, we could easily perform DIVA test using the commercial NP-cELISA tests against HPAIV and HI assay against NDV. These results strongly suggest that utilization of chimeric VLP vaccine in poultry species would be a promising strategy for the better control of HPAI and ND simultaneously.

  5. Conformational flexibility of bacterial RNA polymerase

    PubMed Central

    Darst, Seth A.; Opalka, Natacha; Chacon, Pablo; Polyakov, Andrey; Richter, Catherine; Zhang, Gongyi; Wriggers, Willy

    2002-01-01

    The structure of Escherichia coli core RNA polymerase (RNAP) was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution of 15 Å. Because of the high sequence conservation between the core RNAP subunits, we were able to interpret the E. coli structure in relation to the high-resolution x-ray structure of Thermus aquaticus core RNAP. A very large conformational change of the T. aquaticus RNAP x-ray structure, corresponding to opening of the main DNA/RNA channel by nearly 25 Å, was required to fit the E. coli map. This finding reveals, at least partially, the range of conformational flexibility of the RNAP, which is likely to have functional implications for the initiation of transcription, where the DNA template must be loaded into the channel. PMID:11904365

  6. Molecular Mechanisms of DNA Polymerase Clamp Loaders

    NASA Astrophysics Data System (ADS)

    Kelch, Brian; Makino, Debora; Simonetta, Kyle; O'Donnell, Mike; Kuriyan, John

    Clamp loaders are ATP-driven multiprotein machines that couple ATP hydrolysis to the opening and closing of a circular protein ring around DNA. This ring-shaped clamp slides along DNA, and interacts with numerous proteins involved in DNA replication, DNA repair and cell cycle control. Recently determined structures of clamp loader complexes from prokaryotic and eukaryotic DNA polymerases have revealed exciting new details of how these complex AAA+ machines perform this essential clamp loading function. This review serves as background to John Kuriyan's lecture at the 2010 Erice School, and is not meant as a comprehensive review of the contributions of the many scientists who have advanced this field. These lecture notes are derived from recent reviews and research papers from our groups.

  7. Dual phase multiplex polymerase chain reaction

    DOEpatents

    Pemov, Alexander; Bavykin, Sergei

    2008-10-07

    Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type--pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional "bridge" amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5.times.10.sup.-12 g (equivalent to 10.sup.2-10.sup.3 genomes) of a bacterial genomic DNA are disclosed.

  8. A Cross-chiral RNA Polymerase Ribozyme

    PubMed Central

    Sczepanski, Jonathan T.; Joyce, Gerald F.

    2014-01-01

    Thirty years ago it was shown that the non-enzymatic, template-directed polymerization of activated mononucleotides proceeds readily in a homochiral system, but is severely inhibited by the presence of the opposing enantiomer.1 This finding poses a severe challenge for the spontaneous emergence of RNA-based life, and has led to the suggestion that either RNA was preceded by some other genetic polymer that is not subject to chiral inhibition2 or chiral symmetry was broken through chemical processes prior to the origin of RNA-based life.3,4 Once an RNA enzyme arose that could catalyze the polymerization of RNA, it would have been possible to distinguish among the two enantiomers, enabling RNA replication and RNA-based evolution to occur. It is commonly thought that the earliest RNA polymerase and its substrates would have been of the same handedness, but this is not necessarily the case. Replicating D-and L-RNA molecules may have emerged together, based on the ability of structured RNAs of one handedness to catalyze the templated polymerization of activated mononucleotides of the opposite handedness. Such a cross-chiral RNA polymerase has now been developed using in vitro evolution. The D-RNA enzyme, consisting of 83 nucleotides, catalyzes the joining of L-mono- or oligonucleotide substrates on a complementary L-RNA template, and similarly for the L-enzyme with D-substrates and a D-template. Chiral inhibition is avoided because the 106-fold rate acceleration of the enzyme only pertains to cross-chiral substrates. The enzyme's activity is sufficient to generate full-length copies of its enantiomer through the templated joining of 11 component oligonucleotides. PMID:25363769

  9. RNA binding and replication by the poliovirus RNA polymerase

    SciTech Connect

    Oberste, M.S.

    1988-01-01

    RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to {sup 32}P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K{sub a} for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 {times} 10{sup 9} M{sup {minus}1}. The polymerase binds to a subgenomic RNAs which contain the 3{prime} end of the genome with a K{sub a} similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3{prime} noncoding region.

  10. Functional oligomerization of poliovirus RNA-dependent RNA polymerase.

    PubMed Central

    Pata, J D; Schultz, S C; Kirkegaard, K

    1995-01-01

    Using a hairpin primer/template RNA derived from sequences present at the 3' end of the poliovirus genome, we investigated the RNA-binding and elongation activities of highly purified poliovirus 3D polymerase. We found that surprisingly high polymerase concentrations were required for efficient template utilization. Binding of template RNAs appeared to be the primary determinant of efficient utilization because binding and elongation activities correlated closely. Using a three-filter binding assay, polymerase binding to RNA was found to be highly cooperative with respect to polymerase concentration. At pH 5.5, where binding was most cooperative, a Hill coefficient of 5 was obtained, indicating that several polymerase molecules interact to retain the 110-nt RNA in a filter-bound complex. Chemical crosslinking with glutaraldehyde demonstrated physical polymerase-polymerase interactions, supporting the cooperative binding data. We propose a model in which poliovirus 3D polymerase functions both as a catalytic polymerase and as a cooperative single-stranded RNA-binding protein during RNA-dependent RNA synthesis. Images FIGURE 1 FIGURE 2 FIGURE 5 FIGURE 6 FIGURE 8 PMID:7489508

  11. Basic mechanism of transcription by RNA polymerase II

    PubMed Central

    Svetlov, Vladimir; Nudler, Evgeny

    2012-01-01

    RNA polymerase II-like enzymes carry out transcription of genomes in Eukaryota, Archaea, and some viruses. They also exhibit fundamental similarity to RNA polymerases from bacteria, chloroplasts, and mitochondria. In this review we take an inventory of recent studiesilluminating different steps of basic transcription mechanism, likely common for most multi-subunit RNA polymerases. Through the amalgamation of structural and computational chemistry data we attempt to highlight the most feasible reaction pathway for the two-metal nucleotidyl transfer mechanism, and to evaluate the way catalysis can be linked to translocation in the mechano-chemical cycle catalyzed by RNA polymerase II. PMID:22982365

  12. High-Throughput Polymerase Fidelity Evolution in Microfluidic Droplets

    NASA Astrophysics Data System (ADS)

    Collins, Jesse; de Paz, Alexandra; Cybulski, Ted; Bhan, Namita; Zhang, Huidan; Weitz, Dave; Tyo, Keith; Kording, Konrad

    Polymerases are technologically important as a tool in molecular biology, and are scientifically important for their role in DNA replication and inheritance. We study large numbers (at least millions) of polymerase mutants by compartmentalizing each gene in a droplet in a microfluidic device. Also in each droplet are in vitro transcription and translation proteins, such that mutant polymerases can be generated to extend their own gene along a known DNA template. Reading the resulting sequence tells us both the mutant gene sequence and the number of and particular errors that the resulting mutant polymerase made during extension. This work is supported by the NIH.

  13. Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases.

    PubMed Central

    Bernad, A; Zaballos, A; Salas, M; Blanco, L

    1987-01-01

    The Bacillus subtilis phage luminal diameter 29 DNA polymerase, involved in protein-primed viral DNA replication, was inhibited by phosphonoacetic acid (PAA), a known inhibitor of alpha-like DNA polymerases, by decreasing the rate of elongation. Three highly conserved regions of amino acid homology, found in several viral alpha-like DNA polymerases and in the luminal diameter 29 DNA polymerase, one of them proposed to be the PAA binding site, were also found in the T4 DNA polymerase. This prokaryotic enzyme was highly sensitive to the drugs aphidicolin and the nucleotide analogues butylanilino dATP (BuAdATP) and butylphenyl dGTP (BuPdGTP), known to be specific inhibitors of eukaryotic alpha-like DNA polymerases. Two potential DNA polymerases from the linear plasmid pGKL1 from yeast and the S1 mitochondrial DNA from maize have been identified, based on the fact that they contain the three conserved regions of amino acid homology. Comparison of DNA polymerases from prokaryotic and eukaryotic origin showed extensive amino acid homology in addition to highly conserved domains. These findings reflect evolutionary relationships between hypothetically unrelated DNA polymerases. Images Fig. 1. Fig. 2. Fig. 4. PMID:3127204

  14. Elongation of primed DNA templates by eukaryotic DNA polymerases.

    PubMed Central

    Ikeda, J E; Longiaru, M; Horwitz, M S; Hurwitz, J

    1980-01-01

    The combined action of DNA polymerase alpha and DNA polymerase beta leads to the synthesis of full-length linear DNA strands with phi X174 DNA templates containing an RNA primer. The reaction can be carried out in two stages. In the first stage, DNA polymerase alpha catalyzes the synthesis of a chain that averaged 230 deoxynucleotides long and was covalently linked to the RNA primer. In the second stage, DNA polymerase beta elongates the DNA strand covalently attached to the RNA primer to full length. With DNA primers, DNA polymerase alpha catalyzes only limited deoxynucleotide addition whereas DNA polymerase beta alone elongates DNA primed templates to full length. DNA polymerase beta can also stimulate the synthesis of adenovirus DNA in vitro in the presence of a cytosol extract from adenovirus-infected cells. In all of these systems, dNMP incorporation catalyzed by DNA polymerase beta was sensitive to N-ethylmaleimide; however, this polymerase activity was resistant to N-ethylmaleimide with poly(rA) x (dT) as the primer template. Images PMID:6160581

  15. Rift Valley Fever Virus Lacking the NSs and NSm Genes Is Highly Attenuated, Confers Protective Immunity from Virulent Virus Challenge, and Allows for Differential Identification of Infected and Vaccinated Animals▿

    PubMed Central

    Bird, Brian H.; Albariño, César G.; Hartman, Amy L.; Erickson, Bobbie Rae; Ksiazek, Thomas G.; Nichol, Stuart T.

    2008-01-01

    Rift Valley fever (RVF) virus is a mosquito-borne human and veterinary pathogen associated with large outbreaks of severe disease throughout Africa and more recently the Arabian peninsula. Infection of livestock can result in sweeping “abortion storms” and high mortality among young animals. Human infection results in self-limiting febrile disease that in ∼1 to 2% of patients progresses to more serious complications including hepatitis, encephalitis, and retinitis or a hemorrhagic syndrome with high fatality. The virus S segment-encoded NSs and the M segment-encoded NSm proteins are important virulence factors. The development of safe, effective vaccines and tools to screen and evaluate antiviral compounds is critical for future control strategies. Here, we report the successful reverse genetics generation of multiple recombinant enhanced green fluorescent protein-tagged RVF viruses containing either the full-length, complete virus genome or precise deletions of the NSs gene alone or the NSs/NSm genes in combination, thus creating attenuating deletions on multiple virus genome segments. These viruses were highly attenuated, with no detectable viremia or clinical illness observed with high challenge dosages (1.0 × 104 PFU) in the rat lethal disease model. A single-dose immunization regimen induced robust anti-RVF virus immunoglobulin G antibodies (titer, ∼1:6,400) by day 26 postvaccination. All vaccinated animals that were subsequently challenged with a high dose of virulent RVF virus survived infection and could be serologically differentiated from naïve, experimentally infected animals by the lack of NSs antibodies. These rationally designed marker RVF vaccine viruses will be useful tools for in vitro screening of therapeutic compounds and will provide a basis for further development of RVF virus marker vaccines for use in endemic regions or following the natural or intentional introduction of the virus into previously unaffected areas. PMID:18199647

  16. Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins.

    PubMed

    Artsimovitch, Irina; Vassylyeva, Marina N; Svetlov, Dmitri; Svetlov, Vladimir; Perederina, Anna; Igarashi, Noriyuki; Matsugaki, Naohiro; Wakatsuki, Soichi; Tahirov, Tahir H; Vassylyev, Dmitry G

    2005-08-12

    Rifamycins, the clinically important antibiotics, target bacterial RNA polymerase (RNAP). A proposed mechanism in which rifamycins sterically block the extension of nascent RNA beyond three nucleotides does not alone explain why certain RNAP mutations confer resistance to some but not other rifamycins. Here we show that unlike rifampicin and rifapentin, and contradictory to the steric model, rifabutin inhibits formation of the first and second phosphodiester bonds. We report 2.5 A resolution structures of rifabutin and rifapentin complexed with the Thermus thermophilus RNAP holoenzyme. The structures reveal functionally important distinct interactions of antibiotics with the initiation sigma factor. Strikingly, both complexes lack the catalytic Mg2+ ion observed in the apo-holoenzyme, whereas an increase in Mg2+ concentration confers resistance to rifamycins. We propose that a rifamycin-induced signal is transmitted over approximately 19 A to the RNAP active site to slow down catalysis. Based on structural predictions, we designed enzyme substitutions that apparently interrupt this allosteric signal.

  17. A role for PCNA2 in translesion synthesis by Arabidopsis thaliana DNA polymerase eta.

    PubMed

    Kunz, Bernard A

    2008-10-01

    Eukaryotic DNA polymerase eta (Poleta) confers ultraviolet (UV) resistance by catalyzing translesion synthesis (TLS) past UV photoproducts. Poleta has been studied extensively in budding yeast and mammalian cells, where its interaction with monoubiquitylated proliferating cell nuclear antigen (PCNA) is necessary for its biological activity. Recently, in collaboration with other investigators, our laboratory demonstrated that Arabidopsis thaliana Poleta is required for UV resistance in plants. Furthermore, the purified enzyme can perform TLS opposite a cyclobutane pyrimidine dimer and interacts with PCNA. Intriguingly, the biological activity of Poleta in a heterologous yeast assay depends on co-expression with Arabidopsis PCNA2 and Poleta sequences implicated in binding PCNA or ubiquitin. We suggest that interaction of Arabidopsis Poleta with ubiquitylated PCNA2 is required for TLS past UV photoproducts by Poleta.

  18. 76 FR 57746 - Conference on the International Conference on Harmonisation Q10 Pharmaceutical Quality System: A...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-16

    ... HUMAN SERVICES Food and Drug Administration Conference on the International Conference on Harmonisation... Systems and Processes for Pharmaceutical Manufacturing; Public Conference AGENCY: Food and Drug Administration, HHS. ACTION: Notice of public conference. The Food and Drug Administration (FDA),...

  19. Control Center Technology Conference Proceedings

    NASA Technical Reports Server (NTRS)

    1991-01-01

    Conference papers and presentations are compiled and cover evolving architectures and technologies applicable to flight control centers. Advances by NASA Centers and the aerospace industry are presented.

  20. SVC 2003 Technical Conference Summary

    SciTech Connect

    Martin, Peter M.

    2003-07-01

    The 46th Annual Technical Conference of the Society of Vacuum Coaters was held in San Francisco May 2-8. All the world events apparently did not affect the attendance or the spirit of the attendees. The Conference was a huge success and very well attended. Many feel that it was the best Techcon yet. This year's Conference really raised the bar for the 47th Annual Technical Conference in Dallas next year. Congratulations go out to the program committee, board of directors, education committee, scholarship committee and Management Plus for a job well done. Excellent accommodations were provided by the San Francisco Marriott.

  1. Rural Energy Conference Project

    SciTech Connect

    Dennis Witmer; Shannon Watson

    2008-12-31

    Alaska remains, even at the beginning of the 21st century, a place with many widely scattered, small, remote communities, well beyond the end of both the road system and the power grid. These communities have the highest energy costs of any place in the United States, despite the best efforts of the utilities that service them. This is due to the widespread dependence on diesel electric generators, which require small capital investments, but recent increases in crude oil prices have resulted in dramatic increases in the cost of power. In the enabling legislation for the Arctic Energy Office in 2001, specific inclusion was made for the study of ways of reducing the cost of electrical power in these remote communities. As part of this mandate, the University of Alaska has, in conjunction with the US Department of Energy, the Denali Commission and the Alaska Energy Authority, organized a series of rural energy conferences, held approximately every 18 months. The goal of these meeting was to bring together rural utility operators, rural community leaders, government agency representatives, equipment suppliers, and researchers from universities and national laboratories to discuss the current state of the art in rural power generation, to discuss current projects, including successes as well as near successes. Many of the conference presenters were from industry and not accustomed to writing technical papers, so the typical method of organizing a conference by requesting abstracts and publishing proceedings was not considered viable. Instead, the organizing committee solicited presentations from appropriate individuals, and requested that (if they were comfortable with computers) prepare Power point presentations that were collected and posted on the web. This has become a repository of many presentations, and may be the best single source of information about current projects in the state of Alaska.

  2. 5 CFR 185.120 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Prehearing conferences. 185.120 Section... FRAUD CIVIL REMEDIES § 185.120 Prehearing conferences. (a) The ALJ may schedule prehearing conferences... conference at a reasonable time in advance of the hearing. (c) The ALJ may use prehearing conferences...

  3. 7 CFR 1753.10 - Preconstruction conference.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 11 2010-01-01 2010-01-01 false Preconstruction conference. 1753.10 Section 1753.10... conference. The borrower shall conduct a conference, attended by the borrower, contractor, and resident... participant with a copy of the conference results. The GFR shall be invited to attend this conference....

  4. 10 CFR 820.22 - Informal conference.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Informal conference. 820.22 Section 820.22 Energy... conference. The Director may convene an informal conference to discuss any situation that might be a... information. The Director may compel a person to attend the conference. This conference will not normally...

  5. 34 CFR 668.87 - Prehearing conference.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 3 2010-07-01 2010-07-01 false Prehearing conference. 668.87 Section 668.87 Education... Proceedings § 668.87 Prehearing conference. (a) A hearing official may convene a prehearing conference if he or she thinks that the conference would be useful, or if the conference is requested by— (1)...

  6. 29 CFR 2570.40 - Conferences.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 9 2010-07-01 2010-07-01 false Conferences. 2570.40 Section 2570.40 Labor Regulations... Transaction Exemption Applications § 2570.40 Conferences. (a) Any conference between the Department and an... conference will be held at the applicant's request. (b) An applicant is entitled to only one conference...

  7. Viral Polymerase-Helicase Complexes Regulate Replication Fidelity To Overcome Intracellular Nucleotide Depletion

    PubMed Central

    Stapleford, Kenneth A.; Rozen-Gagnon, Kathryn; Das, Pratyush Kumar; Saul, Sirle; Poirier, Enzo Z.; Blanc, Hervé; Vidalain, Pierre-Olivier; Merits, Andres

    2015-01-01

    /protease that conferred increased fidelity and yet could not operate in the same manner as high-fidelity polymerases. We show that the alphavirus helicase is a key component of the fidelity-regulating machinery. Our data show that the RNA mutagenic activity of compounds such as ribavirin is coupled to and potentiated by nucleotide depletion and that RNA viruses can fine-tune their replication fidelity when faced with an intracellular environment depleted of nucleotides. PMID:26311883

  8. LEAP 1992: Conference summary

    SciTech Connect

    Dover, C.B.

    1992-12-01

    We present a summary of the many new results in antiproton ([bar p]) physics presented at the LEAP '92 conference, in the areas of meson spectroscopy, [bar N]N scattering, annihilation and spin observables, strangeness and charm production, [bar N] annihilation in nuclei, atomic physics with very low energy [bar p]'s, the exploration of fundamental symmetries and interactions with [bar p] (CP, T, CPT, gravitation), and the prospects for new [bar p] facilities at ultralow energies or energies above the LEAR regime ([ge] 2 GeV/c).

  9. LEAP 1992: Conference summary

    SciTech Connect

    Dover, C.B.

    1992-12-01

    We present a summary of the many new results in antiproton ({bar p}) physics presented at the LEAP `92 conference, in the areas of meson spectroscopy, {bar N}N scattering, annihilation and spin observables, strangeness and charm production, {bar N} annihilation in nuclei, atomic physics with very low energy {bar p}`s, the exploration of fundamental symmetries and interactions with {bar p} (CP, T, CPT, gravitation), and the prospects for new {bar p} facilities at ultralow energies or energies above the LEAR regime ({ge} 2 GeV/c).

  10. Aerospace Environmental Technology Conference

    NASA Technical Reports Server (NTRS)

    Whitaker, A. F. (Editor)

    1995-01-01

    The mandated elimination of CFC's, Halons, TCA, and other ozone depleting chemicals and specific hazardous materials has required changes and new developments in aerospace materials and processes. The aerospace industry has been involved for several years in providing product substitutions, redesigning entire production processes, and developing new materials that minimize or eliminate damage to the environment. These activities emphasize replacement cleaning solvents and their application verifications, compliant coatings including corrosion protection systems, and removal techniques, chemical propulsion effects on the environment, and the initiation of modifications to relevant processing and manufacturing specifications and standards. The Executive Summary of this Conference is published as NASA CP-3297.

  11. Networks Technology Conference

    NASA Technical Reports Server (NTRS)

    Tasaki, Keiji K. (Editor)

    1993-01-01

    The papers included in these proceedings represent the most interesting and current topics being pursued by personnel at GSFC's Networks Division and supporting contractors involved in Space, Ground, and Deep Space Network (DSN) technical work. Although 29 papers are represented in the proceedings, only 12 were presented at the conference because of space and time limitations. The proceedings are organized according to five principal technical areas of interest to the Networks Division: Project Management; Network Operations; Network Control, Scheduling, and Monitoring; Modeling and Simulation; and Telecommunications Engineering.

  12. Three-dimensional mapping of differential amino acids of human, murine, canine and equine TLR4/MD-2 receptor complexes conferring endotoxic activation by lipid A, antagonism by Eritoran and species-dependent activities of Lipid IVA in the mammalian LPS sensor system.

    PubMed

    Scior, Thomas; Lozano-Aponte, Jorge; Figueroa-Vazquez, Vianihuini; Yunes-Rojas, Julian A; Zähringer, Ulrich; Alexander, Christian

    2013-01-01

    A literature review concerning the unexpected species differences of the vertebrate innate immune response to lipid IVA was published in CSBJ prior to the present computational study to address the unpaired activity-sequence correlation of prototypic E. coli -type lipid A and its precursor lipid IVA regarding human, murine, equine and canine species. To this end, their sequences and structures of hitherto known Toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD-2) complexes were aligned and their differential side chain patterns studied. If required due to the lack of the corresponding X-ray crystallographic data, three-dimensional models of TLR4/MD-2/ligand complexes were generated using mono and dimeric crystal structures as templates and in silico docking of the prototypic ligands lipid A, lipid IVA and Eritoran. All differential amino acids were mapped to pinpoint species dependency on an atomic scale, i.e. the possible concert of mechanistically relevant side chains. In its most abstract and general form the three-dimensional (3D-) models devise a triangular interface or "wedge" where molecular interactions between TLR4, MD-2 and ligand itself take place. This study identifies two areas in the wedge related to either agonism or antagonism reflecting why ligands like lipid IVA can possess a species dependent dual activity. Lipid IVA represents an imperfect (underacylated and backbone-flipped), low affinity ligand of mammalian TLR4/MD-2 complexes. Its specific but weak antagonistic activity in the human system is in particular due to the loss of phosphate attraction in the wedge-shaped region conferred by nonhomologous residue changes when compared to crystal and modeled structures of the corresponding murine and equine TLR4/MD-2 complexes. The counter-TLR4/MD-2 unit was also taken into account since agonist-mediated dimerization in a defined m-shaped complex composed of two TLR4/MD-2/agonist subunits triggers intracellular signaling during the

  13. National Conference[s] on Career Education: Final Report.

    ERIC Educational Resources Information Center

    Clark, Joseph F.; And Others

    The report describes a series of conferences whose objective was to orient selected educational leaders to the implications of preparing educational personnel with a career education perspective. The first 32 pages of the report discuss project objectives and procedures, and detail participant profiles, pre- and post-conference career education…

  14. Molecular characterization of a positively photoregulated nuclear gene for a chloroplast RNA polymerase sigma factor in Cyanidium caldarium.

    PubMed Central

    Liu, B; Troxler, R F

    1996-01-01

    We have cloned the gene for a putative chloroplast RNA polymerase sigma factor from the unicellular rhodophyte Cyanidium caldarium. This gene contains an open reading frame encoding a protein of 609 amino acids with domains highly homologous to all four conserved regions found in bacterial and cyanobacterial sigma 70-type subunits. When Southern blots of genomic DNA were hybridized to the "rpoD box" oligonucleotide probe, up to six hybridizing hands were observed. Transcripts of the sigma factor gene were undetectable in RNA from dark-grown cells but were abundant in the poly(A)+ fraction of RNA from illuminated cells. The sigma factor gene was expressed in Escherichia coli, and antibodies against the expressed sigma factor fusion protein cross-reacted with a 55-kDa protein in partially purified chloroplast RNA polymerase. Antibodies directed against a cyanobacterial RNA polymerase sigma factor also cross-reacted with a 55-kDa protein in the same enzyme preparation. Immunoprecipitation experiments showed that this enzyme preparation contains proteins with the same molecular weights as the alpha, beta, beta', and beta" subunits of chloroplast RNA polymerase in higher plants. This study identifies a gene for a plastid RNA polymerase sigma factor and indicates that there may be a family of nuclear-encoded sigma factors that recognize promoters in subsets of plastid genes and regulate differential gene expression at the transcriptional level. Images Fig. 1 Fig. 4 Fig. 6 PMID:8622935

  15. A general strategy for expanding polymerase function by droplet microfluidics

    PubMed Central

    Larsen, Andrew C.; Dunn, Matthew R.; Hatch, Andrew; Sau, Sujay P.; Youngbull, Cody; Chaput, John C.

    2016-01-01

    Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson–Crick base pairing. PMID:27044725

  16. A general strategy for expanding polymerase function by droplet microfluidics.

    PubMed

    Larsen, Andrew C; Dunn, Matthew R; Hatch, Andrew; Sau, Sujay P; Youngbull, Cody; Chaput, John C

    2016-04-05

    Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson-Crick base pairing.

  17. RNA polymerase backtracking in gene regulation and genome instability.

    PubMed

    Nudler, Evgeny

    2012-06-22

    RNA polymerase is a ratchet machine that oscillates between productive and backtracked states at numerous DNA positions. Since its first description 15 years ago, backtracking--the reversible sliding of RNA polymerase along DNA and RNA--has been implicated in many critical processes in bacteria and eukaryotes, including the control of transcription elongation, pausing, termination, fidelity, and genome instability.

  18. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  19. Problem-Solving Test: Real-Time Polymerase Chain Reaction

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Terms to be familiar with before you start to solve the test: polymerase chain reaction, DNA amplification, electrophoresis, breast cancer, "HER2" gene, genomic DNA, "in vitro" DNA synthesis, template, primer, Taq polymerase, 5[prime][right arrow]3[prime] elongation activity, 5[prime][right arrow]3[prime] exonuclease activity, deoxyribonucleoside…

  20. A Practical Polymerase Chain Reaction Laboratory for Introductory Biology Classes.

    ERIC Educational Resources Information Center

    Bowlus, R. David; Grether, Susan C.

    1996-01-01

    Presents a polymerase chain reaction (PCR) laboratory exercise that can be performed by introductory biology students in 1 45- to 55-minute class period. Includes a general description of the polymerase chain reaction, materials needed, procedure, and details of interest to teachers. (JRH)

  1. 2008 Gordon Research Conference on Catalysis [Conference summary report

    SciTech Connect

    Soled, Stuart L.; Gray, Nancy Ryan

    2009-01-01

    The GRC on Catalysis is one of the most prestigious catalysis conferences as it brings together leading researchers from around the world to discuss their latest, most exciting work in catalysis. The 2008 conference will continue this tradition. The conference will cover a variety of themes including new catalytic materials, theoretical and experimental approaches to improve understanding of kinetics and transport phenomena, and state of the art nanoscale characterization probes to monitor active sites. The conference promotes interactions among established researchers and young scientists. It provides a venue for students to meet, talk to and learn from some of the world leading researchers in the area. It also gives them a platform for displaying their own work during the poster sessions. The informal nature of the meeting, excellent quality of the presentations and posters, and ability to meet many outstanding colleagues makes this an excellent conference.

  2. History of NAMES Conferences

    NASA Astrophysics Data System (ADS)

    Filippov, Lev

    2013-03-01

    -Russian International Centre was demonstrated. By the high standards of the reports presented, as well as by its overall organization, the second Seminar met the standards of an international conference. Reviews of state-of-the-art developments in materials science were given by leading scientists from Moscow and from the Lorraine region. The three days of the seminar were structured into four main themes: Functional Materials Coatings, Films and Surface Engineering Nanomaterials and Nanotechnologies The Environment and three Round Table discussions: Defining practical means of carrying out Franco-Russian collaborations in technology transfer and innovation Materials science ARCUS: Lorraine-Russian collaboration in materials science and the environment 32 oral and 25 poster presentations within four sections were given by a total of 110 participants. NAMES 2007, the 3rd Franco-Russian Seminar on New Achievements in Materials and Environmental Sciences, took place in Metz, France on 7-9 November 2007. The conference highlights fundamentals and development of the five main themes connected to the Lorraine-Russia ARCUS project with possible extension to other topics. The five main subjects included in the ARCUS project are: Bulk-surface-interface material sciences Nanomaterials and nanotechnologies Environment and natural resources Plasma physics—ITER project Vibrational dynamics The first, second and third NAMES conferences were financially supported by the following organizations: Ambassade de France à Moscou Communauté Urbaine du Grand Nancy Région Lorraine Conseil Général de Meurthe et Moselle Institut National Polytechnique de Lorraine Université de Metz Université Henry Poincaré CNRS ANVAR Federal Agency on Science and Innovations of the Ministry of Education and Science of the Russian Federation Moscow Committee on Science and Technologies Moscow Institute of Steel and Alloys (Technological University) The 4th conference is supported by the Ministry of Foreign Affairs of

  3. Calendar of Conferences

    NASA Astrophysics Data System (ADS)

    1996-08-01

    8 - 18 August 1996 International Summer School on Plasma Physics and Technology La Jolla, CA, USA Contact: Mr V Stefan, Institute for Advanced Physics Studies, PO Box 2964, La Jolla, CA 92038, USA. Tel +1-619-456-5737. 26 - 30 August 1996 Joint Varenna - Lausanne International Workshop on Theory of Fusion Plasmas Villa Monastero, Varenna, Italy Contact: Centro di Cultura Villa Monastero, 1 Piazza Venini, 22050 Varenna (Lecco), Italy. Tel +39-341-831261, Fax +39-341-831281. Application and abstract deadline: 15 June 1996. 2 - 5 September 1996 EU - US Workshop on Transport in Fusion Plasmas Villa Monastero, Varenna, Italy Further information: G Gorini, ISPP, 16 Via Celoria, I-20133 Milano, Italy. Tel +39-2-2392637, Fax +39-2-2392205, E-mail ggorini@mi.infn.it. Administrative contact: Centro di Cultura Villa Monastero, 1 Piazza Venini, 22050 Varenna (Lecco), Italy. Tel +39-341-831261, Fax +39-341-831281. Application and abstract deadline: 15 June 1996. 9 - 13 September 1996 International Conference on Plasma Physics Nagoya, Japan Contact: Conference Secretariat, c/o Prof. Hiromu Momota, National Institute for Fusion Science, Nagoya 464-01, Japan. Tel +81-52-789-4260, Fax +81-52-789-1037, E-mail icpp96@nifs.ac.jp. Abstract deadline: 31 March 1996. 16 - 20 September 1996 19th Symposium on Fusion Technology Lisbon, Portugal Contact: Professor Carlos Varandas, Centro de Fusão Nuclear, 1096 Lisboa Codex, Portugal. Fax +351-1-8417819, E-mail cvarandas@cfn.ist.utl.pt. General information will be available via WWW with URL http://www.cfn.ist.utl.pt. 25 - 29 September 1996 Summer University of Plasma Physics Garching, Germany Contact: Ms Ch Stahlberg, Max-Planck-Institut für PlasmaPhysik, Boltzmannstr 2, D-85748 Garching, Germany. Tel +49-89-3299-2232, Fax +49-89-3299-1001. 11 - 15 November 1996 38th Annual Meeting of the Division of Plasma Physics, APS Denver, CO, USA Contact: Dr Richard Hazeltine, University of Texas

  4. [Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report

    SciTech Connect

    Not Available

    1992-01-01

    This project focuses on the DNA polymerase and accessory proteins of phage T7 for use in DNA sequence analysis. T7 DNA polymerase (gene 5 protein) interacts with accessory proteins for the acquisition of properties such as processivity that are necessary for DNA replication. One goal is to understand these interactions in order to modify the proteins to increase their usefulness with DNA sequence analysis. Using a genetically modified gene 5 protein lacking 3' to 5' exonuclease activity we have found that in the presence of manganese there is no discrimination against dideoxynucleotides, a property that enables novel approaches to DNA sequencing using automated technology. Pyrophosphorolysis can create problems in DNA sequence determination, a problem that can be eliminated by the addition of pyrophosphatase. Crystals of the gene 5 protein/thioredoxin complex have now been obtained and X-ray diffraction analysis will be undertaken once their quality has been improved. Amino acid changes in gene 5 protein have been identified that alter its interaction with thioredoxin. Characterization of these proteins should help determine how thioredoxin confers processivity on polymerization. We have characterized the 17 DNA binding protein, the gene 2.5 protein, and shown that it interacts with gene 5 protein and gene 4 protein. The gene 2.5 protein mediates homologous base pairing and strand uptake. Gene 5.5 protein interacts with E. coli Hl protein and affects gene expression. Biochemical and genetic studies on the T7 56-kDa gene 4 protein, the helicase, are focused on its physical interaction with T7 DNA polymerase and the mechanism by which the hydrolysis of nucleoside triphosphates fuels its unidirectional translocation on DNA.

  5. [Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report

    SciTech Connect

    Not Available

    1992-12-31

    This project focuses on the DNA polymerase and accessory proteins of phage T7 for use in DNA sequence analysis. T7 DNA polymerase (gene 5 protein) interacts with accessory proteins for the acquisition of properties such as processivity that are necessary for DNA replication. One goal is to understand these interactions in order to modify the proteins to increase their usefulness with DNA sequence analysis. Using a genetically modified gene 5 protein lacking 3` to 5` exonuclease activity we have found that in the presence of manganese there is no discrimination against dideoxynucleotides, a property that enables novel approaches to DNA sequencing using automated technology. Pyrophosphorolysis can create problems in DNA sequence determination, a problem that can be eliminated by the addition of pyrophosphatase. Crystals of the gene 5 protein/thioredoxin complex have now been obtained and X-ray diffraction analysis will be undertaken once their quality has been improved. Amino acid changes in gene 5 protein have been identified that alter its interaction with thioredoxin. Characterization of these proteins should help determine how thioredoxin confers processivity on polymerization. We have characterized the 17 DNA binding protein, the gene 2.5 protein, and shown that it interacts with gene 5 protein and gene 4 protein. The gene 2.5 protein mediates homologous base pairing and strand uptake. Gene 5.5 protein interacts with E. coli Hl protein and affects gene expression. Biochemical and genetic studies on the T7 56-kDa gene 4 protein, the helicase, are focused on its physical interaction with T7 DNA polymerase and the mechanism by which the hydrolysis of nucleoside triphosphates fuels its unidirectional translocation on DNA.

  6. Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

    PubMed

    Serebryannyy, Leonid A; Parilla, Megan; Annibale, Paolo; Cruz, Christina M; Laster, Kyle; Gratton, Enrico; Kudryashov, Dmitri; Kosak, Steven T; Gottardi, Cara J; de Lanerolle, Primal

    2016-09-15

    Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.

  7. DNA polymerase as a molecular motor and pump.

    PubMed

    Sengupta, Samudra; Spiering, Michelle M; Dey, Krishna K; Duan, Wentao; Patra, Debabrata; Butler, Peter J; Astumian, R Dean; Benkovic, Stephen J; Sen, Ayusman

    2014-03-25

    DNA polymerase is responsible for synthesizing DNA, a key component in the running of biological machinery. Using fluorescence correlation spectroscopy, we demonstrate that the diffusive movement of a molecular complex of DNA template and DNA polymerase enhances during nucleotide incorporation into the growing DNA template. The diffusion coefficient of the complex also shows a strong dependence on its inorganic cofactor, Mg2+ ions. When exposed to gradients of either nucleotide or cofactor concentrations, an ensemble of DNA polymerase complex molecules shows collective movement toward regions of higher concentrations. By immobilizing the molecular complex on a patterned gold surface, we demonstrate the fabrication of DNA polymerase-powered fluid pumps. These miniature pumps are capable of transporting fluid and tracer particles in a directional manner with the pumping speed increasing in the presence of the cofactor. The role of DNA polymerase as a micropump opens up avenues for designing miniature fluid pumps using enzymes as engines.

  8. Role of polymerase η in mitochondrial mutagenesis of Saccharomyces cerevisiae

    SciTech Connect

    Chatterjee, Nimrat; Pabla, Ritu; Siede, Wolfram

    2013-02-08

    Highlights: ► DNA polymerase η is detectable in mitochondria of budding yeast. ► Pol η reduces UV-induced mitochondrial base pair substitutions and frameshifts. ► For UV-induced base pair substitutions, Pol η and Pol ζ interact epistatically. -- Abstract: DNA polymerase η mostly catalyzes an error-free bypass of the most frequent UV lesions, pyrimidine dimers of the cyclobutane-type. In addition to its nuclear localization, we show here for the first time its mitochondrial localization in budding yeast. In mitochondria, this polymerase improves bypass replication fidelity opposite UV damage as shown in base pair substitution and frameshift assays. For base pair substitutions, polymerase η appears to be related in function and epistatic to DNA polymerase ζ which, however, plays the opposite role in the nucleus.

  9. 38 CFR 39.33 - Conferences.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... THE ESTABLISHMENT, EXPANSION, AND IMPROVEMENT, OR OPERATION AND MAINTENANCE, OF VETERANS CEMETERIES Establishment, Expansion, and Improvement Projects Grant Requirements and Procedures § 39.33 Conferences. (a) Predesign conference. A predesign conference is required for all Establishment, Expansion, and...

  10. 38 CFR 39.33 - Conferences.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... THE ESTABLISHMENT, EXPANSION, AND IMPROVEMENT, OR OPERATION AND MAINTENANCE, OF VETERANS CEMETERIES Establishment, Expansion, and Improvement Projects Grant Requirements and Procedures § 39.33 Conferences. (a) Predesign conference. A predesign conference is required for all Establishment, Expansion, and...

  11. 38 CFR 39.33 - Conferences.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... STATES FOR ESTABLISHMENT, EXPANSION, AND IMPROVEMENT, OR OPERATION AND MAINTENANCE, OF VETERANS CEMETERIES Establishment, Expansion, and Improvement Projects Grant Requirements and Procedures § 39.33 Conferences. (a) Predesign conference. A predesign conference is required for all Establishment,...

  12. 38 CFR 39.33 - Conferences.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... THE ESTABLISHMENT, EXPANSION, AND IMPROVEMENT, OR OPERATION AND MAINTENANCE, OF VETERANS CEMETERIES Establishment, Expansion, and Improvement Projects Grant Requirements and Procedures § 39.33 Conferences. (a) Predesign conference. A predesign conference is required for all Establishment, Expansion, and...

  13. The polymerase slips and PIPO exists

    PubMed Central

    White, K Andrew

    2015-01-01

    Plant viruses that contain plus-sensed single-stranded RNA genomes are highly abundant in nature. As the equivalents of large mRNAs, these viral genomes utilize a wide variety of gene expression strategies for the production of their encoded proteins. The potyviruses, which are among the most agriculturally important members in this category, contain a single large open reading frame (ORF) coding for a polyprotein that is processed into functional units. For many years, the products derived from the full-length polyprotein were thought to be the only functional viral proteins. However, this notion was dispelled when an additional essential viral ORF, PIPO, was discovered encoded in an alternative reading frame. Since then, the PIPO protein—P3N-PIPO, which mediates virus movement in plants—has been intensively studied, but its mode of expression remained elusive until now. Two articles, one in this issue of EMBO Reports, now report that slippage of the viral polymerase during viral genome replication is responsible for shifting PIPO into a translated reading frame, thereby allowing for production of P3N-PIPO 1,2. This mechanism of gene expression represents a novel strategy for plant viruses. PMID:26160653

  14. Conserved Endonuclease Function of Hantavirus L Polymerase

    PubMed Central

    Rothenberger, Sylvia; Torriani, Giulia; Johansson, Maria U.; Kunz, Stefan; Engler, Olivier

    2016-01-01

    Hantaviruses are important emerging pathogens belonging to the Bunyaviridae family. Like other segmented negative strand RNA viruses, the RNA-dependent RNA polymerase (RdRp) also known as L protein of hantaviruses lacks an intrinsic “capping activity”. Hantaviruses therefore employ a “cap snatching” strategy acquiring short 5′ RNA sequences bearing 5′cap structures by endonucleolytic cleavage from host cell transcripts. The viral endonuclease activity implicated in cap snatching of hantaviruses has been mapped to the N-terminal domain of the L protein. Using a combination of molecular modeling and structure–function analysis we confirm and extend these findings providing evidence for high conservation of the L endonuclease between Old and New World hantaviruses. Recombinant hantavirus L endonuclease showed catalytic activity and a defined cation preference shared by other viral endonucleases. Based on the previously reported remarkably high activity of hantavirus L endonuclease, we established a cell-based assay for the hantavirus endonuclase function. The robustness of the assay and its high-throughput compatible format makes it suitable for small molecule drug screens to identify novel inhibitors of hantavirus endonuclease. Based on the high degree of similarity to RdRp endonucleases, some candidate inhibitors may be broadly active against hantaviruses and other emerging human pathogenic Bunyaviruses. PMID:27144576

  15. Transcriptional proofreading in dense RNA polymerase traffic

    NASA Astrophysics Data System (ADS)

    Sahoo, Mamata; Klumpp, Stefan

    2011-12-01

    The correction of errors during transcription involves the diffusive backward translocation (backtracking) of RNA polymerases (RNAPs) on the DNA. A trailing RNAP on the same template can interfere with backtracking as it progressively restricts the space that is available for backward translocation and thereby ratchets the backtracked RNAP forward. We analyze the resulting negative impact on proofreading theoretically using a driven lattice gas model of transcription under conditions of dense RNAP traffic. The fraction of errors that are corrected is calculated exactly for the case of a single RNAP; for multi-RNAP transcription, we use simulations and an analytical approximation and find a decrease with increasing traffic density. Moreover, we ask how the parameters of the system have to be set to keep down the impact of the interference of a trailing RNAP. Our analysis uncovers a surprisingly simple picture of the design of the error correction system: its efficiency is essentially determined by the rate for the initial backtracking step, while the value of the cleavage rate ensures that the correction mechanism remains efficient at high transcription rates. Finally, we argue that our analysis can also be applied to cases with transcription-translation coupling where the leading ribosome on the transcript assumes the role of the trailing RNAP.

  16. Solving the RNA polymerase I structural puzzle

    PubMed Central

    Moreno-Morcillo, María; Taylor, Nicholas M. I.; Gruene, Tim; Legrand, Pierre; Rashid, Umar J.; Ruiz, Federico M.; Steuerwald, Ulrich; Müller, Christoph W.; Fernández-Tornero, Carlos

    2014-01-01

    Knowing the structure of multi-subunit complexes is critical to understand basic cellular functions. However, when crystals of these complexes can be obtained they rarely diffract beyond 3 Å resolution, which complicates X-ray structure determination and refinement. The crystal structure of RNA polymerase I, an essential cellular machine that synthesizes the precursor of ribosomal RNA in the nucleolus of eukaryotic cells, has recently been solved. Here, the crucial steps that were undertaken to build the atomic model of this multi-subunit enzyme are reported, emphasizing how simple crystallographic experiments can be used to extract relevant biological information. In particular, this report discusses the combination of poor molecular replacement and experimental phases, the application of multi-crystal averaging and the use of anomalous scatterers as sequence markers to guide tracing and to locate the active site. The methods outlined here will likely serve as a reference for future structural determination of large complexes at low resolution. PMID:25286842

  17. Integrated polymerase chain reaction/electrophoresis instrument

    DOEpatents

    Andresen, Brian D.

    2000-01-01

    A new approach and instrument for field identification of micro-organisms and DNA fragments using a small and disposable device containing integrated polymerase chain reaction (PCR) enzymatic reaction wells, attached capillary electrophoresis (CE) channels, detectors, and read-out all on/in a small hand-held package. The analysis instrument may be made inexpensively, for example, of plastic, and thus is disposable, which minimizes cross contamination and the potential for false positive identification between samples. In addition, it is designed for multiple users with individual applications. The integrated PCR/CE is manufactured by the PCR well and CE channels are "stamped" into plastic depressions where conductive coatings are made in the wells and ends of the CE microchannels to carry voltage and current to heat the PCR reaction mixtures and simultaneously draw DNA bands up the CE channels. Light is transmitted through the instrument at appropriate points and detects PCR bands and identifies DNA fragments by size (retention time) and quantifies each by the amount of light generated as each phototransistor positioned below each CE channel detects a passing band. The instrument is so compact that at least 100 PCR/CE reactions/analyses can be performed easily on one detection device.

  18. Actinobaculum suis Detection Using Polymerase Chain Reaction

    PubMed Central

    Amigo, Cristina Román; de Gobbi, Debora Dirani Sena; Gomes, Vasco Túlio de Moura; Perina, Danilo do Prado; Nogueira de Lima Filsner, Pedro Henrique; Costa, Barbara Letícia Pereira; Spindola, Maria Garcia; Ferreira, Thais Sebastiana Porfida; Brandão, Paulo Eduardo; Moreno, Andrea Micke

    2012-01-01

    Actinobaculum suis is an important agent related to urinary infection in swine females. Due to its fastidious growth characteristics, the isolation of this anaerobic bacterium is difficult, thus impairing the estimation of its prevalence. The purpose of this study was to develop and test a polymerase chain reaction (PCR) for the detection and identification of A. suis and then compare these results with traditional isolation methods. Bacterial isolation and PCR were performed on one hundred and ninety-two urine samples from sows and forty-five preputial swabs from boars. The results indicate that this PCR was specific for A. suis, presenting a detection limit between 1.0 × 101 CFU/mL and 1.0 × 102 CFU/mL. A. suis frequencies, as measured by PCR, were 8.9% (17/192) in sow urine samples and 82.2% (37/45) in preputial swabs. Assessed using conventional culturing techniques, none of the urine samples were positive for A. suis; however, A. suis was detected in 31.1% (14/45) of the swabs. This PCR technique was shown to be an efficient method for the detection of A. suis in urine and preputial swabs. PMID:23346017

  19. Adenovirus DNA polymerase is a phosphoprotein.

    PubMed

    Ramachandra, M; Nakano, R; Mohan, P M; Rawitch, A B; Padmanabhan, R

    1993-01-05

    Biological activities of many of the eukaryotic DNA replication proteins are modulated by protein phosphorylation. Investigations of the phosphorylation of adenovirus DNA polymerase (AdPol) have been difficult mainly because of its low level of synthesis in adenovirus-infected HeLa cells. However, when AdPol was overproduced using the recombinant vaccinia virus (RV-AdPol) and the baculovirus expression systems, or by a large scale metabolic labeling of adenovirus 2-infected HeLa cells (native AdPol), in vivo phosphorylation of AdPol could be demonstrated. Phosphoamino acid analysis of [32P]AdPol indicated the presence of phosphoserine independent of the source of AdPol. Comparison of tryptic peptide maps of native AdPol and RV-AdPol revealed that the majority of phosphopeptides were common. Fractionation by high performance liquid chromatography and sequencing of one of the major phosphopeptides revealed serine 67 as a site of phosphorylation. Interestingly, this site is located close to the nuclear localization signal of AdPol and has a consensus substrate recognition sequence for histone H1 (cdc2-related) kinases and mitogen-activated protein kinases. Dephosphorylation of AdPol with calf intestinal alkaline phosphatase resulted in significant decrease in its activity in the in vitro DNA replication initiation assay, suggesting that phosphorylation is important for its biological activity.

  20. Conference Report: Improving College Teaching.

    ERIC Educational Resources Information Center

    Mayo, C. Douglas; Claxton, Charles S.

    The conference report includes an explanation of the conference plan, a description of the highlights, and descriptions of each session. Among the session topics are: faculty development; instructional improvement in the community college, with emphasis on developmental education; conditions that enhance learning; improving the lecture; evaluating…

  1. The Second National Conference Summary.

    ERIC Educational Resources Information Center

    Chai, Carolyn

    In this summary of the 1978 National Conference on Asians in America and Asian Americans, conference proceedings, as well as papers and panel discussions, are briefly outlined. Workshops on foreign policy, immigration, Asian identity, education and employment, Indo-Chinese in the United States, teaching English to immigrants, racism and…

  2. Beating the Futures Conference Game.

    ERIC Educational Resources Information Center

    Simmonds, W. H. Clive

    1983-01-01

    Conference survival tips include: (1) noting the different kinds of speakers, their strengths and weaknesses; (2) discovering the reasons for often apparent cross-talk and misunderstanding; and (3) using the concept of futuring to gain ideas as to what can be done after the conference ends. (RM)

  3. SLA at 100: Conference Preview

    ERIC Educational Resources Information Center

    Blumenstein, Lynn

    2009-01-01

    When School Library Association (SLA) convenes its annual conference in Washington, DC, June 14-17, 2009, the association will be celebrating its 100th birthday. This occasion allows for grand gestures--the SLA Salutes! Awards and Leadership Reception will be held in the Library of Congress's Great Hall. The conference also draws upon Washington…

  4. Vague Language in Conference Abstracts

    ERIC Educational Resources Information Center

    Cutting, Joan

    2012-01-01

    This study examined abstracts for a British Association for Applied Linguistics conference and a Sociolinguistics Symposium, to define the genre of conference abstracts in terms of vague language, specifically universal general nouns (e.g. people) and research general nouns (e.g. results), and to discover if the language used reflected the level…

  5. THE PREPLANNING TITLE VII CONFERENCE.

    ERIC Educational Resources Information Center

    KIEFFER, ROBERT DE

    THIS REPORT IS A TRANSCRIPT OF PROCEEDINGS OF A PLANNING CONFERENCE HELD AT BOULDER, COLORADO IN SEPTEMBER 1958 FOR THE PURPOSE OF DESIGNING A LARGER CONFERENCE IN APRIL 1960. SPECIFICALLY THIS WOULD DEAL WITH MORE EFFECTIVE UTILIZATION OF TELEVISION, RADIO, MOTION PICTURES AND RELATED MEDIA FOR EDUCATIONAL PURPOSES IN THE MOUNTAIN PLAINS STATES…

  6. Conference Connections: Rewiring the Circuit

    ERIC Educational Resources Information Center

    Siemens, George; Tittenberger, Peter; Anderson, Terry

    2008-01-01

    Increased openness, two-way dialogue, and blurred distinctions between experts and amateurs have combined with numerous technology tools for dialogue, personal expression, networking, and community formation to "remake" conferences, influencing not only how attendees participate in but also how organizers host conferences today. (Contains 31…

  7. Student-Led Portfolio Conferences.

    ERIC Educational Resources Information Center

    Paulson, F. Leon; Paulson, Pearl R.

    In at least one Oregon school system, student-led conferences have begun to replace traditional report cards. When conferences are well done, parents believe they have learned more about their child's learning and progress than they would through a traditional report card. There is an important additional benefit in that students can rise to the…

  8. Structural Determinant for Switching between the Polymerase and Exonuclease Modes in the PCNA-Replicative DNA Polymerase Complex

    NASA Astrophysics Data System (ADS)

    Nishida, Hirokazu; Mayanagi, Kouta; Ishino, Yoshizumi; Morikawa, Kosuke

    Proliferating cell nuclear antigen (PCNA) is responsible for the processivity of DNA polymerase. We determined the crystal structure of Pyrococcus furiosus DNA polymerase (PfuPol) complexed with a cognate monomeric PCNA, which allowed us to construct a convincing model of the polymerase-PCNA ring interaction. Electron microscopy analyses confirmed that this complex structure exists among the multiple functional configurations in solution. Together with data from mutational analyses, this structural study indicated that the novel interaction between a stretched loop of PCNA and the PfuPol Thumb domain is quite important, in addition to the authentic PCNA-polymerase recognition site (PIP box). A comparison of the present structures with the previously reported structures of polymerases complexed with DNA suggested that the second interaction site plays a crucial role in switching between the polymerase and exonuclease modes, by stabilizing only the polymerase mode. This proposed mechanism of fidelity control of replicative DNA polymerases was supported by experiments, in which a mutation within the second interaction site caused an enhancement in the exonuclease activity in the presence of PCNA.

  9. Structural determinant for switching between the polymerase and exonuclease modes in the PCNA-replicative DNA polymerase complex

    PubMed Central

    Nishida, Hirokazu; Mayanagi, Kouta; Kiyonari, Shinichi; Sato, Yuichi; Oyama, Takuji; Ishino, Yoshizumi; Morikawa, Kosuke

    2009-01-01

    Proliferating cell nuclear antigen (PCNA) is responsible for the processivity of DNA polymerase. We determined the crystal structure of Pyrococcus furiosus DNA polymerase (PfuPol) complexed with the cognate monomeric PCNA, which allowed us to construct a convincing model of the polymerase-PCNA ring interaction, with unprecedented configurations of the two molecules. Electron microscopic analyses indicated that this complex structure exists in solution. Our structural study revealed that an interaction occurs between a stretched loop of PCNA and the PfuPol Thumb domain, in addition to the authentic PCNA-polymerase recognition site (PIP box). Comparisons of the present structure with the previously reported structures of polymerases complexed with DNA, suggested that the second interaction plays a crucial role in switching between the polymerase and exonuclease modes, by inducing a PCNA-polymerase complex configuration that favors synthesis over editing. This putative mechanism for fidelity control of replicative DNA polymerases is supported by experiments, in which mutations at the second interaction site caused enhancements in the exonuclease activity in the presence of PCNA. PMID:19934045

  10. Summary of 1990 Code Conference

    SciTech Connect

    Cooper, R.K.; Chan, Kwok-Chi D.

    1990-01-01

    The Conference on Codes and the Linear Accelerator Community was held in Los Alamos in January 1990, and had approximately 100 participants. This conference was the second in a series which has as its goal the exchange of information about codes and code practices among those writing and actually using these codes for the design and analysis of linear accelerators and their components. The first conference was held in San Diego in January 1988, and concentrated on beam dynamics codes and Maxwell solvers. This most recent conference concentrated on 3-D codes and techniques to handle the large amounts of data required for three-dimensional problems. In addition to descriptions of codes, their algorithms and implementations, there were a number of paper describing the use of many of the codes. Proceedings of both these conferences are available. 3 refs., 2 tabs.

  11. Testing promoter activity in the trypanosome genome: isolation of a metacyclic-type VSG promoter, and unexpected insights into RNA polymerase II transcription.

    PubMed

    McAndrew, M; Graham, S; Hartmann, C; Clayton, C

    1998-09-01

    In trypanosomes, most genes are arranged in polycistronic transcription units. Individual mRNAs are generated by 5'-trans splicing and 3' polyadenylation. Remarkably, no regulation of RNA polymerase II transcription has been detected although many RNAs are differentially expressed during kinetoplastid life cycles. Demonstration of specific class II promoters is complicated by the difficulty in distinguishing between genuine promoter activity and stimulation of trans splicing. Using vectors that were designed to allow the detection of low promoter activities in a transcriptionally silent chromosomal context, we isolated a novel trypanosome RNA polymerase I promoter. We were however unable to detect class II promoter activity in any tested DNA fragment. We also integrated genes which were preceded by a T3 promoter into the genome of cells expressing bacteriophage T3 polymerase: surprisingly, transcription was alpha-amanitin sensitive. One possible interpretation of these results is that in trypanosomes, RNA polymerase II initiation is favored by genomic accessibility and double-strand melting.

  12. Conserved Overlapping Gene Arrangement, Restricted Expression, and Biochemical Activities of DNA Polymerase ν (POLN)*

    PubMed Central

    Takata, Kei-ichi; Tomida, Junya; Reh, Shelley; Swanhart, Lisa M.; Takata, Minoru; Hukriede, Neil A.; Wood, Richard D.

    2015-01-01

    DNA polymerase ν (POLN) is one of 16 DNA polymerases encoded in vertebrate genomes. It is important to determine its gene expression patterns, biological roles, and biochemical activities. By quantitative analysis of mRNA expression, we found that POLN from the zebrafish Danio rerio is expressed predominantly in testis. POLN is not detectably expressed in zebrafish embryos or in mouse embryonic stem cells. Consistent with this, injection of POLN-specific morpholino antisense oligonucleotides did not interfere with zebrafish embryonic development. Analysis of transcripts revealed that vertebrate POLN has an unusual gene expression arrangement, sharing a first exon with HAUS3, the gene encoding augmin-like complex subunit 3. HAUS3 is broadly expressed in embryonic and adult tissues, in contrast to POLN. Differential expression of POLN and HAUS3 appears to arise by alternate splicing of transcripts in mammalian cells and zebrafish. When POLN was ectopically overexpressed in human cells, it specifically coimmunoprecipitated with the homologous recombination factors BRCA1 and FANCJ, but not with previously suggested interaction partners (HELQ and members of the Fanconi anemia core complex). Purified zebrafish POLN protein is capable of thymine glycol bypass and strand displacement, with activity dependent on a basic amino acid residue known to stabilize the primer-template. These properties are conserved with the human enzyme. Although the physiological function of pol ν remains to be clarified, this study uncovers distinctive aspects of its expression control and evolutionarily conserved properties of this DNA polymerase. PMID:26269593

  13. Involvement of DNA polymerase beta overexpression in the malignant transformation induced by benzo[a]pyrene

    PubMed Central

    Zhao, Wei; Wu, Mei; Lai, Yanhao; Deng, Wenwen; Liu, Yuan; Zhang, Zunzhen

    2014-01-01

    Objective To explore the relationship between DNA polymerase β (pol β) overexpression and benzo[a]pyrene (BaP) carcinogenesis. Methods Firstly, mouse embryonic fibroblasts that express wild-type level of DNA polymerase β (pol β cell) and high level of pol β (pol β oe cell) were treated by various concentrations of BaP to determine genetic instability induced by BaP under differential expression levels of pol β. Secondly, malignant transformation of pol β cells by low concentration of BaP (20 μM) was determined by soft agar colony formation assay and transformation focus assay. Thirdly, the mRNA and protein levels of BaP-transformed pol β cells (named pol β-T cells) was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot, and the genetic instability of these cells were examined by HPRT gene mutation assay and random amplified polymorphic DNA (RAPD) assay. Results Pol β cells were successfully transformed into malignant pol β-T cells by an exposure to low concentration of BaP for 6 months. Pol β-T cells exhibited increased levels of pol β gene expression, HPRT gene mutation frequency and polymorphisms of RAPD products that were comparable to those of pol β oe cells. Conclusion Pol β overexpression and its-associated genetic instability may play a key role in BaP carcinogenesis. PMID:23652152

  14. Eastern Pacific Ocean Conference

    NASA Astrophysics Data System (ADS)

    The promotion of interaction among investigators of all oceanographic disciplines studying the eastern Pacific Ocean was the goal of the 1990 Eastern Pacific Ocean Conference (EPOC), held October 17-19 on the snow-covered slopes of Mt. Hood, Oreg. Thirty oceanographers representing all disciplines attended.Dick Barber, Duke University Marine Lab, Beaufort, N.C., chaired a session on the eastern equatorial Pacific Ocean, emphasizing issues related to biological activity. Steve Ramp of the Naval Postgraduate School in Montery, Calif., chaired a session on recent results from northern and central California experiments. On October 19, following an early morning earthquake, a business meeting and discussions regarding a collaboration in future experiments were held.

  15. ISMB Conference Proceedings

    SciTech Connect

    Teresa, Gaasterand; Martin, Vingron

    2011-07-01

    This special issue comprises the papers accepted for presentation at the 19th Annual International Conference on Intelligent Systems for Molecular Biology, joint with the 10th European Conference on Computational Biology, an official conference of the International Society for Computational Biology (ISCB; http://www.iscb.org). ISMB/ECCB 2011 (http://www.iscb.org/ismb2011/) will take place in Vienna, Austria, from July 17 through July 19, 2011; preceded during July 14–16 by eight 1- or 2- day Special Interest Group (SIG) meetings, three satellite meetings and nine half-day tutorials; and followed by two additional satellite meetings. The 48 papers in this volume were selected from 258 submitted papers. Submitted papers were assigned to 13 areas. Area Chairs led each topic area by selecting their area's program committee and overseeing the reviewing process. Many Area Chairs were new compared to 2010, and two completely new areas were added in 2011, ‘Data Visualization’ and ‘Mass Spectrometry and Proteomics’. Six papers for which Area Chairs were in conflict were reviewed under a ‘Conflicts Management’ section headed by the Proceedings Chairs; one such paper was accepted in ‘Bioimaging’. Areas, co-chairs and acceptance information are listed in Table 1. Compared to prior years, five mature topic areas had steady submissions, ‘Evolution and Comparative Genomics’, ‘Gene Regulation and Transcriptomics’, ‘Protein Structure and Function’, ‘Sequence Analysis’, ‘Text Mining’. Two areas newer to ISMB were underrepresented this year, ‘Bioimaging’ and ‘Disease Models and Epidemiology’. One area doubled, ‘Applied Bioinformatics’, renamed from last year's ‘Other Bioinformatics Applications’; and one tripled, ‘Protein Interactions and Molecular Networks’. Across the areas, 326 members of the bioinformatics community provided reviews. Most papers received three reviews and several received four or more. There was

  16. Computational Biology Support: RECOMB Conference Series (Conference Support)

    SciTech Connect

    Michael Waterman

    2006-06-15

    This funding was support for student and postdoctoral attendance at the Annual Recomb Conference from 2001 to 2005. The RECOMB Conference series was founded in 1997 to provide a scientific forum for theoretical advances in computational biology and their applications in molecular biology and medicine. The conference series aims at attracting research contributions in all areas of computational molecular biology. Typical, but not exclusive, the topics of interest are: Genomics, Molecular sequence analysis, Recognition of genes and regulatory elements, Molecular evolution, Protein structure, Structural genomics, Gene Expression, Gene Networks, Drug Design, Combinatorial libraries, Computational proteomics, and Structural and functional genomics. The origins of the conference came from the mathematical and computational side of the field, and there remains to be a certain focus on computational advances. However, the effective use of computational techniques to biological innovation is also an important aspect of the conference. The conference had a growing number of attendees, topping 300 in recent years and often exceeding 500. The conference program includes between 30 and 40 contributed papers, that are selected by a international program committee with around 30 experts during a rigorous review process rivaling the editorial procedure for top-rate scientific journals. In previous years papers selection has been made from up to 130--200 submissions from well over a dozen countries. 10-page extended abstracts of the contributed papers are collected in a volume published by ACM Press and Springer, and are available at the conference. Full versions of a selection of the papers are published annually in a special issue of the Journal of Computational Biology devoted to the RECOMB Conference. A further point in the program is a lively poster session. From 120-300 posters have been presented each year at RECOMB 2000. One of the highlights of each RECOMB conference is a

  17. Conference Report: Power and Energy Society Annual Conference

    NASA Astrophysics Data System (ADS)

    Yorino, Naoto; Mori, Hiroyuki

    The 19th Power & Energy Society Annual Conference was held on September 24-26, 2008 at Hiroshima University. The total number of technical papers was 415 and 53 sessions (52 oral sessions and 1 poster session) were organized. A panel discussion, a special lecture, technical exhibitions and technical tours were also organized. All events were very well attended and the final enrollment attained to 954 registrations. The conference has been successfully closed by the great contribution of all participants. In this article, the outline of the conference is reported.

  18. Theoretical analysis of transcription process with polymerase stalling

    NASA Astrophysics Data System (ADS)

    Li, Jingwei; Zhang, Yunxin

    2015-05-01

    Experimental evidence shows that in gene transcription RNA polymerase has the possibility to be stalled at a certain position of the transcription template. This may be due to the template damage or protein barriers. Once stalled, polymerase may backtrack along the template to the previous nucleotide to wait for the repair of the damaged site, simply bypass the barrier or damaged site and consequently synthesize an incorrect messenger RNA, or degrade and detach from the template. Thus, the effective transcription rate (the rate to synthesize correct product mRNA) and the transcription effectiveness (the ratio of the effective transcription rate to the effective transcription initiation rate) are both influenced by polymerase stalling events. So far, no theoretical model has been given to discuss the gene transcription process including polymerase stalling. In this study, based on the totally asymmetric simple exclusion process, the transcription process including polymerase stalling is analyzed theoretically. The dependence of the effective transcription rate, effective transcription initiation rate, and transcription effectiveness on the transcription initiation rate, termination rate, as well as the backtracking rate, bypass rate, and detachment (degradation) rate when stalling, are discussed in detail. The results showed that backtracking restart after polymerase stalling is an ideal mechanism to increase both the effective transcription rate and the transcription effectiveness. Without backtracking, detachment of stalled polymerase can also help to increase the effective transcription rate and transcription effectiveness. Generally, the increase of the bypass rate of the stalled polymerase will lead to the decrease of the effective transcription rate and transcription effectiveness. However, when both detachment rate and backtracking rate of the stalled polymerase vanish, the effective transcription rate may also be increased by the bypass mechanism.

  19. Theoretical analysis of transcription process with polymerase stalling.

    PubMed

    Li, Jingwei; Zhang, Yunxin

    2015-05-01

    Experimental evidence shows that in gene transcription RNA polymerase has the possibility to be stalled at a certain position of the transcription template. This may be due to the template damage or protein barriers. Once stalled, polymerase may backtrack along the template to the previous nucleotide to wait for the repair of the damaged site, simply bypass the barrier or damaged site and consequently synthesize an incorrect messenger RNA, or degrade and detach from the template. Thus, the effective transcription rate (the rate to synthesize correct product mRNA) and the transcription effectiveness (the ratio of the effective transcription rate to the effective transcription initiation rate) are both influenced by polymerase stalling events. So far, no theoretical model has been given to discuss the gene transcription process including polymerase stalling. In this study, based on the totally asymmetric simple exclusion process, the transcription process including polymerase stalling is analyzed theoretically. The dependence of the effective transcription rate, effective transcription initiation rate, and transcription effectiveness on the transcription initiation rate, termination rate, as well as the backtracking rate, bypass rate, and detachment (degradation) rate when stalling, are discussed in detail. The results showed that backtracking restart after polymerase stalling is an ideal mechanism to increase both the effective transcription rate and the transcription effectiveness. Without backtracking, detachment of stalled polymerase can also help to increase the effective transcription rate and transcription effectiveness. Generally, the increase of the bypass rate of the stalled polymerase will lead to the decrease of the effective transcription rate and transcription effectiveness. However, when both detachment rate and backtracking rate of the stalled polymerase vanish, the effective transcription rate may also be increased by the bypass mechanism.

  20. Novel triterpenoids inhibit both DNA polymerase and DNA topoisomerase.

    PubMed Central

    Mizushina, Y; Iida, A; Ohta, K; Sugawara, F; Sakaguchi, K

    2000-01-01

    As described previously, we found that new triterpenoid compounds, designated fomitellic acids A and B, which selectively inhibit the activities of mammalian DNA polymerases alpha and beta [Mizushina, Tanaka, Kitamura, Tamai, Ikeda, Takemura, Sugawara, Arai, Matsukage, Yoshida and Sakaguchi (1998) Biochem. J. 330, 1325-1332; Tanaka, Kitamura, Mizushina, Sugawara and Sakaguchi (1998) J. Nat. Prod. 61, 193-197] and that a known triterpenoid, ursolic acid, is an inhibitor of human DNA topoisomerases I and II (A. Iida, Y. Mizushina and K. Sakaguchi, unpublished work). Here we report that all of these triterpenoids are potent inhibitors of calf DNA polymerase alpha, rat DNA polymerase beta and human DNA topoisomerases I and II, and show moderate inhibitory effects on plant DNA polymerase II and human immunodeficiency virus reverse transcriptase. However, these compounds did not influence the activities of prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I or other DNA metabolic enzymes such as human telomerase, T7 RNA polymerase and bovine deoxyribonuclease I. These triterpenoids were not only mammalian DNA polymerase inhibitors but also inhibitors of DNA topoisomerases I and II even though the enzymic characteristics of DNA polymerases and DNA topoisomerases, including their modes of action, amino acid sequences and three-dimensional structures, differed markedly. These triterpenoids did not bind to DNA, suggesting that they act directly on these enzymes. Because the three-dimensional structures of fomitellic acids were shown by computer simulation to be very similar to that of ursolic acid, the DNA-binding sites of both enzymes, which compete for the inhibitors, might be very similar. Fomitellic acid A and ursolic acid prevented the growth of NUGC cancer cells, with LD(50) values of 38 and 30 microM respectively. PMID:10970789

  1. Differential gear

    SciTech Connect

    Shibuya, K.; Hamada, T.; Masuda, K.; Shimada, K.

    1989-05-02

    A differential gear for permitting a difference in rotational speed between two output shafts is described, the differential gear including an input shaft and two output shafts. The improvement consists of means for limiting the difference in rotational speed between the two output shafts in response to the rotational speed of the input shaft, the rotational speed limiting means comprising a differential casing coupled to the input shaft and adapted to be rotated by the input shaft, a differential pinion shaft radially extending within the differential casing and rotatably mounted at its opposite ends in the differential casing. A plurality of differential pinion gears rotatably mounted on the differential pinion shaft is also included, and also a pair of side gears having a rotational axis common to that of the differential casing, wherein the side gears mesh with the differential pinion gears and the two output shafts are fixed to the side gears, the means for limiting the difference in rotational speed between the two output shafts comprising a weight means radially movable in the differential casing, the weight means limiting the difference in rotational speed between the two output shafts in response to the centrifugal force applied to the weight means, the weight means being slidably mounted on the differential pinion shaft and being biased radially inwardly.

  2. 33rd Actinide Separations Conference

    SciTech Connect

    McDonald, L M; Wilk, P A

    2009-05-04

    Welcome to the 33rd Actinide Separations Conference hosted this year by the Lawrence Livermore National Laboratory. This annual conference is centered on the idea of networking and communication with scientists from throughout the United States, Britain, France and Japan who have expertise in nuclear material processing. This conference forum provides an excellent opportunity for bringing together experts in the fields of chemistry, nuclear and chemical engineering, and actinide processing to present and discuss experiences, research results, testing and application of actinide separation processes. The exchange of information that will take place between you, and other subject matter experts from around the nation and across the international boundaries, is a critical tool to assist in solving both national and international problems associated with the processing of nuclear materials used for both defense and energy purposes, as well as for the safe disposition of excess nuclear material. Granlibakken is a dedicated conference facility and training campus that is set up to provide the venue that supports communication between scientists and engineers attending the 33rd Actinide Separations Conference. We believe that you will find that Granlibakken and the Lake Tahoe views provide an atmosphere that is stimulating for fruitful discussions between participants from both government and private industry. We thank the Lawrence Livermore National Laboratory and the United States Department of Energy for their support of this conference. We especially thank you, the participants and subject matter experts, for your involvement in the 33rd Actinide Separations Conference.

  3. Control of Transcriptional Elongation by RNA Polymerase II: A Retrospective.

    PubMed

    Brannan, Kris; Bentley, David L

    2012-01-01

    The origins of our current understanding of control of transcription elongation lie in pioneering experiments that mapped RNA polymerase II on viral and cellular genes. These studies first uncovered the surprising excess of polymerase molecules that we now know to be situated at the at the 5' ends of most genes in multicellular organisms. The pileup of pol II near transcription start sites reflects a ubiquitous bottle-neck that limits elongation right at the start of the transcription elongation. Subsequent seminal work identified conserved protein factors that positively and negatively control the flux of polymerase through this bottle-neck, and make a major contribution to control of gene expression.

  4. Optical tweezers studies of transcription by eukaryotic RNA polymerases.

    PubMed

    Lisica, Ana; Grill, Stephan W

    2017-02-21

    Transcription is the first step in the expression of genetic information and it is carried out by large macromolecular enzymes called RNA polymerases. Transcription has been studied for many years and with a myriad of experimental techniques, ranging from bulk studies to high-resolution transcript sequencing. In this review, we emphasise the advantages of using single-molecule techniques, particularly optical tweezers, to study transcription dynamics. We give an overview of the latest results in the single-molecule transcription field, focusing on transcription by eukaryotic RNA polymerases. Finally, we evaluate recent quantitative models that describe the biophysics of RNA polymerase translocation and backtracking dynamics.

  5. RNA polymerase activity in purified virions of avian reticuloendotheliosis viruses.

    PubMed Central

    Mizutani, S; Temin, H M

    1976-01-01

    An RNA polymerase activity that synthesizes a U-rich RNA hydrogen bonded to a large viral RNA molecule was found in the cores of virions of avian reticuloendotheliosis viruses (REV). The RNA polymerase activity was separable from the DNA polymerase activity of REV virions. The 5'-terminus of the newly synthesized RNA was A. In addition, a tRNA nucleotidyl transferase activity, which added -CpCpA ends to tRNA, appears to be present in the REV virions. Images PMID:183017

  6. PREFACE: The Irago Conference 2012

    NASA Astrophysics Data System (ADS)

    Sandhu, Adarsh; Okada, Hiroshi

    2013-04-01

    The Irago Conference 2012 - 360 degree outlook on critical scientific and technological challenges for a sustainable society Organized by the Electronics-Inspired Interdisciplinary Research Institute (EIIRIS) at Toyohashi University of Technology, the Irago Conference, held recently (15-16 November) in Aichi, Japan, aimed to enhance mutual understanding between scientists, engineers and policymakers. Over 180 participants tackled topics ranging from energy and natural resources to public health and disaster prevention. The 360-degree outlook of the conference impressed speakers and guests. ''This conference has been extremely informative,'' noted Robert Gellar from the University of Tokyo. ''A unique conference with experts from a range of backgrounds,'' agreed Uracha Ruktanonchai from the National Nanotechnology Center (NANOTEC) in Thailand. Similarly, G P Li, professor of electrical engineering and computer science at the University of California Irvine commented that he had been ''able to think the unthinkable'' as a range of topics came together. The conference was streamed live on Ustream to ensure that researchers from across the world could benefit from thought-provoking presentations examining global issues such as energy, disaster mitigation and nanotechnology. ''This was wonderful,'' said Oussama Khatib from Stanford University, ''A good recipe of speakers from such a range of backgrounds.'' Manuscripts submitted to the organizers were peer-reviewed, and the papers in this proceedings were accepted for Journal of Physics: Conference Series. In addition to the formal speaker programme, graduate-student sessions provided a platform for graduate students to describe their latest findings as oral presentations. A series of excursions to relevant locations, such as the Tahara megasolar region under construction and a local car-manufacturing factory, gave participants the opportunity to further consider practical applications of their research in industry

  7. Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies

    PubMed Central

    Macao, Bertil; Grönke, Sebastian; Siibak, Triinu; Stewart, James B; Baggio, Francesca; Dols, Jacqueline; Partridge, Linda; Falkenberg, Maria; Wredenberg, Anna; Larsson, Nils-Göran

    2016-01-01

    Replication errors are the main cause of mtDNA mutations and a compelling approach to decrease mutation levels would therefore be to increase the fidelity of the catalytic subunit (POLγA) of the mtDNA polymerase. Here we genomically engineered the tamas locus, encoding fly POLγA, and introduced alleles expressing exonuclease- (exo-) and polymerase-deficient (pol-) POLγA versions. The exo- mutant leads to accumulation of point mutations and linear deletions of mtDNA, whereas pol- mutants cause mtDNA depletion. The mutant tamas alleles are developmentally lethal but can complement each other in trans resulting in viable flies with clonally expanded mtDNA mutations. Reconstitution of human mtDNA replication in vitro confirms that replication is a highly dynamic process where POLγA goes on and off the template to allow complementation during proofreading and elongation. The created fly models are valuable tools to study germ line transmission of mtDNA and the pathophysiology of POLγA mutation disease. PMID:26554610

  8. Hypermutagenesis in mutA cells is mediated by mistranslational corruption of polymerase, and is accompanied by replication fork collapse.

    PubMed

    Al Mamun, Abu Amar M; Gautam, Satyendra; Humayun, M Zafri

    2006-12-01

    Elevated mistranslation induces a mutator response termed translational stress-induced mutagenesis (TSM) that is mediated by an unidentified modification of DNA polymerase III. Here we address two questions: (i) does TSM result from direct polymerase corruption, or from an indirect pathway triggered by increased protein turnover? (ii) Why are homologous recombination functions required for the expression of TSM under certain conditions, but not others? We show that replication of bacteriophage T4 in cells expressing the mutA allele of the glyVtRNA gene (Asp-Gly mistranslation), leads to both increased mutagenesis, and to an altered mutational specificity, results that strongly support mistranslational corruption of DNA polymerase. We also show that expression of mutA, which confers a recA-dependent mutator phenotype, leads to increased lambdoid prophage induction (selectable in vivo expression technology assay), suggesting that replication fork collapse occurs more frequently in mutA cells relative to control cells. No such increase in prophage induction is seen in cells expressing alaVGlu tRNA (Glu-->Ala mistranslation), in which the mutator phenotype is recA-independent. We propose that replication fork collapse accompanies episodic hypermutagenic replication cycles in mutA cells, requiring homologous recombination functions for fork recovery, and therefore, for mutation recovery. These findings highlight hitherto under-appreciated links among translation, replication and recombination, and suggest that translational fidelity, which is affected by genetic and environmental signals, is a key modulator of replication fidelity.

  9. Mechanics of Textile Composites Conference

    SciTech Connect

    Poe, C.C. Jr.; Harris, C.E.

    1995-10-01

    This document is a compilation of papers presented at the Mechanics of Textile Composites Conference in Hampton, Virginia, December 6-8, 1994. This conference was the culmination of a 3-year program that was initiated by NASA late in 1990 to develop mechanics of textile composites in support of the NASA Advanced Composites Technology Program (ACT). The goal of the program was to develop mathematical models of textile preform materials and test methods to facilitate structural analysis and design. Participants in the program were from NASA, academia, and industry. Separate abstracts have been submitted to the database for articles from this conference.

  10. DOE Workshop at Tapia Conference

    SciTech Connect

    Taylor, Valerie

    2015-02-19

    The DE-SC0013568 DOE Grant, in the amount of $11,822.79, was used to support five doctoral students from underrepresented groups to attend the 2015 Richard Tapia Celebration of Diversity in Computing Conference, held February 18-21 in Boston, MA. Each scholarship was approximately $1200 to cover conference registration, travel, and lodging for the duration of the conference. The remaining $5,822.79 was used to support a DOE Breakfast Workshop during breakfast on Thursday, February 19. The Breakfast supported approximately 140 graduate students from underrepresented groups to learn about the different career opportunities at the different DOE National Laboratories.

  11. Third International Satellite Conference on Mathematical Methods in Physics

    NASA Astrophysics Data System (ADS)

    The aim of the Conference is to present the latest advances in Mathematical Methods to researchers, post-docs and graduated students acting in the areas of Physics of Particles and Fields, Mathematical Physics and Applied Mathematics. Topics: Methods of Spectral and Group Theory, Differential and Algebraic Geometry and Topology in Field Theory, Quantum Gravity, String Theory and Cosmology. http://www.uel.br/eventos/isc/

  12. Complete replication in vitro of tobacco mosaic virus RNA by a template-dependent, membrane-bound RNA polymerase.

    PubMed Central

    Osman, T A; Buck, K W

    1996-01-01

    A crude membrane-bound RNA polymerase, obtained by differential centrifugation of extracts of tomato leaves infected with tobacco mosaic tobamovirus (tomato strain L) TMV-L), was purified by sucrose density gradient centrifugation. Removal of the endogenous RNA template with micrococcal nuclease rendered the polymerase template dependent and template specific. The polymerase was primer independent and able to initiate RNA synthesis on templates containing the 3'-terminal sequences of the TMV-L positive or negative strands. TMV-vulgare RNA was a less efficient template, while RNAs of cucumber mosaic cucumovirus and red clover necrotic mosaic dianthovirus, or 5'-terminal sequences of TMV-L positive or negative strands, did not act as templates for the polymerase. A main product of the reaction with TMV-L genomic RNA as a template, carried out in the presence of [alpha-32P]UTP, was genomic-length single-stranded RNA. This was shown to be the positive strand and uniformly labelled along its length, demonstrating complete replication of TMV-L RNA. Genomic-length double-stranded RNA, labelled in both strands, and small amounts of RNAs corresponding to the single- and double-stranded forms of the coat protein subgenomic mRNA were also formed. Antibodies to N-terminal and C-terminal portions of the 126-kDa protein detected the 126-kDa protein and the 183-kDa readthrough protein in purified RNA polymerase preparations, whereas antibodies to the readthrough portion of the 183-kDa protein detected only the 183-kDa protein. All three antibodies inhibited the template-dependent RNA polymerase, but none of them had any effect on the template-bound enzyme. PMID:8709249

  13. PREFACE: Quark Matter 2006 Conference

    NASA Astrophysics Data System (ADS)

    Ma, Yu-Gang; Wang, En-Ke; Cai, Xu; Huang, Huan-Zhong; Wang, Xin-Nian; Zhu, Zhi-Yuan

    2007-07-01

    The Quark Matter 2006 conference was held on 14 20 November 2006 at the Shanghai Science Hall of the Shanghai Association of Sciences and Technology in Shanghai, China. It was the 19th International Conference on Ultra-Relativistic Nucleus Nucleus Collisions. The conference was organized jointly by SINAP (Shanghai Institute of Applied Physics, Chinese Academy of Sciences (CAS)) and CCNU (Central China Normal University, Wuhan). Over 600 scientists from 32 countries in five continents attended the conference. This is the first time that China has hosted such a premier conference in the field of relativistic heavy-ion collisions, an important event for the Chinese high energy nuclear physics community. About one half of the conference participants are junior scientists—a clear indication of the vigor and momentum for this field, in search of the fundamental nature of the nuclear matter at extreme conditions. Professor T D Lee, honorary chair of the conference and one of the founders of the quark matter research, delivered an opening address with his profound and philosophical remarks on the recent discovery of the nature of strongly-interacting quark-gluon-plasma (sQGP). Professor Hongjie Xu, director of SINAP, gave a welcome address to all participants on behalf of the two hosting institutions. Dr Peiwen Ji, deputy director of the Mathematics and Physics Division of the Natural Science Foundation of China (NSFC), also addressed the conference participants and congratulated them on the opening of the conference. Professor Mianheng Jiang, vice president of the Chinese Academy of Sciences (CAS), gave a concise introduction about the CAS as the premier research institution in China. He highlighted continued efforts at CAS to foster international collaborations between China and other nations. The Quark Matter 2006 conference is an example of such a successful collaboration between high energy nuclear physicists in China and other nations all over the world. The

  14. The origin and early evolution of nucleic acid polymerases

    NASA Technical Reports Server (NTRS)

    Lazcano, A.; Cappello, R.; Valverde, V.; Llaca, V.; Oro, J.

    1992-01-01

    The hypothesis that vestiges of the ancestral RNA-dependent RNA polymerase involved in the replication of RNA genomes of Archean cells are present in the eubacterial RNA-polymerase beta-prime subunit and its homologues is discussed. It is shown that, in the DNA-dependent RNA polymerases from three cellular lineages, a very conserved sequence of eight amino acids, also found in a small RNA-binding site previously described for the E. coli polynucleotide phosphorylase and the S1 ribosomal protein, is present. The optimal conditions for the replicase activity of the avian-myeloblastosis-virus reverse transcriptase are presented. The evolutionary significance of the in vitro modifications of substrate and template specificities of RNA polymerases and reverse transcriptases is discussed.

  15. PCNA tool belts and polymerase bridges form during translesion synthesis

    PubMed Central

    Boehm, Elizabeth M.; Spies, Maria; Washington, M. Todd

    2016-01-01

    Large multi-protein complexes play important roles in many biological processes, including DNA replication and repair, transcription, and signal transduction. One of the challenges in studying such complexes is to understand their mechanisms of assembly and disassembly and their architectures. Using single-molecule total internal reflection (TIRF) microscopy, we have examined the assembly and disassembly of the multi-protein complex that carries out translesion synthesis, the error-prone replication of damaged DNA. We show that the ternary complexes containing proliferating cell nuclear antigen (PCNA) and two non-classical DNA polymerases, Rev1 and DNA polymerase η, have two architectures: PCNA tool belts and Rev1 bridges. Moreover, these complexes are dynamic and their architectures can interconvert without dissociation. The formation of PCNA tool belts and Rev1 bridges and the ability of these complexes to change architectures are likely means of facilitating selection of the appropriate non-classical polymerase and polymerase-switching events. PMID:27325737

  16. The origin and early evolution of nucleic acid polymerases

    NASA Astrophysics Data System (ADS)

    Lazcano, A.; Llaca, V.; Cappello, R.; Valverde, V.; Oro, J.

    The hypothesis that vestiges of the ancestral RNA-dependent RNA polymerase involved in the replication of RNA genomes of Archean cells are present in the cubacterial RNA polymerase β' subunit and its homologues is discussed. We show that in the DNA-dependent RNA polymerases from the three cellular lineages a very conserved sequence of eight amino acids also found in a small RNA-binding site previously described for the E. coli polynucleotide phosphorylase and the S1 ribosomal protein is present. The optimal conditions for the replicase activity of the avian myeloblastosis virus reverse transcriptase are presented. The evolutionary significance of the in vitro modifications of substrate and template specificities of RNA polymerases and reverse transcriptases is also discussed.

  17. Engineered DNA polymerase improves PCR results for plastid DNA1

    PubMed Central

    Schori, Melanie; Appel, Maryke; Kitko, AlexaRae; Showalter, Allan M.

    2013-01-01

    • Premise of the study: Secondary metabolites often inhibit PCR and sequencing reactions in extractions from plant material, especially from silica-dried and herbarium material. A DNA polymerase that is tolerant to inhibitors improves PCR results. • Methods and Results: A novel DNA amplification system, including a DNA polymerase engineered via directed evolution for improved tolerance to common plant-derived PCR inhibitors, was evaluated and PCR parameters optimized for three species. An additional 31 species were then tested with the engineered enzyme and optimized protocol, as well as with regular Taq polymerase. • Conclusions: PCR products and high-quality sequence data were obtained for 96% of samples for rbcL and 79% for matK, compared to 29% and 21% with regular Taq polymerase. PMID:25202519

  18. Tetrahydrobenzothiophene inhibitors of hepatitis C virus NS5B polymerase.

    PubMed

    Laporte, M G; Lessen, T A; Leister, L; Cebzanov, D; Amparo, E; Faust, C; Ortlip, D; Bailey, T R; Nitz, T J; Chunduru, S K; Young, D C; Burns, C J

    2006-01-01

    A novel series of selective HCV NS5B RNA dependent RNA polymerase inhibitors has been disclosed. These compounds contain an appropriately substituted tetrahydrobenzothiophene scaffold. This communication will detail the SAR and activities of this series.

  19. Proofreading genotyping assays mediated by high fidelity exo+ DNA polymerases.

    PubMed

    Zhang, Jia; Li, Kai; Pardinas, Jose R; Sommer, Steve S; Yao, Kai-Tai

    2005-02-01

    DNA polymerases with 3'-5' proofreading function mediate high fidelity DNA replication but their application for mutation detection was almost completely neglected before 1998. The obstacle facing the use of exo(+) polymerases for mutation detection could be overcome by primer-3'-termini modification, which has been tested using allele-specific primers with 3' labeling, 3' exonuclease-resistance and 3' dehydroxylation modifications. Accordingly, three new types of single nucleotide polymorphism (SNP) assays have been developed to carry out genome-wide genotyping making use of the fidelity advantage of exo(+) polymerases. Such SNP assays might also provide a novel approach for re-sequencing and de novo sequencing. These new mutation detection assays are widely adaptable to a variety of platforms, including real-time PCR, multi-well plate and microarray technologies. Application of exo(+) polymerases to genetic analysis could accelerate the pace of personalized medicine.

  20. 17 CFR 201.221 - Prehearing conference.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 2 2010-04-01 2010-04-01 false Prehearing conference. 201.221... Rules of Practice Initiation of Proceedings and Prehearing Rules § 201.221 Prehearing conference. (a) Purposes of conference. The purposes of a prehearing conference include, but are not limited to:...

  1. 49 CFR 511.21 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 6 2010-10-01 2010-10-01 false Prehearing conferences. 511.21 Section 511.21...; Interlocutory Appeals; Summary Judgment; Settlement § 511.21 Prehearing conferences. (a) When held. (1) A prehearing conference shall be held in person or by conference telephone call, except in...

  2. 10 CFR 205.171 - Conferences.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 3 2010-01-01 2010-01-01 false Conferences. 205.171 Section 205.171 Energy DEPARTMENT OF ENERGY OIL ADMINISTRATIVE PROCEDURES AND SANCTIONS Conferences, Hearings, and Public Hearings § 205.171 Conferences. (a) The DOE in its discretion may direct that a conference be convened, on its own initiative...

  3. 24 CFR 26.39 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 1 2010-04-01 2010-04-01 false Prehearing conferences. 26.39... § 26.39 Prehearing conferences. (a) The ALJ may schedule prehearing conferences as appropriate. (b) Upon the motion of any party or sua sponte, the ALJ may schedule a prehearing conference at...

  4. 14 CFR 302.414 - Prehearing conference.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Prehearing conference. 302.414 Section 302... Prehearing conference. A prehearing conference may be held in an enforcement proceeding whenever the... prehearing conference is held, it shall be conducted in accordance with § 302.22....

  5. 20 CFR 498.206 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false Prehearing conferences. 498.206 Section 498... RECOMMENDED EXCLUSIONS § 498.206 Prehearing conferences. (a) The ALJ will schedule at least one prehearing conference, and may schedule additional prehearing conferences as appropriate, upon reasonable notice to...

  6. 33 CFR 20.501 - Conferences.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Conferences. 20.501 Section 20... PRACTICE, PROCEDURE, AND EVIDENCE FOR FORMAL ADMINISTRATIVE PROCEEDINGS OF THE COAST GUARD Conferences and Settlements § 20.501 Conferences. (a) Any party may by motion request a conference. (b) The ALJ may direct...

  7. 47 CFR 1.956 - Settlement conferences.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 1 2010-10-01 2010-10-01 false Settlement conferences. 1.956 Section 1.956... Applications and Proceedings Application Requirements and Procedures § 1.956 Settlement conferences. Parties... their attorneys to appear before it for a conference. (a) The purposes of such conferences are: (1)...

  8. 28 CFR 71.19 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Prehearing conferences. 71.19 Section 71... § 71.19 Prehearing conferences. (a) The ALJ may schedule prehearing conferences as appropriate. (b) Upon the motion of any party, the ALJ shall schedule at least one prehearing conference at a...

  9. 42 CFR 3.512 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Prehearing conferences. 3.512 Section 3.512 Public... ORGANIZATIONS AND PATIENT SAFETY WORK PRODUCT Enforcement Program § 3.512 Prehearing conferences. (a) The ALJ must schedule at least one prehearing conference, and may schedule additional prehearing conferences...

  10. 27 CFR 70.418 - Conferences.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 2 2010-04-01 2010-04-01 false Conferences. 70.418... Beer § 70.418 Conferences. Any person desiring a conference with TTB, relative to any matter arising in connection with such person's operations, will be accorded such a conference upon request. No...

  11. 2 CFR 801.1112 - Conference.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 2 Grants and Agreements 1 2010-01-01 2010-01-01 false Conference. 801.1112 Section 801.1112 Grants... Subpart for OMB Guidance at 2 CFR Part 180). § 801.1112 Conference. Upon receipt of a request for a conference, the official imposing the sanction shall arrange such a conference with the participant...

  12. 20 CFR 405.330 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false Prehearing conferences. 405.330 Section 405... INITIAL DISABILITY CLAIMS Administrative Law Judge Hearing § 405.330 Prehearing conferences. (a)(1) The... conference if he or she finds that such a conference would facilitate the hearing or the decision on...

  13. 10 CFR 1003.61 - Conferences.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Conferences. 1003.61 Section 1003.61 Energy DEPARTMENT OF ENERGY (GENERAL PROVISIONS) OFFICE OF HEARINGS AND APPEALS PROCEDURAL REGULATIONS Conferences and Hearings § 1003.61 Conferences. (a) The OHA in its discretion may direct that a conference be convened,...

  14. 10 CFR 1013.19 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Prehearing conferences. 1013.19 Section 1013.19 Energy... conferences. (a) The ALJ may schedule prehearing conferences as appropriate. (b) Upon the motion of any party, the ALJ shall schedule at least one prehearing conference at a reasonable time in advance of...

  15. 20 CFR 405.366 - Posthearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false Posthearing conferences. 405.366 Section 405... INITIAL DISABILITY CLAIMS Administrative Law Judge Hearing § 405.366 Posthearing conferences. (a) The... conference to facilitate the hearing decision. A posthearing conference normally will be held by...

  16. 22 CFR 521.19 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 22 Foreign Relations 2 2010-04-01 2010-04-01 true Prehearing conferences. 521.19 Section 521.19... § 521.19 Prehearing conferences. (a) The ALJ may schedule prehearing conferences as appropriate. (b) Upon the motion of any party, the ALJ shall schedule at least one prehearing conference at a...

  17. RNA polymerase II transcription on the fast lane.

    PubMed

    Marcello, Alessandro

    2012-01-01

    Transcription by RNA polymerase II is the process that copies DNA into RNA leading to the expression of a specific gene. Averaged estimates of polymerase elongation rates in mammalian cells have been shown to vary between 1 and 4 kilobases per minute. However, recent advances in live cell imaging allowed direct measurements of RNA biogenesis from a single gene exceeded 50 kb·min(-1) . This unexpected finding opens novel and intriguing perspectives on the control of metazoan transcription.

  18. Deoxyribonucleic acid polymerase III of Escherichia coli. Purification and properties.

    PubMed

    Livingston, D M; Hinkle, D C; Richardson, C C

    1975-01-25

    DNA polymerase III has been purified 4,500-fold from the Escherichis coli mutant, HMS83, which lacks DNA polymerases I and II. When subjected to disc gel electrophoresis, the most purified fraction exhibits a single major protein band from which enzymatic activity may be recovered. Polyacrylamide gel electrophoresis under denaturing conditions produces two protein bands with molecular weights of 140,000 and 40,000. The sedimentation coefficient of the enzyme is 7.0 S, and the Stokes radius is 62 A. Taken together these tow parameters indicate a native molecular weight of 180,000. Purified DNA polymerase III catalyzes the polymerization of nucleotides into DNA when provided with both a DNA template and a complementary primer strand. The newly synthesized DNA is covalently attached to the 3' terminus of the primer strand. Because the extent of polymerization is only 10 to 100 nucleotides, the best substrates are native DNA molecules with small single-stranded regions. The most purified enzyme preparation is devoid of endonuclease activities. In addition to the two exonuclease activities described in the accompanying paper, purified polymerase III also catalyzes pyrophosphorolysis and the exchange of pyrophosphate into deoxynucleoside triphosphates. DNA polymerase III has also been isolated from wild type E. coli containing the other two known DNA polymerases. Futhermore, the enzyme purified from three different polC mutants exhibits altered polymerase III activity, confirming that polC is the structural gene for DNA polymerase III (Gefter, M., Hirota, Y., Kornberb, T., Wechsler, J., and Barnoux, C. (1971) Proc. Natl. Acad. Sci. U. S. A. 68, 3150-3153).

  19. Kinetics and thermodynamics of DNA polymerases with exonuclease proofreading

    NASA Astrophysics Data System (ADS)

    Gaspard, Pierre

    2016-04-01

    Kinetic theory and thermodynamics are applied to DNA polymerases with exonuclease activity, taking into account the dependence of the rates on the previously incorporated nucleotide. The replication fidelity is shown to increase significantly thanks to this dependence at the basis of the mechanism of exonuclease proofreading. In particular, this dependence can provide up to a 100-fold lowering of the error probability under physiological conditions. Theory is compared with numerical simulations for the DNA polymerases of T7 viruses and human mitochondria.

  20. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2004-10-12

    The present invention relates to 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  1. Interstitial contacts in an RNA-dependent RNA polymerase lattice

    PubMed Central

    Tellez, Andres B.; Wang, Jing; Tanner, Elizabeth J.; Spagnolo, Jeannie F.; Kirkegaard, Karla; Bullitt, Esther

    2011-01-01

    Catalytic activities can be facilitated by ordered enzymatic arrays that co-localize and orient enzymes and their substrates. The purified RNA-dependent RNA polymerase from poliovirus self-assembles to form two-dimensional lattices, possibly facilitating the assembly of viral RNA replication complexes on the cytoplasmic face of intracellular membranes. Creation of a two-dimensional lattice requires at least two different molecular contacts between polymerase molecules. One set of polymerase contacts, between the ‘thumb’ domain of one polymerase and the back of the ‘palm’ domain of another, has been previously defined. To identify the second interface needed for lattice formation and to test its function in viral RNA synthesis, a hybrid approach of both electron microscopic and biochemical evaluation of wild-type and mutant viral polymerases was used to evaluate computationally generated models of this second interface. A unique solution satisfied all constraints and predicted a two-dimensional structure formed from antiparallel arrays of polymerase fibers that use contacts from the flexible amino-terminal region of the protein. Enzymes that contained mutations in this newly defined interface did not form lattices and altered the structure of wild-type lattices. When reconstructed into virus, mutations that disrupt lattice assembly exhibited growth defects, synthetic lethality, or both, supporting the function of the oligomeric lattice in infected cells. Understanding the structure of polymerase lattices within the multimeric RNA-dependent RNA polymerase complex should faciliate antiviral drug design and provide a precedent for other positive-strand RNA viruses. PMID:21839092

  2. The DNA-polymerase-X family: controllers of DNA quality?

    PubMed

    Ramadan, Kristijan; Shevelev, Igor; Hübscher, Ulrich

    2004-12-01

    Synthesis of the genetic material of the cell is achieved by a large number of DNA polymerases. Besides replicating the genome, they are involved in DNA-repair processes. Recent studies have indicated that certain DNA-polymerase-X-family members can synthesize unusual DNA structures, and we propose that these DNA structures might serve as 'flag wavers' for the induction of DNA-repair and/or DNA-damage-checkpoint pathways.

  3. 10. international mouse genome conference

    SciTech Connect

    Meisler, M.H.

    1996-12-31

    Ten years after hosting the First International Mammalian Genome Conference in Paris in 1986, Dr. Jean-Louis Guenet presided over the Tenth Conference at the Pasteur Institute, October 7--10, 1996. The 1986 conference was a satellite to the Human Gene Mapping Workshop and had approximately 50 attendees. The 1996 meeting was attended by 300 scientists from around the world. In the interim, the number of mapped loci in the mouse increased from 1,000 to over 20,000. This report contains a listing of the program and its participants, and two articles that review the meeting and the role of the laboratory mouse in the Human Genome project. More than 200 papers were presented at the conference covering the following topics: International mouse chromosome committee meetings; Mutant generation and identification; Physical and genetic maps; New technology and resources; Chromatin structure and gene regulation; Rate and hamster genetic maps; Informatics and databases; and Quantitative trait analysis.

  4. 2009 Community Involvement Training Conference

    EPA Pesticide Factsheets

    A dynamic training conference that brings together more than 450 people from EPA and the Agency’s partners and stakeholders who plan and implement environmental community involvement, partnership, stewardship, outreach, and education programs.

  5. EPA Community Involvement Training Conferences

    EPA Pesticide Factsheets

    A dynamic training conference that brings together more than 450 people from EPA and the Agency’s partners and stakeholders who plan and implement environmental community involvement, partnership, stewardship, outreach, and education programs.

  6. 2013 Community Involvement Training Conference

    EPA Pesticide Factsheets

    A dynamic training conference that brings together more than 450 people from EPA and the Agency’s partners and stakeholders who plan and implement environmental community involvement, partnership, stewardship, outreach, and education programs.

  7. 2011 Community Involvement Training Conference

    EPA Pesticide Factsheets

    A dynamic training conference that brings together more than 450 people from EPA and the Agency’s partners and stakeholders who plan and implement environmental community involvement, partnership, stewardship, outreach, and education programs.

  8. Climate Leadership Awards and Conference

    EPA Pesticide Factsheets

    The fifth annual Climate Leadership Awards Dinner will be held on March 9, 2016 during the 2016 Climate Leadership Conference, and publicly recognize individuals and organizations for their outstanding leadership in reducing greenhouse gas emissions.

  9. 2015 Community Involvement Training Conference

    EPA Pesticide Factsheets

    A dynamic training conference that brings together more than 450 people from EPA and the Agency’s partners and stakeholders who plan and implement environmental community involvement, partnership, stewardship, outreach, and education programs.

  10. 2007 Community Involvement Training Conference

    EPA Pesticide Factsheets

    A dynamic training conference that brings together more than 450 people from EPA and the Agency’s partners and stakeholders who plan and implement environmental community involvement, partnership, stewardship, outreach, and education programs.

  11. International Conference on Human Relations.

    DTIC Science & Technology

    2007-11-02

    Festinger , Sherif, Ex, Rohrer, Pinto, Singh, and Mailhiot. One outcome of the Conference was the formation of a working committee to establish an International Documentation and Information Center for the field of Human Relations.

  12. Eighteen Years of the Great Lakes Regional Counseling Psychology Conference: Revisiting the Need for Regional Conferences

    ERIC Educational Resources Information Center

    Delgado-Romero, Edward A.; Bowman, Sharon L.; Gerstein, Lawrence H.

    2006-01-01

    The Great Lakes Regional Conference on Counseling Psychology is the only conference to continuously fulfill the 1987 mandate issued by Division 17 for regional counseling conferences. The rationale for regional conferences is reviewed, and the 18-year history of the Great Lakes Regional Conference is examined. The authors conclude by noting the…

  13. 9. international mouse genome conference

    SciTech Connect

    1995-12-31

    This conference was held November 12--16, 1995 in Ann Arbor, Michigan. The purpose of this conference was to provide a multidisciplinary forum for exchange of state-of-the-art information on genetic mapping in mice. This report contains abstracts of presentations, focusing on the following areas: mutation identification; comparative mapping; informatics and complex traits; mutagenesis; gene identification and new technology; and genetic and physical mapping.

  14. NASA Microgravity Materials Science Conference

    NASA Technical Reports Server (NTRS)

    Gillies, D. C. (Compiler); McCauley, D. E. (Compiler)

    1999-01-01

    The Microgravity Materials Science Conference was held July 14-16, 1998 at the Von Braun Center in Huntsville, AL. It was organized by the Microgravity Materials Science Discipline Working Group, sponsored by the Microgravity Research Division at NASA Headquarters, and hosted by the NASA Marshall Space Flight Center and the Alliance for Microgravity Materials Science and Applications. It was the third NASA conference of this type in the microgravity materials science discipline. The microgravity science program sponsored approximately 125 investigations and 100 principal investigators in FY98, almost all of whom made oral or poster presentations at this conference. The conference's purpose was to inform the materials science community of research opportunities in reduced gravity in preparation for a NASA Research Announcement scheduled for release in late 1998 by the Microgravity Research Division at NASA Headquarters. The conference was aimed at materials science researchers from academia, industry, and government. A tour of the Marshall Space Flight Center microgravity research facilities was held on July 16, 1998. This volume is comprised of the research reports submitted by the principal investigators after the conference.

  15. NASA Microgravity Materials Science Conference

    NASA Technical Reports Server (NTRS)

    Szofran, Frank R. (Compiler); McCauley, D. (Compiler); Walker, C. (Compiler)

    1996-01-01

    The Microgravity Materials Science Conference was held June 10-11, 1996 at the Von Braun Civic Center in Huntsville, AL. It was organized by the Microgravity Materials Science Discipline Working Group, sponsored by the Microgravity Science and Applications Division at NASA Headquarters, and hosted by the NASA Marshall Space Flight Center and the Alliance for Microgravity Materials Science and Applications (AMMSA). It was the second NASA conference of this type in the microgravity materials science discipline. The microgravity science program sponsored approximately 80 investigations and 69 principal investigators in FY96, all of whom made oral or poster presentations at this conference. The conference's purpose was to inform the materials science community of research opportunities in reduced gravity in preparation for a NASA Research Announcement (NRA) scheduled for release in late 1996 by the Microgravity Science and Applications Division at NASA Headquarters. The conference was aimed at materials science researchers from academia, industry, and government. A tour of the MSFC microgravity research facilities was held on June 12, 1996. This volume is comprised of the research reports submitted by the principal investigators after the conference and presentations made by various NASA microgravity science managers.

  16. An autoradiographic demonstration of nuclear DNA replication by DNA polymerase alpha and of mitochondrial DNA synthesis by DNA polymerase gamma.

    PubMed Central

    Geuskens, M; Hardt, N; Pedrali-Noy, G; Spadari, S

    1981-01-01

    The incorporation of thymidine into the DNA of eukaryotic cells is markedly depressed, but not completely inhibited, by aphidicolin, a highly specific inhibitor of DNA polymerase alpha. An electron microscope autoradiographic analysis of the synthesis of nuclear and mitochondrial DNA in vivo in Concanavalin A stimulated rabbit spleen lymphocytes and in Hamster cell cultures, in the absence and in the presence of aphidicolin, revealed that aphidicolin inhibits the nuclear but not the mitochondrial DNA replication. We therefore conclude that DNA polymerase alpha performs the synchronous bidirectional replication of nuclear DNA and that DNA polymerase gamma, the only DNA polymerase present in the mitochondria, performs the "strand displacement" DNA synthesis of these organelles. Images PMID:6262734

  17. PREFACE: XXI Fluid Mechanics Conference

    NASA Astrophysics Data System (ADS)

    Szmyd, Janusz S.; Fornalik-Wajs, Elzbieta; Jaszczur, Marek

    2014-08-01

    This Conference Volume contains the papers presented at the 21st Fluid Mechanics Conference (XXI FMC) held at AGH - University of Science and Technology in Krakow, Poland, 15-18 June 2014, and accepted for Proceedings published in the Journal of Physics: Conference Series. The Fluid Mechanics Conferences have been taking place every two years since 1974, a total of forty years. The 21st Fluid Mechanics Conference (XXI FMC) is being organized under the auspices of the Polish Academy of Sciences, Committee of Mechanics. The goal of this conference is to provide a forum for the exposure and exchange of ideas, methods and results in fluid mechanics. Conference topics include, but are not limited to Aerodynamics, Atmospheric Science, Bio-Fluids, Combustion and Reacting Flows, Computational Fluid Dynamics, Experimental Fluid Mechanics, Flow Machinery, General Fluid Dynamics, Hydromechanics, Heat and Fluid Flow, Measurement Techniques, Micro- and Nano- Flow, Multi-Phase Flow, Non-Newtonian Fluids, Rotating and Stratified Flows, Turbulence. Within the general subjects of this conference, the Professor Janusz W. Elsner Competition for the best fluid mechanics paper presented during the Conference is organized. Authors holding a M.Sc. or a Ph.D. degree and who are not older than 35 years of age may enter the Competition. Authors with a Ph.D. degree must present individual papers; authors with a M.Sc. degree may present papers with their supervisor as coauthor, including original results of experimental, numerical or analytic research. Six state-of-the-art keynote papers were delivered by world leading experts. All contributed papers were peer reviewed. Recommendations were received from the International Scientific Committee, reviewers and the advisory board. Accordingly, of the 163 eligible extended abstracts submitted, after a review process by the International Scientific Committee, 137 papers were selected for presentation at the 21st Fluid Mechanics Conference, 68

  18. Norovirus Polymerase Fidelity Contributes to Viral Transmission In Vivo

    PubMed Central

    Thorne, Lucy; Ghurburrun, Elsa

    2016-01-01

    ABSTRACT Intrahost genetic diversity and replication error rates are intricately linked to RNA virus pathogenesis, with alterations in viral polymerase fidelity typically leading to attenuation during infections in vivo. We have previously shown that norovirus intrahost genetic diversity also influences viral pathogenesis using the murine norovirus model, as increasing viral mutation frequency using a mutagenic nucleoside resulted in clearance of a persistent infection in mice. Given the role of replication fidelity and genetic diversity in pathogenesis, we have now investigated whether polymerase fidelity can also impact virus transmission between susceptible hosts. We have identified a high-fidelity norovirus RNA-dependent RNA polymerase mutant (I391L) which displays delayed replication kinetics in vivo but not in cell culture. The I391L polymerase mutant also exhibited lower transmission rates between susceptible hosts than the wild-type virus and, most notably, another replication defective mutant that has wild-type levels of polymerase fidelity. These results provide the first experimental evidence that norovirus polymerase fidelity contributes to virus transmission between hosts and that maintaining diversity is important for the establishment of infection. This work supports the hypothesis that the reduced polymerase fidelity of the pandemic GII.4 human norovirus isolates may contribute to their global dominance. IMPORTANCE Virus replication fidelity and hence the intrahost genetic diversity of viral populations are known to be intricately linked to viral pathogenesis and tropism as well as to immune and antiviral escape during infection. In this study, we investigated whether changes in replication fidelity can impact the ability of a virus to transmit between susceptible hosts by the use of a mouse model for norovirus. We show that a variant encoding a high-fidelity polymerase is transmitted less efficiently between mice than the wild-type strain. This

  19. Norovirus Polymerase Fidelity Contributes to Viral Transmission In Vivo.

    PubMed

    Arias, Armando; Thorne, Lucy; Ghurburrun, Elsa; Bailey, Dalan; Goodfellow, Ian

    2016-01-01

    Intrahost genetic diversity and replication error rates are intricately linked to RNA virus pathogenesis, with alterations in viral polymerase fidelity typically leading to attenuation during infections in vivo. We have previously shown that norovirus intrahost genetic diversity also influences viral pathogenesis using the murine norovirus model, as increasing viral mutation frequency using a mutagenic nucleoside resulted in clearance of a persistent infection in mice. Given the role of replication fidelity and genetic diversity in pathogenesis, we have now investigated whether polymerase fidelity can also impact virus transmission between susceptible hosts. We have identified a high-fidelity norovirus RNA-dependent RNA polymerase mutant (I391L) which displays delayed replication kinetics in vivo but not in cell culture. The I391L polymerase mutant also exhibited lower transmission rates between susceptible hosts than the wild-type virus and, most notably, another replication defective mutant that has wild-type levels of polymerase fidelity. These results provide the first experimental evidence that norovirus polymerase fidelity contributes to virus transmission between hosts and that maintaining diversity is important for the establishment of infection. This work supports the hypothesis that the reduced polymerase fidelity of the pandemic GII.4 human norovirus isolates may contribute to their global dominance. IMPORTANCE Virus replication fidelity and hence the intrahost genetic diversity of viral populations are known to be intricately linked to viral pathogenesis and tropism as well as to immune and antiviral escape during infection. In this study, we investigated whether changes in replication fidelity can impact the ability of a virus to transmit between susceptible hosts by the use of a mouse model for norovirus. We show that a variant encoding a high-fidelity polymerase is transmitted less efficiently between mice than the wild-type strain. This constitutes

  20. Genetic evidence that both dNTP-stabilized and strand slippage mechanisms may dictate DNA polymerase errors within mononucleotide microsatellites.

    PubMed

    Baptiste, Beverly A; Jacob, Kimberly D; Eckert, Kristin A

    2015-05-01

    Mononucleotide microsatellites are tandem repeats of a single base pair, abundant within coding exons and frequent sites of mutation in the human genome. Because the repeated unit is one base pair, multiple mechanisms of insertion/deletion (indel) mutagenesis are possible, including strand-slippage, dNTP-stabilized, and misincorportion-misalignment. Here, we examine the effects of polymerase identity (mammalian Pols α, β, κ, and η), template sequence, dNTP pool size, and reaction temperature on indel errors during in vitro synthesis of mononucleotide microsatellites. We utilized the ratio of insertion to deletion errors as a genetic indicator of mechanism. Strikingly, we observed a statistically significant bias toward deletion errors within mononucleotide repeats for the majority of the 28 DNA template and polymerase combinations examined, with notable exceptions based on sequence and polymerase identity. Using mutator forms of Pol β did not substantially alter the error specificity, suggesting that mispairing-misalignment mechanism is not a primary mechanism. Based on our results for mammalian DNA polymerases representing three structurally distinct families, we suggest that dNTP-stabilized mutagenesis may be an alternative mechanism for mononucleotide microsatellite indel mutation. The change from a predominantly dNTP-stabilized mechanism to a strand-slippage mechanism with increasing microsatellite length may account for the differential rates of tandem repeat mutation that are observed genome-wide.

  1. Mutation at the Polymerase Active Site of Mouse DNA Polymerase δ Increases Genomic Instability and Accelerates Tumorigenesis▿

    PubMed Central

    Venkatesan, Ranga N.; Treuting, Piper M.; Fuller, Evan D.; Goldsby, Robert E.; Norwood, Thomas H.; Gooley, Ted A.; Ladiges, Warren C.; Preston, Bradley D.; Loeb, Lawrence A.

    2007-01-01

    Mammalian DNA polymerase δ (Pol δ) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol δ. Homozygous Pold1L604G/L604G and Pold1L604K/L604K mice died in utero. However, heterozygous animals were viable and displayed no overall increase in disease incidence, indicative of efficient compensation for the defective mutant polymerase. The life spans of wild-type and heterozygous Pold1+/L604G mice did not differ, while that of Pold1+/L604K mice was reduced by 18%. Cultured embryonic fibroblasts from the heterozygous strains exhibited comparable increases in both spontaneous mutation rate and chromosome aberrations. We observed no significant increase in cancer incidence; however, Pold1+/L604K mice bearing histologically diagnosed tumors died at a younger median age than wild-type mice. Our results indicate that heterozygous mutation at L604 in the polymerase active site of DNA polymerase δ reduces life span, increases genomic instability, and accelerates tumorigenesis in an allele-specific manner, novel findings that have implications for human cancer. PMID:17785453

  2. EDITORIAL: International MEMS Conference 2006

    NASA Astrophysics Data System (ADS)

    Tay, Francis E. H.; Jianmin, Miao; Iliescu, Ciprian

    2006-04-01

    The International MEMS conference (iMEMS2006) organized by the Institute of Bioengineering and Nanotechnology and Nanyang Technological University aims to provide a platform for academicians, professionals and industrialists in various related fields from all over the world to share and learn from each other. Of great interest is the incorporation of the theme of life sciences application using MEMS. It is the desire of this conference to initiate collaboration and form network of cooperation. This has continued to be the objective of iMEMS since its inception in 1997. The technological advance of MEMS over the past few decades has been truly exciting in terms of development and applications. In order to participate in this rapid development, a conference involving delegates from within the MEMS community and outside the community is very meaningful and timely. With the receipt of over 200 articles, delegates related to MEMS field from all over the world will share their perspectives on topics such as MEMS/MST Design, MEMS Teaching and Education, MEMS/MST Packaging, MEMS/MST Fabrication, Microsystems Applications, System Integration, Wearable Devices, MEMSWear and BioMEMS. Invited speakers and delegates from outside the field have also been involved to provide challenges, especially in the life sciences field, for the MEMS community to potentially address. The proceedings of the conference will be published as an issue in the online Journal of Physics: Conference Series and this can reach a wider audience and will facilitate the reference and citation of the work presented in the conference. We wish to express our deep gratitude to the International Scientific Committee members and the organizing committee members for contributing to the success of this conference. We would like to thank all the delegates, speakers and sponsors from all over the world for presenting and sharing their perspectives on topics related to MEMS and the challenges that MEMS can

  3. Differential games.

    NASA Technical Reports Server (NTRS)

    Varaiya, P. P.

    1972-01-01

    General discussion of the theory of differential games with two players and zero sum. Games starting at a fixed initial state and ending at a fixed final time are analyzed. Strategies for the games are defined. The existence of saddle values and saddle points is considered. A stochastic version of a differential game is used to examine the synthesis problem.

  4. Differentiated Staffing.

    ERIC Educational Resources Information Center

    Allen, Dwight W.; Kline, Lloyd W.

    The traditional educational structure requires the teacher to be part bookkeeper, part clerical assistant, and part psychologist, among other roles, while his salary scale is based on length of service. Differentiated staffing offers ways of changing this pattern. The details of differentiated duties are largely a matter of local option and…

  5. Mitochondrial DNA Polymerase POLG1 Disease Mutations and Germline Variants Promote Tumorigenic Properties.

    PubMed

    Singh, Bhupendra; Owens, Kjerstin M; Bajpai, Prachi; Desouki, Mohamed Mokhtar; Srinivasasainagendra, Vinodh; Tiwari, Hemant K; Singh, Keshav K

    2015-01-01

    Germline mutations in mitochondrial DNA polymerase gamma (POLG1) induce mitochondrial DNA (mtDNA) mutations, depletion, and decrease oxidative phosphorylation. Earlier, we identified somatic mutations in POLG1 and the contribution of these mutations in human cancer. However, a role for germline variations in POLG1 in human cancers is unknown. In this study, we examined a role for disease associated germline variants of POLG1, POLG1 gene expression, copy number variation and regulation in human cancers. We analyzed the mutations, expression and copy number variation in POLG1 in several cancer databases and validated the analyses in primary breast tumors and breast cancer cell lines. We discovered 5-aza-2'-deoxycytidine led epigenetic regulation of POLG1, mtDNA-encoded genes and increased mitochondrial respiration. We conducted comprehensive race based bioinformatics analyses of POLG1 gene in more than 33,000 European-Americans and 5,000 African-Americans. We identified a mitochondrial disease causing missense variation in polymerase domain of POLG1 protein at amino acid 1143 (E1143G) to be 25 times more prevalent in European-Americans (allele frequency 0.03777) when compared to African-American (allele frequency 0.00151) population. We identified T251I and P587L missense variations in exonuclease and linker region of POLG1 also to be more prevalent in European-Americans. Expression of these variants increased glucose consumption, decreased ATP production and increased matrigel invasion. Interestingly, conditional expression of these variants revealed that matrigel invasion properties conferred by these germline variants were reversible suggesting a role of epigenetic regulators. Indeed, we identified a set of miRNA whose expression was reversible after variant expression was turned off. Together, our studies demonstrate altered genetic and epigenetic regulation of POLG1 in human cancers and suggest a role for POLG1 germline variants in promoting tumorigenic

  6. Stable interactions between DNA polymerase δ catalytic and structural subunits are essential for efficient DNA repair.

    PubMed

    Brocas, Clémentine; Charbonnier, Jean-Baptiste; Dhérin, Claudine; Gangloff, Serge; Maloisel, Laurent

    2010-10-05

    Eukaryotic DNA polymerase δ (Pol δ) activity is crucial for chromosome replication and DNA repair and thus, plays an essential role in genome stability. In Saccharomyces cerevisiae, Pol δ is a heterotrimeric complex composed of the catalytic subunit Pol3, the structural B subunit Pol31, and Pol32, an additional auxiliary subunit. Pol3 interacts with Pol31 thanks to its C-terminal domain (CTD) and this interaction is of functional importance both in DNA replication and DNA repair. Interestingly, deletion of the last four C-terminal Pol3 residues, LSKW, in the pol3-ct mutant does not affect DNA replication but leads to defects in homologous recombination and in break-induced replication (BIR) repair pathways. The defect associated with pol3-ct could result from a defective interaction between Pol δ and a protein involved in recombination. However, we show that the LSKW motif is required for the interaction between Pol3 C-terminal end and Pol31. This loss of interaction is relevant in vivo since we found that pol3-ct confers HU sensitivity on its own and synthetic lethality with a POL32 deletion. Moreover, pol3-ct shows genetic interactions, both suppression and synthetic lethality, with POL31 mutant alleles. Structural analyses indicate that the B subunit of Pol δ displays a major conserved region at its surface and that pol31 alleles interacting with pol3-ct, correspond to substitutions of Pol31 amino acids that are situated in this particular region. Superimposition of our Pol31 model on the 3D architecture of the phylogenetically related DNA polymerase α (Pol α) suggests that Pol3 CTD interacts with the conserved region of Pol31, thus providing a molecular basis to understand the defects associated with pol3-ct. Taken together, our data highlight a stringent dependence on Pol δ complex stability in DNA repair.

  7. ASM Conference on Prokaryotic Development

    SciTech Connect

    Kaplan, H. B.

    2005-07-13

    Support was provided by DOE for the 2nd ASM Conference on Prokaryotic Development. The final conference program and abstracts book is attached. The conference presentations are organized around topics that are central to the current research areas in prokaryotic development. The program starts with topics that involve relatively simple models systems and ends with systems that are more complex. The topics are: i) the cell cycle, ii) the cytoskeleton, iii) morphogenesis, iv) developmental transcription, v) signaling, vi) multicellularity, and vii) developmental diversity and symbiosis. The best-studied prokaryotic development model systems will be highlighted at the conference through research presentations by leaders in the field. Many of these systems are also model systems of relevance to the DOE mission including carbon sequestration (Bradyrizobium, Synechococcus), energy production (Anabaena, Rhodobacter) and bioremediation (Caulobacter, Mesorhizobium). In addition, many of the highlighted organisms have important practical applications; the actinomycetes and myxobacteria produce antimicrobials that are of commercial interest. It is certain that the cutting-edge science presented at the conference will be applicable to the large group of bacteria relevant to the DOE mission.

  8. 5-methylcytosine-sensitive variants of Thermococcus kodakaraensis DNA polymerase

    PubMed Central

    Huber, Claudia; von Watzdorf, Janina; Marx, Andreas

    2016-01-01

    DNA methylation of cytosine in eukaryotic cells is a common epigenetic modification, which plays an important role in gene expression and thus affects various cellular processes like development and carcinogenesis. The occurrence of 5-methyl-2′-deoxycytosine (5mC) as well as the distribution pattern of this epigenetic marker were shown to be crucial for gene regulation and can serve as important biomarkers for diagnostics. DNA polymerases distinguish little, if any, between incorporation opposite C and 5mC, which is not surprising since the site of methylation is not involved in Watson–Crick recognition. Here, we describe the development of a DNA polymerase variant that incorporates the canonical 2′-deoxyguanosine 5′-monophosphate (dGMP) opposite C with higher efficiency compared to 5mC. The variant of Thermococcus kodakaraensis (KOD) exo- DNA polymerase was discovered by screening mutant libraries that were built by rational design. We discovered that an amino acid substitution at a single site that does not directly interact with the templating nucleobase, may alter the ability of the DNA polymerase in processing C in comparison to 5mC. Employing these findings in combination with a nucleotide, which is fluorescently labeled at the terminal phosphate, indicates the potential use of the mutant DNA polymerase in the detection of 5mC. PMID:27651460

  9. Amplification of RNA by an RNA polymerase ribozyme

    PubMed Central

    Horning, David P.; Joyce, Gerald F.

    2016-01-01

    In all extant life, genetic information is stored in nucleic acids that are replicated by polymerase proteins. In the hypothesized RNA world, before the evolution of genetically encoded proteins, ancestral organisms contained RNA genes that were replicated by an RNA polymerase ribozyme. In an effort toward reconstructing RNA-based life in the laboratory, in vitro evolution was used to improve dramatically the activity and generality of an RNA polymerase ribozyme by selecting variants that can synthesize functional RNA molecules from an RNA template. The improved polymerase ribozyme is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA. Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR. Thus, the two prerequisites of Darwinian life—the replication of genetic information and its conversion into functional molecules—can now be accomplished with RNA in the complete absence of proteins. PMID:27528667

  10. PCR performance of a thermostable heterodimeric archaeal DNA polymerase.

    PubMed

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

  11. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    PubMed Central

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

  12. World Food Conference: Issues and Preparation for Conference,

    DTIC Science & Technology

    1974-01-17

    AD-AO&I 2-95 DEPARTMENT OF STATE WASHINGTON 0 C OFFICE OF EkeTERNA-TP F/6 2/1! I WORLD FOOD CONFERENCE: ISSUES AND PREPARtATION FOR CONFERENCE. (U) M...1,136 million tons, a fall of 40 million tons, ( 5 %) from the production of the proceeding year. Nevertheless an increase in world demand to 1,174...grains, as estimated by the USDA is likely to be 1,230 million tons,(only 5 % less than projected for 1980 by the FAO) and world demand, 1,232 million

  13. Core-sigma interaction: probing the interaction of the bacteriophage T4 gene 55 promoter recognition protein with E.coli RNA polymerase core.

    PubMed Central

    Léonetti, J P; Wong, K; Geiduschek, E P

    1998-01-01

    The bacterial RNA polymerase sigma subunits are key participants in the early steps of RNA synthesis, conferring specificity of promoter recognition, facilitating promoter opening and promoter clearance, and responding to diverse transcriptional regulators. The T4 gene 55 protein (gp55), the sigma protein of the bacteriophage T4 late genes, is one of the smallest and most divergent members of this family. Protein footprinting was used to identify segments of gp55 that become buried upon binding to RNA polymerase core, and are therefore likely to constitute its interface with the core enzyme. Site-directed mutagenesis in two parts of this contact surface generated gene 55 proteins that are defective in polymerase-binding to different degrees. Alignment with the sequences of the sigma proteins and with a recently determined structure of a large segment of sigma70 suggests that the gp55 counterpart of sigma70 regions 2.1 and 2.2 is involved in RNA polymerase core binding, and that sigma70 and gp55 may be structurally similar in this region. The diverse phenotypes of the mutants implicate this region of gp55 in multiple aspects of sigma function. PMID:9482743

  14. Differentiating Knowledge, Differentiating (Occupational) Practice

    ERIC Educational Resources Information Center

    Hordern, Jim

    2016-01-01

    This paper extends arguments for differentiating knowledge into conceptualisations of occupational practice. It is argued that specialised forms of knowledge and practice require recognition and differentiation in ways that many contemporary approaches to practice theory deny. Drawing on Hager's interpretation of MacIntyre, it is suggested that…

  15. Inaugural AGU Science Policy Conference

    NASA Astrophysics Data System (ADS)

    Uhlenbrock, Kristan

    2012-01-01

    AGU will present its inaugural Science Policy Conference, 30 April to 3 May 2012, at the Ronald Reagan Building and International Trade Center, located in downtown Washington, D. C. This conference will bring together leading scientists, policy makers, industry professionals, press, and other stakeholders to discuss natural hazards, natural resources, oceans, and Arctic science and the role these sciences play in serving communities. To bridge the science and policy fields, AGU plans to host this conference every 2 years and focus on the applications of Earth and space sciences to serve local and national communities. "Our nation faces a myriad of challenges such as the sustainability of our natural resources, current and future energy needs, and the ability to mitigate and adapt to natural and manmade hazards," said Michael McPhaden, president of AGU. "It is essential that policies to address these challenges be built on a solid foundation of credible scientific knowledge."

  16. Message from the Conference Chairs

    NASA Astrophysics Data System (ADS)

    Krishna, Sanjay; Perera, Unil

    2015-05-01

    We were very excited to host the 8th International Workshop on Quantum Structure Infrared Photodetectors (QSIP 2014), in picturesque Santa Fe, New Mexico from June 29th-July 3rd, 2014. This followed successful QSIP conferences at Dana Point (2000), Torino (2002), Kananaskis (2004), Kandy (2006), Yosimite (2009), Istanbul (2010) and Corsica (2012). The QSIP workshop is a high level scientific conference that aims to bring together scientists, engineers, industrial organizations, students and users in order to discuss recent advances, and to share the "State of the Art" in this field. QSIP conferences provide an international forum for attendees to present and discuss progress in infrared device physics and modeling, materials growth and processing issues, focal plane array development and characterization.

  17. 9th Caribbean Geological Conference

    NASA Astrophysics Data System (ADS)

    Draper, Gren

    The ninth in a series of Caribbean Geological Conferences, which are held every 3 or 4 years, took place in Santo Domingo, capital of the Dominican Republic, from the 15th to 26th of August 1980. The conference, which was sponsored by the government of the Dominican Republic and the Universidad Catolica Madre y Maestra, was preceded by 2 days of field trips and was opened by President Antonio Guzman on the evening of the 17th of August. Generous support was provided by Alcoa Exploration Co., Falconbridge Dominicana, and Rosario Dominicana.Geologists and geophysicists from 25 countries presented about 130 papers on a wide variety of topics ranging from geophysics to paleontology. While the whole Caribbean area was discussed, there was special emphasis on the northern Caribbean and Hispaniola, as befitted the site of the conference. The contribution of workers from the Dirección General de Mineriá was particularly notable.

  18. 2002 Microgravity Materials Science Conference

    NASA Technical Reports Server (NTRS)

    Gillies, Donald (Editor); Ramachandran, Narayanan (Editor); Murphy, Karen (Editor); McCauley, Dannah (Editor); Bennett, Nancy (Editor)

    2003-01-01

    The 2002 Microgravity Materials Science Conference was held June 25-26, 2002, at the Von Braun Center, Huntsville, Alabama. Organized by the Microgravity Materials Science Discipline Working Group, sponsored by the Physical Sciences Research Division, NASA Headquarters, and hosted by NASA Marshall Space Flight Center and member institutions under the Cooperative Research in Biology and Materials Science (CORBAMS) agreement, the conference provided a forum to review the current research and activities in materials science, discuss the envisioned long-term goals, highlight new crosscutting research areas of particular interest to the Physical Sciences Research Division, and inform the materials science community of research opportunities in reduced gravity. An abstracts book was published and distributed at the conference to the approximately 240 people attending, who represented industry, academia, and other NASA Centers. This CD-ROM proceedings is comprised of the research reports submitted by the Principal Investigators in the Microgravity Materials Science program.

  19. PCNA Mono-ubiquitination and Activation of Translesion DNA Polymerases by DNA Polymerase α

    PubMed Central

    Suzuki, Motoshi; Niimi, Atsuko; Limsirichaikul, Siripan; Tomida, Shuta; Miao, Huang Qin; Izuta, Shunji; Usukura, Jiro; Itoh, Yasutomo; Hishida, Takashi; Akashi, Tomohiro; Nakagawa, Yoshiyuki; Kikuchi, Akihiko; Pavlov, Youri; Murate, Takashi; Takahashi, Takashi

    2014-01-01

    SUMMARY Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol α, PCNA was spontaneously mono-ubiquitinated. Pol α L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol α errors, pol ζ participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol δ mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol η) suppressed this defect. These data suggest that nucleotide misincorporation by pol α induces exposure of single-stranded DNA, PCNA mono-ubiquitination, and activates TLS pols. PMID:19279190

  20. Backtracking dynamics of RNA polymerase: pausing and error correction

    NASA Astrophysics Data System (ADS)

    Sahoo, Mamata; Klumpp, Stefan

    2013-09-01

    Transcription by RNA polymerases is frequently interrupted by pauses. One mechanism of such pauses is backtracking, where the RNA polymerase translocates backward with respect to both the DNA template and the RNA transcript, without shortening the transcript. Backtracked RNA polymerases move in a diffusive fashion and can return to active transcription either by diffusive return to the position where backtracking was initiated or by cleaving the transcript. The latter process also provides a mechanism for proofreading. Here we present some exact results for a kinetic model of backtracking and analyse its impact on the speed and the accuracy of transcription. We show that proofreading through backtracking is different from the classical (Hopfield-Ninio) scheme of kinetic proofreading. Our analysis also suggests that, in addition to contributing to the accuracy of transcription, backtracking may have a second effect: it attenuates the slow down of transcription that arises as a side effect of discriminating between correct and incorrect nucleotides based on the stepping rates.

  1. Direct Characterization of Transcription Elongation by RNA Polymerase I

    PubMed Central

    Ucuncuoglu, Suleyman; Engel, Krysta L.; Purohit, Prashant K.; Dunlap, David D.; Schneider, David A.

    2016-01-01

    RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for more than 60% of transcription in a growing cell. Despite this fundamental role that directly impacts cell growth and proliferation, the kinetics of transcription by Pol I are poorly understood. This study provides direct characterization of S. Cerevisiae Pol I transcription elongation using tethered particle microscopy (TPM). Pol I was shown to elongate at an average rate of approximately 20 nt/s. However, the maximum speed observed was, in average, about 60 nt/s, comparable to the rate calculated based on the in vivo number of active genes, the cell division rate and the number of engaged polymerases observed in EM images. Addition of RNA endonucleases to the TPM elongation assays enhanced processivity. Together, these data suggest that additional transcription factors contribute to efficient and processive transcription elongation by RNA polymerase I in vivo. PMID:27455049

  2. RNA polymerase IV functions in paramutation in Zea mays.

    PubMed

    Erhard, Karl F; Stonaker, Jennifer L; Parkinson, Susan E; Lim, Jana P; Hale, Christopher J; Hollick, Jay B

    2009-02-27

    Plants have distinct RNA polymerase complexes (Pol IV and Pol V) with largely unknown roles in maintaining small RNA-associated gene silencing. Curiously, the eudicot Arabidopsis thaliana is not affected when either function is lost. By use of mutation selection and positional cloning, we showed that the largest subunit of the presumed maize Pol IV is involved in paramutation, an inherited epigenetic change facilitated by an interaction between two alleles, as well as normal maize development. Bioinformatics analyses and nuclear run-on transcription assays indicate that Pol IV does not engage in the efficient RNA synthesis typical of the three major eukaryotic DNA-dependent RNA polymerases. These results indicate that Pol IV employs abnormal RNA polymerase activities to achieve genome-wide silencing and that its absence affects both maize development and heritable epigenetic changes.

  3. In-ice evolution of RNA polymerase ribozyme activity

    PubMed Central

    Attwater, James; Wochner, Aniela; Holliger, Philipp

    2014-01-01

    Mechanisms of molecular self-replication have the potential to shed light upon the origins of life. In particular, self-replication through RNA-catalysed templated RNA synthesis is thought to have supported a primordial ‘RNA World’. However, existing polymerase ribozymes lack the capacity to synthesise RNAs approaching their own size. Here we report the in vitro evolution of such catalysts directly in the RNA-stabilising medium of water-ice, which yielded RNA polymerase ribozymes specifically adapted to sub-zero temperatures and able to synthesise RNA in ices at temperatures as low as −19°C. Combination of cold-adaptive mutations with a previously described 5′ extension operating at ambient temperatures enabled the design of a first polymerase ribozyme capable of catalysing the accurate synthesis of an RNA sequence longer than itself (adding up to 206 nucleotides), an important stepping stone towards RNA self-replication. PMID:24256864

  4. Modulation of RNA polymerase assembly dynamics in transcriptional regulation

    PubMed Central

    Gorski, Stanislaw A.; Snyder, Sara K.; John, Sam; Grummt, Ingrid; Misteli, Tom

    2008-01-01

    The interaction of transcription factors with target genes is highly dynamic. Whether the dynamic nature of these interactions is merely an intrinsic property of transcriptions factors or serves a regulatory role is unknown. Here, we have used single cell fluorescence imaging combined with computational modeling and chromatin immunoprecipitation to analyze transcription complex dynamics in gene regulation during the cell cycle in living cells. We demonstrate a link between the dynamics of RNA polymerase I (RNA pol I) assembly and transcriptional output. We show that transcriptional upregulation is accompanied by prolonged retention of RNA pol I components at the promoter, resulting in longer promoter dwell time, and an increase in the steady state population of assembling polymerase. As a consequence, polymerase assembly efficiency, and ultimately, an rate of entry into processive elongation are elevated. Our results show that regulation of rDNA transcription in vivo occurs via modulation of the efficiency of transcription complex subunit capture and assembly. PMID:18498750

  5. Creative template-dependent synthesis by human polymerase mu

    PubMed Central

    Moon, Andrea F.; Gosavi, Rajendrakumar A.; Kunkel, Thomas A.; Pedersen, Lars C.; Bebenek, Katarzyna

    2015-01-01

    Among the many proteins used to repair DNA double-strand breaks by nonhomologous end joining (NHEJ) are two related family X DNA polymerases, Pol λ and Pol µ. Which of these two polymerases is preferentially used for filling DNA gaps during NHEJ partly depends on sequence complementarity at the break, with Pol λ and Pol µ repairing complementary and noncomplementary ends, respectively. To better understand these substrate preferences, we present crystal structures of Pol µ on a 2-nt gapped DNA substrate, representing three steps of the catalytic cycle. In striking contrast to Pol λ, Pol µ “skips” the first available template nucleotide, instead using the template base at the 5′ end of the gap to direct nucleotide binding and incorporation. This remarkable divergence from canonical 3′-end gap filling is consistent with data on end-joining substrate specificity in cells, and provides insights into polymerase substrate choices during NHEJ. PMID:26240373

  6. Structural Basis of RNA Polymerase I Transcription Initiation.

    PubMed

    Engel, Christoph; Gubbey, Tobias; Neyer, Simon; Sainsbury, Sarah; Oberthuer, Christiane; Baejen, Carlo; Bernecky, Carrie; Cramer, Patrick

    2017-03-23

    Transcription initiation at the ribosomal RNA promoter requires RNA polymerase (Pol) I and the initiation factors Rrn3 and core factor (CF). Here, we combine X-ray crystallography and cryo-electron microscopy (cryo-EM) to obtain a molecular model for basal Pol I initiation. The three-subunit CF binds upstream promoter DNA, docks to the Pol I-Rrn3 complex, and loads DNA into the expanded active center cleft of the polymerase. DNA unwinding between the Pol I protrusion and clamp domains enables cleft contraction, resulting in an active Pol I conformation and RNA synthesis. Comparison with the Pol II system suggests that promoter specificity relies on a distinct "bendability" and "meltability" of the promoter sequence that enables contacts between initiation factors, DNA, and polymerase.

  7. Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II.

    PubMed Central

    Archambault, J; Lacroute, F; Ruet, A; Friesen, J D

    1992-01-01

    Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII. Images PMID:1508210

  8. Yeast Cells Expressing the Human Mitochondrial DNA Polymerase Reveal Correlations between Polymerase Fidelity and Human Disease Progression*

    PubMed Central

    Qian, Yufeng; Kachroo, Aashiq H.; Yellman, Christopher M.; Marcotte, Edward M.; Johnson, Kenneth A.

    2014-01-01

    Mutations in the human mitochondrial polymerase (polymerase-γ (Pol-γ)) are associated with various mitochondrial disorders, including mitochondrial DNA (mtDNA) depletion syndrome, Alpers syndrome, and progressive external opthamalplegia. To correlate biochemically quantifiable defects resulting from point mutations in Pol-γ with their physiological consequences, we created “humanized” yeast, replacing the yeast mtDNA polymerase (MIP1) with human Pol-γ. Despite differences in the replication and repair mechanism, we show that the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and other components of the replisome. We also examined the effects of four disease-related point mutations (S305R, H932Y, Y951N, and Y955C) and an exonuclease-deficient mutant (D198A/E200A). In haploid cells, each mutant results in rapid mtDNA depletion, increased mutation frequency, and mitochondrial dysfunction. Mutation frequencies measured in vivo equal those measured with purified enzyme in vitro. In heterozygous diploid cells, wild-type Pol-γ suppresses mutation-associated growth defects, but continuous growth eventually leads to aerobic respiration defects, reduced mtDNA content, and depolarized mitochondrial membranes. The severity of the Pol-γ mutant phenotype in heterozygous diploid humanized yeast correlates with the approximate age of disease onset and the severity of symptoms observed in humans. PMID:24398692

  9. Mechanics of Textile Composites Conference

    NASA Technical Reports Server (NTRS)

    Poe, Clarence C. (Editor); Harris, Charles E. (Editor)

    1995-01-01

    This document is a compilation of papers presented at the Mechanics of Textile Composites Conference in Hampton, Virginia, December 6-8, 1994. This conference was the culmination of a 3-year program that was initiated by NASA late in 1990 to develop mechanics of textile composites in support of the NASA Advance Composites Technology Program (ACT). The goal of the program was to develop mathematical models of textile preform materials and test methods to facilitate structural analysis and design. Participants in the program were from NASA, academia, and industry.

  10. Mechanics of Textile Composites Conference

    SciTech Connect

    Poe, C.C.; Harris, C.E.

    1995-10-01

    This document is a compilation of papers presented at the Mechanics of Textile Composites Conference in Hampton, Virginia, December 6-8, 1994. This conference was the culmination of a 3-year program that was initiated by NASA late in 1990 to develop mechanics of textile composites in support of the NASA Advance Composites Technology Program (ACT). The goal of the program was to develop mathematical models of textile preform materials and test methods to facilitate structural analysis and design. Participants in the program were from NASA, academia, and industry. Separate abstracts were prepared for articles from this document.

  11. Chapman Conference on Rainfall Fields

    NASA Astrophysics Data System (ADS)

    Gupta, V. K.

    The Chapman Conference on Rainfall Fields, sponsored by AGU, was the first of its kind; it was devoted to strengthening scientific interaction between the North American and Latin American geophysics communities. It was hosted by Universidad Simon Bolivar and Instituto Internacional de Estudios Avanzados, in Caracas, Venezuela, during March 24-27, 1986. A total of 36 scientists from Latin America, the United States, Canada, and Europe participated. The conference, which was convened by I. Rodriguez-Iturbe (Universidad Simon Bolivar) and V. K. Gupta (University of Mississippi, University), brought together hydrologists, meteorologists, and mathematicians/statisticians in the name of enhancing an interdisciplinary focus on rainfall research.

  12. First Stars III Conference Summary

    NASA Astrophysics Data System (ADS)

    O'Shea, B. W.; McKee, C. F.; Heger, A.; Abel, T.

    2008-03-01

    The understanding of the formation, life, and death of Population III stars, as well as the impact that these objects had on later generations of structure formation, is one of the foremost issues in modern cosmological research and has been an active area of research during the past several years. We summarize the results presented at "First Stars III," a conference sponsored by Los Alamos National Laboratory, the Kavli Institute for Particle Astrophysics and Cosmology, and the Joint Institute for Nuclear Astrophysics. This conference, the third in a series, took place in July 2007 at the La Fonda Hotel in Santa Fe, New Mexico, U.S.A.

  13. Ethnic minority energy conference: report

    SciTech Connect

    Not Available

    1984-01-01

    The report of a 1977 energy conference sponsored by the National Association for the Advancement of Colored People summarizes the basic concern that US energy policy was not addressing the importance of full employment or the impact of rising energy costs on the poor. Conference speakers spoke of the social and economic changes that are needed if minorities are to participate in the economics of the technological age. These include better educational opportunities and cooperation between civil rights groups and energy planners. Other topics were venture opportunities for minorities in energy-related fields and opportunities for minority advocacy and energy efficiency actions.

  14. Conference Proceedings: Health Manpower Planning Conference. May, 1971.

    ERIC Educational Resources Information Center

    Hospital Educational and Research Foundation of Pennsylvania, Camp Hill.

    A two-day Health Manpower Planning Conference was held at State College, Pennsylvania, in 1971. The speakers represented a variety of organizations with responsibility for aspects of manpower planning and others not normally involved in planning. Program content was structured in such a way as to broaden the context within which manpower issues…

  15. Nineteenth annual actinide separations conference: Conference program and abstracts

    SciTech Connect

    Bronson, M.

    1995-12-31

    This report contains the abstracts from the conference presentations. Sessions were divided into the following topics: Waste treatment; Spent fuel treatment; Issues and responses to Defense Nuclear Facility Safety Board 94-1; Pyrochemical technologies; Disposition technologies; and Aqueous separation technologies.

  16. Inter Association Child Care Conference. Conference Proceedings 1979.

    ERIC Educational Resources Information Center

    Austin, David, Ed.

    This publication of the proceedings of the Inter Association Child Care Conference includes a debate for and against professionalization in the field of child care. A section on meeting the treatment needs of children through educational preparation of child care practitioners discusses background factors, levels of education for practitioners,…

  17. Kinetics and thermodynamics of exonuclease-deficient DNA polymerases

    NASA Astrophysics Data System (ADS)

    Gaspard, Pierre

    2016-04-01

    A kinetic theory is developed for exonuclease-deficient DNA polymerases, based on the experimental observation that the rates depend not only on the newly incorporated nucleotide, but also on the previous one, leading to the growth of Markovian DNA sequences from a Bernoullian template. The dependencies on nucleotide concentrations and template sequence are explicitly taken into account. In this framework, the kinetic and thermodynamic properties of DNA replication, in particular, the mean growth velocity, the error probability, and the entropy production are calculated analytically in terms of the rate constants and the concentrations. Theory is compared with numerical simulations for the DNA polymerases of T7 viruses and human mitochondria.

  18. Gene and library synthesis without amplification: polymerase step reaction (PSR).

    PubMed

    Lee, Zhuo-Bin; Firnhaber, Christopher; Clarke, Jesse; DeDecker, Brian S

    2015-09-01

    Current gene synthesis methods often incorporate a PCR amplification step in order to yield final material sufficient for resolution from multiple off-products. These amplification steps can cause stochastic sampling effects that propagate errors in gene synthesis or decrease variability when applied to the construction of randomized libraries. We have developed a simple DNA polymerase-based gene synthesis reaction, polymerase step reaction (PSR), that assembles DNA oligonucleotides in a unidirectional fashion without the need for amplification. We demonstrate that PSR is efficient, with little off-product production, no detectable error propagation, and maximized variability in the synthesis of a phage display library.

  19. DIFFERENTIAL ANALYZER

    DOEpatents

    Sorensen, E.G.; Gordon, C.M.

    1959-02-10

    Improvements in analog eomputing machines of the class capable of evaluating differential equations, commonly termed differential analyzers, are described. In general form, the analyzer embodies a plurality of basic computer mechanisms for performing integration, multiplication, and addition, and means for directing the result of any one operation to another computer mechanism performing a further operation. In the device, numerical quantities are represented by the rotation of shafts, or the electrical equivalent of shafts.

  20. MassTag Polymerase Chain Reaction for Differential Diagnosis of Viral Hemorrhagic Fevers

    DTIC Science & Technology

    2006-04-01

    and Sabiá virus (Arenaviridae); Rift Valley fever virus (RVFV), Crimean-Congo hemorrhagic fever virus (CCHFV), and hantaviruses (Bunyaviridae); and...agents are encountered out of their natu- ral geographic context. Vaccines have been developed for YFV, RVFV, Junín virus, KFDV, and hantaviruses (3–7...239–61. 6. Hooper JW, Li D. Vaccines against hantaviruses . Curr Top Microbiol Immunol. 2001;256:171–91. 7. Dandawate CN, Desai GB, Achar TR, Banerjee K

  1. Dynamics of interaction of RNA polymerase II with nucleosomes. II. During read-through and elongation.

    PubMed Central

    Bhargava, P.

    1993-01-01

    The sulfhydryl-specific fluorescence probe 1,5-IAEDANS (5-(2-((iodoacetyl)amino)ethyl)amino-naphthalene-1-sulfonic acid) was attached to the single cysteine of H3, and reconstituted fluorescent mononucleosomes were used as the template for in vitro transcription by the yeast RNA polymerase II (pol II). DNase I digestion analysis revealed that transcription of nucleosomes by pol II resulted in an overall loosening of the structure. Monitoring the transcription event by steady-state fluorescence analysis showed that nucleosomes only partially open during transcription. This opening is transient in nature, and nucleosomes close back as soon as the pol II falls off the template. Thus, using the technique of fluorescence spectroscopy, partial opening of nucleosome structure could be differentiated from complete dissociation into free DNA and histone octamer, a distinction that may not be possible by techniques like gel electrophoresis. Time-resolved fluorescence emission spectroscopy suggested that during read-through of the template by the pol II, histone octamers do not fall off the DNA. Only minor conformational changes within the histone octamer take place to accommodate the transcribing polymerase. PMID:8298468

  2. A Good Day Out? Conferences and Learning.

    ERIC Educational Resources Information Center

    Richards, Mary

    1993-01-01

    Attendance at conferences provides opportunities for developing listening, notetaking, and questioning skills as well as confidence and self-esteem. Planning conferences provides other experiential learning through such roles as entrepreneur, administrator, troubleshooter, and organizer. (SK)

  3. Conferences in Context: Status, Communication, and Evaluation

    ERIC Educational Resources Information Center

    Wagner, L. Wesley; Brinkerhoff, Merlin

    1975-01-01

    The relationship between managers and three variables are examined: time commitments of managers to staff conferences, status or rank of managers, and the quality of communicative exchange in conference settings. (Author/DEP)

  4. 7 CFR 1.167 - Conference.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... that conducting the conference by audio-visual telecommunication: (i) Is necessary to prevent prejudice... in the conference: (i) Is necessary to prevent prejudice to a party; (ii) Is necessary because of...

  5. 1995 International Sherwood Fusion Theory Conference

    SciTech Connect

    1995-07-01

    This book is a guide to the 1995 International Sherwood Fusion Theory Conference. It consists largely of abstracts of the oral and poster presentations that were to be made, and gives some general information about the conference and its schedule.

  6. 15 CFR 25.19 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...) Upon the motion of any party, the ALJ shall schedule at least one prehearing conference at a reasonable time in advance of the hearing. (c) The ALJ may use prehearing conferences to discuss the following:...

  7. 45 CFR 79.19 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... the motion of any party, the ALJ shall schedule at least one prehearing conference at a reasonable time in advance of the hearing. (c) The ALJ may use prehearing conferences to discuss the following:...

  8. 48 CFR 242.503 - Postaward conferences.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 3 2010-10-01 2010-10-01 false Postaward conferences. 242.503 Section 242.503 Federal Acquisition Regulations System DEFENSE ACQUISITION REGULATIONS SYSTEM... 242.503 Postaward conferences....

  9. 48 CFR 242.503 - Postaward conferences.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 3 2014-10-01 2014-10-01 false Postaward conferences. 242.503 Section 242.503 Federal Acquisition Regulations System DEFENSE ACQUISITION REGULATIONS SYSTEM... 242.503 Postaward conferences....

  10. 38 CFR 59.123 - Conference.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... recommend that a conference (such as a design development conference) be held in VA Central Office in Washington, DC, to provide an opportunity for the State and its architects to discuss requirements for...

  11. Technology Utilization Conference Series, volume 1

    NASA Technical Reports Server (NTRS)

    1975-01-01

    The design, development, and results of a series of technology utilization conferences are presented. The conference series represents the development of a viable and successful means of encouraging the transfer of technology to the minority business community.

  12. Apollo 11 Lunar Science Conference

    ERIC Educational Resources Information Center

    Cochran, Wendell

    1970-01-01

    Report of a conference called to discuss the findings of 142 scientists from their investigations of samples of lunar rock and soil brought back by the Apollo 11 mission. Significant findings reported include the age and composition of the lunar samples, and the absence of water and organic matter. Much discussed was the origin and structure of…

  13. Economic Downturn Limits Conference Travel

    ERIC Educational Resources Information Center

    Young, Jeffrey R.

    2009-01-01

    Attendance is down at many academic and professional conferences in higher education this year, and next year's numbers are expected to be far worse, as campus budgets take further beatings. With many colleges limiting travel to professors or administrators who are speaking at events they are attending, will anyone be left in the audience? A new…

  14. The United Nations Water Conference

    ERIC Educational Resources Information Center

    United Nations and Water, 1977

    1977-01-01

    This Water Conference adapted a set of detailed action recommendations on various aspects of water resources development and management and decided that this set of recommendations would be known as the "Mar del Plata Action Plan." This article presents an abridged version of selected recommendations in the Action Plan. (Author/MA)

  15. NOLPE Conference Proceedings, November 1968.

    ERIC Educational Resources Information Center

    National Organization on Legal Problems of Education, Topeka, KS.

    This booklet contains the texts of speeches given at the 1968 National Organization on Legal Problems of Education conference on school law. The topics covered in the speeches include student rights, dress codes, defacto segregation, education and religious freedom, doctoral dissertations in school law, master contracts, and teacher strikes. (JF)

  16. Ozone Conference II: Abstract Proceedings

    SciTech Connect

    1999-11-01

    Ozone Conference II: Pre- and Post-Harvest Applications Two Years After Gras, was held September 27-28, 1999 in Tulare, California. This conference, sponsored by EPRI's Agricultural Technology Alliance and Southern California Edison's AgTAC facility, was coordinated and organized by the on-site ATA-AgTAC Regional Center. Approximately 175 people attended the day-and-a-half conference at AgTAC. During the Conference twenty-two presentations were given on ozone food processing and agricultural applications. Included in the presentations were topics on: (1) Ozone fumigation; (2) Ozone generation techniques; (3) System and design applications; (4) Prewater treatment requirements; (5) Poultry water reuse; (6) Soil treatments with ozone gas; and (7) Post-harvest aqueous and gaseous ozone research results. A live videoconference between Tulare and Washington, D.C. was held to discuss the regulators' view from inside the beltway. Attendees participated in two Roundtable Question and Answer sessions and visited fifteen exhibits and demonstrations. The attendees included university and governmental researchers, regulators, consultants and industry experts, technology developers and providers, and corporate and individual end-users. This report is comprised of the Abstracts of each presentation, biographical sketches for each speaker and a registration/attendees list.

  17. National Environmental Manpower Planning Conference.

    ERIC Educational Resources Information Center

    Environmental Career Center, Inc., Washington, DC.

    The national planning conference was held to acquaint State and local environmental agencies with available resources and Federal/State activities related to the development and utilization of an environmental workforce. The 200 participants and 48 speakers represented Federal, State, local, and private agencies as well as professional…

  18. Columbine Author Speaks at Conference

    ERIC Educational Resources Information Center

    Cook, Andrea J.

    2004-01-01

    Brooks Brown, the author of No Easy Answers: The Truth Behind Death at Columbine High School, spoke at the 11th annual Reclaiming Youth International No Disposable Kids conference at Rushmore Plaza Holiday Inn. Brown, who worked with Michael Moore on his Academy Award winning documentary, "Bowling for Columbine," said the letters he…

  19. IEEE Conference Publications in Libraries.

    ERIC Educational Resources Information Center

    Johnson, Karl E.

    1984-01-01

    Conclusions of surveys (63 libraries, OCLC database, University of Rhode Island users) assessing handling of Institute of Electrical and Electronics Engineers (IEEE) conference publications indicate that most libraries fully catalog these publications using LC cataloging, and library patrons frequently require series access to publications. Eight…

  20. 2001 MAPLD International Conference Proceedings

    NASA Technical Reports Server (NTRS)

    2002-01-01

    This CD contains the proceedings from the '2001 MAPLD International Conference', held on 11-13 September 2001 at Johns Hopkins University. Sessions included the following: (1) Applications: Military and Aerospace; (2) Design 1: Processors, Logic, and Programmable Devices; (3) Reliability: Devices and The Effects of the Radiation Environment; (4) Design 2: Systems; and (5) Design 3: Fault Tolerance.

  1. What Good Are Conferences, Anyway?

    ERIC Educational Resources Information Center

    Pietro, David C.

    1996-01-01

    According to Frederick Herzberg's studies of employee motivation, humans are driven by motivating factors that allow them to grow psychologically and hygiene factors that help them meet physical needs. Good education conferences can enhance both factors by helping principals refocus their energies, exchange ideas with trusted colleagues, and view…

  2. SLA's Conference Wrap-Up.

    ERIC Educational Resources Information Center

    Shepherd, Susan J., Comp.

    1987-01-01

    Encapsulates a wrap-up session on technical programs presented at the Special Libraries Association's June 1987 conference, including presentations in the areas of information technology, management, education, and international issues. Ethics, value, leadership, international issues, image, and risk are identified as major themes of the…

  3. 78 FR 23786 - Settlement Conference

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-22

    ... noticing a recent order convening a settlement conference between GameFly, Inc. and the Postal Service.... Introduction II. Background III. GameFly Motion, Postal Service Reply, and GameFly Response IV. Analysis V... Court's mandate, on March 7, 2013, GameFly, Inc. (GameFly) filed a motion requesting the Commission...

  4. Space Station Freedom Utilization Conference

    NASA Technical Reports Server (NTRS)

    1992-01-01

    The topics addressed in Space Station Freedom Utilization Conference are: (1) space station freedom overview and research capabilities; (2) space station freedom research plans and opportunities; (3) life sciences research on space station freedom; (4) technology research on space station freedom; (5) microgravity research and biotechnology on space station freedom; and (6) closing plenary.

  5. 22 CFR 224.19 - Prehearing conferences.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 22 Foreign Relations 1 2011-04-01 2011-04-01 false Prehearing conferences. 224.19 Section 224.19 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT IMPLEMENTATION OF THE PROGRAM FRAUD CIVIL REMEDIES ACT § 224.19 Prehearing conferences. (a) The ALJ may schedule prehearing conferences as...

  6. AFA Support for Research: Journals and Conferences.

    ERIC Educational Resources Information Center

    Sillars, Malcolm O.; Zarefsky, David

    2000-01-01

    Claims the American Forensic Association's (AFA) publications and conferences represent its principal commitment to research. Presents a review of the journals and conferences under AFA sponsorship. Reviews the strong commitment to research on the part of the AFA, particularly as it is manifested in the association's journal and in conferences.…

  7. Teacher Centering: A National Institute. Conference Report.

    ERIC Educational Resources Information Center

    Tague, Linda Clark, Ed.; And Others

    This report is organized around six chapters: (1) "How This Institute Came About"; (2) "Agenda"; (3) "Teacher Centering in 1976: The Real Experience"; (4) "Description of Teacher Centers"; (5) "Conference Reactions"; and (6) "Conference Directory of Participants." The first chapter discusses the sponsors, and organizers of the conference. Chapter…

  8. Proceedings Region East Perceptual Motor Conference.

    ERIC Educational Resources Information Center

    American Alliance for Health, Physical Education, and Recreation, Washington, DC.

    This book of conference proceeding presents speeches and panel discussions from the Region East Perceptual-Motor Conference. The purpose of the conference was to seek an understanding of children and their perceptual-motor development through (a) exchange of knowledge and practices in perceptual-motor development, (b) examination of program…

  9. 10 CFR 13.19 - Prehearing conferences.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 1 2013-01-01 2013-01-01 false Prehearing conferences. 13.19 Section 13.19 Energy NUCLEAR REGULATORY COMMISSION PROGRAM FRAUD CIVIL REMEDIES § 13.19 Prehearing conferences. (a) The ALJ may schedule prehearing conferences as appropriate. (b) Upon the motion of any party, the ALJ shall schedule at least...

  10. 43 CFR 4.430 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 43 Public Lands: Interior 1 2010-10-01 2010-10-01 false Prehearing conferences. 4.430 Section 4... Questions of Fact § 4.430 Prehearing conferences. (a) The administrative law judge may, in his discretion... representatives to appear at a specified time and place for a prehearing conference to consider: (1)...

  11. 49 CFR 386.55 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 5 2010-10-01 2010-10-01 false Prehearing conferences. 386.55 Section 386.55... General Rules and Hearings § 386.55 Prehearing conferences. (a) Convening. At any time before the hearing... parties or their counsel to participate with him/her in a prehearing conference to consider the...

  12. 31 CFR 16.19 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Prehearing conferences. 16.19 Section... THE PROGRAM FRAUD CIVIL REMEDIES ACT OF 1986 § 16.19 Prehearing conferences. (a) The ALJ may schedule prehearing conferences as appropriate. (b) Upon the motion of any party, the ALJ shall schedule at least...

  13. 29 CFR 1921.9 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 7 2010-07-01 2010-07-01 false Prehearing conferences. 1921.9 Section 1921.9 Labor... WORKERS' COMPENSATION ACT Prehearing Procedures § 1921.9 Prehearing conferences. (a) Upon his own motion... with him for a conference to consider: (1) Simplification of the issues; (2) Necessity or...

  14. 16 CFR 1025.21 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Prehearing conferences. 1025.21 Section 1025... Prehearing conferences. (a) When held. Except when the presiding officer determines that unusual circumstances would render it impractical or valueless, a prehearing conference shall be held in person or...

  15. 22 CFR 906.5 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 22 Foreign Relations 2 2010-04-01 2010-04-01 true Prehearing conferences. 906.5 Section 906.5 Foreign Relations FOREIGN SERVICE GRIEVANCE BOARD HEARINGS § 906.5 Prehearing conferences. (a) The Board may in its discretion order a prehearing conference of the parties (which may be presided over by...

  16. 42 CFR 422.1028 - Prehearing conference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Prehearing conference. 422.1028 Section 422.1028....1028 Prehearing conference. (a) At any time before the hearing, the ALJ may call a prehearing conference for the purpose of delineating the issues in controversy, identifying the evidence and...

  17. 14 CFR 1264.118 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 5 2010-01-01 2010-01-01 false Prehearing conferences. 1264.118 Section... PROGRAM FRAUD CIVIL PENALTIES ACT OF 1986 § 1264.118 Prehearing conferences. (a) The presiding officer may schedule prehearing conferences as appropriate. (b) Upon the motion of any party, the presiding...

  18. 12 CFR 263.96 - Conferences.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Conferences. 263.96 Section 263.96 Banks and... PRACTICE FOR HEARINGS Practice Before the Board § 263.96 Conferences. (a) General. The Board's staff may.... If a conference results in a stipulation in connection with a proceeding in which the individual...

  19. 13 CFR 134.221 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Prehearing conferences. 134.221... conferences. Prior to a hearing, the Judge, at his or her own initiative, or upon the motion of any party, may... matter which may assist in the efficient, prompt, and fair determination of the case. The conference...

  20. 20 CFR 725.416 - Conferences.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 3 2010-04-01 2010-04-01 false Conferences. 725.416 Section 725.416... AND HEALTH ACT, AS AMENDED Adjudication of Claims by the District Director § 725.416 Conferences. (a..., the district director may conduct an informal conference in any claim where it appears that...

  1. 40 CFR 209.20 - Conferences.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Conferences. 209.20 Section 209.20... Orders Issued Under Section 11(d) of the Noise Control Act § 209.20 Conferences. (a) At the discretion of the administrative law judge, conferences may be held prior to or during any hearing....

  2. 42 CFR 1005.6 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 5 2010-10-01 2010-10-01 false Prehearing conferences. 1005.6 Section 1005.6... AUTHORITIES APPEALS OF EXCLUSIONS, CIVIL MONEY PENALTIES AND ASSESSMENTS § 1005.6 Prehearing conferences. (a) The ALJ will schedule at least one prehearing conference, and may schedule additional...

  3. 29 CFR 1903.20 - Informal conferences.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 5 2010-07-01 2010-07-01 false Informal conferences. 1903.20 Section 1903.20 Labor... INSPECTIONS, CITATIONS AND PROPOSED PENALTIES § 1903.20 Informal conferences. At the request of an affected... conference for the purpose of discussing any issues raised by an inspection, citation, notice of...

  4. 28 CFR 68.13 - Conferences.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Conferences. 68.13 Section 68.13 Judicial... IMMIGRATION-RELATED EMPLOYMENT PRACTICES, AND DOCUMENT FRAUD § 68.13 Conferences. (a) Purpose and scope. (1... parties or their counsel to participate in a prehearing conference at any reasonable time prior to...

  5. 12 CFR 19.198 - Conferences.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 1 2010-01-01 2010-01-01 false Conferences. 19.198 Section 19.198 Banks and... and Representational Practice Before the OCC; Standards of Conduct § 19.198 Conferences. (a) General... suspension has been commenced. If a conference results in a stipulation in connection with a proceeding...

  6. 24 CFR 180.440 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 1 2010-04-01 2010-04-01 false Prehearing conferences. 180.440... Prior to Hearing § 180.440 Prehearing conferences. (a) Before the commencement of or during the course of the hearing, the ALJ may direct the parties to participate in a conference to expedite the...

  7. 32 CFR 644.391 - Predisposal conference.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 4 2010-07-01 2010-07-01 true Predisposal conference. 644.391 Section 644.391... ESTATE HANDBOOK Disposal Predisposal Action § 644.391 Predisposal conference. (a) Where a substantial Army installation, or portion thereof, is involved, the DE will convene a predisposal conference...

  8. 45 CFR 1606.7 - Informal conference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 4 2010-10-01 2010-10-01 false Informal conference. 1606.7 Section 1606.7 Public... DEBARMENT PROCEDURES; RECOMPETITION § 1606.7 Informal conference. (a) A recipient may submit a request for an informal conference within 30 days of its receipt of the proposed decision. (b) Within 5 days...

  9. 36 CFR 1150.61 - Prehearing conference.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Prehearing conference. 1150... COMPLIANCE BOARD PRACTICE AND PROCEDURES FOR COMPLIANCE HEARINGS Prehearing Conferences and Discovery § 1150.61 Prehearing conference. (a) At any time before a hearing, the judge on his/her own motion or...

  10. 20 CFR 355.19 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Prehearing conferences. 355.19 Section 355.19... REGULATIONS UNDER THE PROGRAM FRAUD CIVIL REMEDIES ACT OF 1986 § 355.19 Prehearing conferences. (a) The ALJ may schedule prehearing conferences as appropriate. (b) Upon the motion of any party, the ALJ...

  11. 49 CFR 1113.4 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 8 2010-10-01 2010-10-01 false Prehearing conferences. 1113.4 Section 1113.4... OF TRANSPORTATION RULES OF PRACTICE ORAL HEARING § 1113.4 Prehearing conferences. (a) Purposes. Upon... for a conference, prior to or during the course of a hearing, or in lieu of personally appearing,...

  12. 24 CFR 1720.260 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 5 2010-04-01 2010-04-01 false Prehearing conferences. 1720.260... Adjudicatory Proceedings Pleadings § 1720.260 Prehearing conferences. (a) Where it will expedite the proceeding... conference to consider: (1) Simplification and clarification of the issues; (2) Necessity or desirability...

  13. 39 CFR 962.10 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 39 Postal Service 1 2010-07-01 2010-07-01 false Prehearing conferences. 962.10 Section 962.10... PROGRAM FRAUD CIVIL REMEDIES ACT § 962.10 Prehearing conferences. (a) At a reasonable time in advance of... by telephone, one or more prehearing conference to discuss the following: (1) Simplification of...

  14. 39 CFR 3001.24 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 39 Postal Service 1 2010-07-01 2010-07-01 false Prehearing conferences. 3001.24 Section 3001.24... Applicability § 3001.24 Prehearing conferences. (a) Initiation and purposes. In any proceeding the Commission or... participants in a proceeding to appear for a prehearing conference for the purposes of considering all...

  15. 24 CFR 26.17 - Prehearing conference.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 1 2010-04-01 2010-04-01 false Prehearing conference. 26.17... Development HEARING PROCEDURES Hearings Before Hearing Officers Discovery § 26.17 Prehearing conference. (a) Prehearing conference. The hearing officer may, sua sponte or at the request of any party, direct counsel...

  16. 15 CFR 785.11 - Prehearing conference.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 15 Commerce and Foreign Trade 2 2010-01-01 2010-01-01 false Prehearing conference. 785.11 Section....11 Prehearing conference. (a) On the ALJ's own motion, or on request of a party, the ALJ may direct the parties to participate in a prehearing conference, either in person or by telephone, to...

  17. 39 CFR 3001.29 - Settlement conferences.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 39 Postal Service 1 2010-07-01 2010-07-01 false Settlement conferences. 3001.29 Section 3001.29... Applicability § 3001.29 Settlement conferences. Any participant in a proceeding may submit offers of settlement or proposals of adjustment at any time and may request a conference between the participants...

  18. 42 CFR 423.1028 - Prehearing conference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Prehearing conference. 423.1028 Section 423.1028... Penalties § 423.1028 Prehearing conference. (a) At any time before the hearing, the ALJ may call a prehearing conference for the purpose of delineating the issues in controversy, identifying the evidence...

  19. 15 CFR 766.12 - Prehearing conference.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 15 Commerce and Foreign Trade 2 2010-01-01 2010-01-01 false Prehearing conference. 766.12 Section... ENFORCEMENT PROCEEDINGS § 766.12 Prehearing conference. (a) The administrative law judge, on the judge's own motion or on request of a party, may direct the parties to participate in a prehearing conference,...

  20. 28 CFR 76.19 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Prehearing conferences. 76.19 Section 76... CIVIL PENALTIES FOR POSSESSION OF CERTAIN CONTROLLED SUBSTANCES § 76.19 Prehearing conferences. (a... or their counsel to participate in a prehearing conference at any reasonable time prior to a...

  1. 31 CFR 15.737-10 - Conferences.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Conferences. 15.737-10 Section 15.737... INTEREST Administrative Enforcement Proceedings § 15.737-10 Conferences. (a) In general. The Director may... administrative disciplinary proceeding has been instituted against him/her. If such conference results in...

  2. 7 CFR 283.29 - Scheduling conference.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 4 2010-01-01 2010-01-01 false Scheduling conference. 283.29 Section 283.29... Procedure for Appeals of QC Claims of Less Than $50,000 § 283.29 Scheduling conference. (a) Time and place. The ALJ shall direct the parties or their counsel to attend a scheduling conference following...

  3. 49 CFR 845.23 - Prehearing conference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 7 2010-10-01 2010-10-01 false Prehearing conference. 845.23 Section 845.23... Prehearing conference. (a) Except as provided in paragraph (d) of this section for expedited hearings, the chairman of the board of inquiry shall hold a prehearing conference with the parties to the hearing at...

  4. 39 CFR 958.10 - Prehearing conferences.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 39 Postal Service 1 2010-07-01 2010-07-01 false Prehearing conferences. 958.10 Section 958.10... conferences. (a) At a reasonable time after issuing the Notice of Docketing and Hearing, and with adequate... prehearing conferences to discuss the following: (1) Simplification of the issues; (2) The necessity...

  5. 46 CFR 502.94 - Prehearing conference.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 9 2010-10-01 2010-10-01 false Prehearing conference. 502.94 Section 502.94 Shipping... Settlement; Prehearing Procedure § 502.94 Prehearing conference. (a)(1) Prior to any hearing, the Commission... prehearing conferences for the purpose of considering any settlement under § 502.91, formulating the...

  6. Ready, Set, Conference! Mastering the Logistics

    ERIC Educational Resources Information Center

    McGuire, Margaret

    2005-01-01

    Planning to have a successful NAEYC Annual Conference experience is similar to preparing for a day of bargain shopping. In this article, the author describes how to get the most out of NAEYC's Annual Conference by analyzing their goals, preparing ahead, and being flexible. As a veteran conference attendee, the author learned from her mistakes and…

  7. [The Third International Conference on Apomixis].

    PubMed

    Sokolv, V A; Tarakanova, T K; Abdyrkhmanova, E A

    2008-11-01

    The materials of the Third International Conference on Apomixis held in Germany are presented. The oral presentations and most posters of the conference are discussed in detail. Based on comparing the materials of three international conferences, the global trends in investigations of apomixis are traced.

  8. 29 CFR 22.19 - Prehearing conferences.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 1 2014-07-01 2013-07-01 true Prehearing conferences. 22.19 Section 22.19 Labor Office of the Secretary of Labor PROGRAM FRAUD CIVIL REMEDIES ACT OF 1986 § 22.19 Prehearing conferences. (a) The ALJ may schedule prehearing conferences as appropriate. (b) Upon the motion of any party, the...

  9. The conferences for undergraduate women in physics

    NASA Astrophysics Data System (ADS)

    Blessing, Susan K.

    2015-12-01

    The American Physical Society Conferences for Undergraduate Women in Physics are the continuation of a grassroots collaborative effort that began in 2006. The goals of the conferences are to increase retention and improve career outcomes of undergraduate women in physics. I describe the conferences, including organization and participant response, and encourage other countries to host similar programs for their undergraduate women.

  10. Women's Participation in Academic Conferences in Israel

    ERIC Educational Resources Information Center

    Eden, Devorah

    2016-01-01

    This article examines the participation of women in academic conferences in Israel, a country in which women are under-represented in academia vertically and horizontally. Data were retrieved from announcements of academic conferences in Israel, for one academic year, covering 56 conferences that attracted 997 participants. Participation was…

  11. A Model for Replication: Conference for Parents.

    ERIC Educational Resources Information Center

    Meriweather, Suzanne; Karnes, Frances A.

    1988-01-01

    The experience of the University of Southern Mississippi in sponsoring "The Parenting the Gifted Children Conference" is described. Discussed are needs assessment, conference format, selection of speakers, budget, public relations, conference evaluation, and physical facilities. Included are a sample evaluation form, a conference…

  12. West Tennessee ACEI 2006 Fall Conference

    ERIC Educational Resources Information Center

    Clifford, Anna; Hailey, Beth

    2007-01-01

    This article presents the West Tennessee ACEI 2006 Fall Conference held at Union University in Jackson, Tennessee on October 14, 2006. The conference theme, Turning the Pages: A Focus on Children's Literature, was emphasized throughout the day. During the conference, the early childhood classroom teachers, preservice teachers, and administrators…

  13. Making Conferences Human Places of Learning

    ERIC Educational Resources Information Center

    Kenny, Michael

    2014-01-01

    Open Space Technology is a cumbersome name for a participative conference model that enables dynamic inclusive engagement and challenges traditional, highly structured hierarchical conference formats. Based on self-organising systems, (Wenger, 1998) Open Space Technology conferences have an open process, start with no agenda and empower the most…

  14. Quantitative Analysis of Transcription Elongation by RNA Polymerase I In Vitro

    PubMed Central

    Schneider, David Alan

    2016-01-01

    The elongation step in transcription has gained attention for its roles in regulation of eukaryotic gene expression and for its influence on RNA processing. Sophisticated genetic analyses have identified factors and/or conditions that may affect transcription elongation rate or processivity; however, differentiation of direct and indirect effects on transcription is difficult using in vivo strategies. Therefore, effective, reproducible in vitro assays have been developed to test whether a given factor or condition can have a direct effect on the kinetics of transcription elongation. We have adapted a fully reconstituted transcription system for RNA polymerase I (Pol I) for kinetic analysis of transcription elongation rate in vitro. The assay described here has proven to be effective in the characterization of defects or enhancement of wild-type transcription elongation by RNA Pol I. Since transcription elongation by RNA Pol I has only recently gained significant attention, this assay will be a valuable resource for years to come. PMID:22113301

  15. The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription.

    PubMed

    Torreira, Eva; Louro, Jaime Alegrio; Pazos, Irene; González-Polo, Noelia; Gil-Carton, David; Duran, Ana Garcia; Tosi, Sébastien; Gallego, Oriol; Calvo, Olga; Fernández-Tornero, Carlos

    2017-03-06

    Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I-Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.

  16. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis.

    PubMed Central

    Telenti, A; Marchesi, F; Balz, M; Bally, F; Böttger, E C; Bodmer, T

    1993-01-01

    A method for the rapid identification of mycobacteria to the species level was developed on the basis of evaluation by the polymerase chain reaction (PCR) of the gene encoding for the 65-kDa protein. The method involves restriction enzyme analysis of PCR products obtained with primers common to all mycobacteria. Using two restriction enzymes, BstEII and HaeIII, medically relevant and other frequent laboratory isolates were differentiated to the species or subspecies level by PCR-restriction enzyme pattern analysis. PCR-restriction enzyme pattern analysis was performed on isolates (n = 330) from solid and fluid culture media, including BACTEC, or from frozen and lyophilized stocks. The procedure does not involve hybridization steps or the use of radioactivity and can be completed within 1 working day. Images PMID:8381805

  17. The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription

    PubMed Central

    Torreira, Eva; Louro, Jaime Alegrio; Pazos, Irene; González-Polo, Noelia; Gil-Carton, David; Duran, Ana Garcia; Tosi, Sébastien; Gallego, Oriol; Calvo, Olga; Fernández-Tornero, Carlos

    2017-01-01

    Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I–Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association. DOI: http://dx.doi.org/10.7554/eLife.20832.001 PMID:28262097

  18. Rapid Detection and Identification of a Pathogen's DNA Using Phi29 DNA Polymerase

    SciTech Connect

    Xu, Y.; Dunn, J.; Gao, S.; Bruno, J. F.; Luft, B. J.

    2008-10-31

    Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.

  19. RNA-Dependent RNA Polymerase Activity in Influenza Virions

    PubMed Central

    Penhoet, Edward; Miller, Henry; Doyle, Michael; Blatti, Stanley

    1971-01-01

    An RNA-dependent RNA polymerase activity has been detected in purified preparations of influenza virus. In contrast to the replicase activity induced in influenza-infected cells, the virion-associated enzyme has an absolute requirement for Mn++. Most of the RNA synthesized in vitro is complementary to virion RNA. PMID:5288388

  20. Stochastic resetting in backtrack recovery by RNA polymerases

    NASA Astrophysics Data System (ADS)

    Roldán, Édgar; Lisica, Ana; Sánchez-Taltavull, Daniel; Grill, Stephan W.

    2016-06-01

    Transcription is a key process in gene expression, in which RNA polymerases produce a complementary RNA copy from a DNA template. RNA polymerization is frequently interrupted by backtracking, a process in which polymerases perform a random walk along the DNA template. Recovery of polymerases from the transcriptionally inactive backtracked state is determined by a kinetic competition between one-dimensional diffusion and RNA cleavage. Here we describe backtrack recovery as a continuous-time random walk, where the time for a polymerase to recover from a backtrack of a given depth is described as a first-passage time of a random walker to reach an absorbing state. We represent RNA cleavage as a stochastic resetting process and derive exact expressions for the recovery time distributions and mean recovery times from a given initial backtrack depth for both continuous and discrete-lattice descriptions of the random walk. We show that recovery time statistics do not depend on the discreteness of the DNA lattice when the rate of one-dimensional diffusion is large compared to the rate of cleavage.

  1. DNA sequencing using electrical conductance measurements of a DNA polymerase

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Shiun; Lee, Chia-Hui; Hung, Meng-Yen; Pan, Hsu-An; Chiou, Jin-Chern; Huang, G. Steven

    2013-06-01

    The development of personalized medicine--in which medical treatment is customized to an individual on the basis of genetic information--requires techniques that can sequence DNA quickly and cheaply. Single-molecule sequencing technologies, such as nanopores, can potentially be used to sequence long strands of DNA without labels or amplification, but a viable technique has yet to be established. Here, we show that single DNA molecules can be sequenced by monitoring the electrical conductance of a phi29 DNA polymerase as it incorporates unlabelled nucleotides into a template strand of DNA. The conductance of the polymerase is measured by attaching it to a protein transistor that consists of an antibody molecule (immunoglobulin G) bound to two gold nanoparticles, which are in turn connected to source and drain electrodes. The electrical conductance of the DNA polymerase exhibits well-separated plateaux that are ~3 pA in height. Each plateau corresponds to an individual base and is formed at a rate of ~22 nucleotides per second. Additional spikes appear on top of the plateaux and can be used to discriminate between the four different nucleotides. We also show that the sequencing platform works with a variety of DNA polymerases and can sequence difficult templates such as homopolymers.

  2. A noncontact temperature measurement method in polymerase chain reaction reactors

    NASA Astrophysics Data System (ADS)

    Sochivko, D. G.; Varlamov, D. A.; Fedorov, A. A.; Kurochkin, V. E.

    2016-04-01

    A new noncontact method for measuring temperatures of liquids, which is based on the fluorescent probes, is proposed. The method is intended for measuring temperatures of reaction media in reactors of devices for polymerase chain reactions in real time and can be used for determining dynamic temperature parameters.

  3. Interactions between the human RNA polymerase II subunits.

    PubMed

    Acker, J; de Graaff, M; Cheynel, I; Khazak, V; Kedinger, C; Vigneron, M

    1997-07-04

    As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione S-transferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.

  4. Continuous in vitro evolution of bacteriophage RNA polymerase promoters

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  5. DNA Polymerase θ: A Unique Multifunctional End-Joining Machine

    PubMed Central

    Black, Samuel J.; Kashkina, Ekaterina; Kent, Tatiana; Pomerantz, Richard T.

    2016-01-01

    The gene encoding DNA polymerase θ (Polθ) was discovered over ten years ago as having a role in suppressing genome instability in mammalian cells. Studies have now clearly documented an essential function for this unique A-family polymerase in the double-strand break (DSB) repair pathway alternative end-joining (alt-EJ), also known as microhomology-mediated end-joining (MMEJ), in metazoans. Biochemical and cellular studies show that Polθ exhibits a unique ability to perform alt-EJ and during this process the polymerase generates insertion mutations due to its robust terminal transferase activity which involves template-dependent and independent modes of DNA synthesis. Intriguingly, the POLQ gene also encodes for a conserved superfamily 2 Hel308-type ATP-dependent helicase domain which likely assists in alt-EJ and was reported to suppress homologous recombination (HR) via its anti-recombinase activity. Here, we review our current knowledge of Polθ-mediated end-joining, the specific activities of the polymerase and helicase domains, and put into perspective how this multifunctional enzyme promotes alt-EJ repair of DSBs formed during S and G2 cell cycle phases. PMID:27657134

  6. Mathematics analysis of polymerase chain reaction kinetic curves.

    PubMed

    Sochivko, D G; Fedorov, A A; Varlamov, D A; Kurochkin, V E; Petrov, R V

    2016-01-01

    The paper reviews different approaches to the mathematical analysis of polymerase chain reaction (PCR) kinetic curves. The basic principles of PCR mathematical analysis are presented. Approximation of PCR kinetic curves and PCR efficiency curves by various functions is described. Several PCR models based on chemical kinetics equations are suggested. Decision criteria for an optimal function to describe PCR efficiency are proposed.

  7. Mapping DNA polymerase errors by single-molecule sequencing

    PubMed Central

    Lee, David F.; Lu, Jenny; Chang, Seungwoo; Loparo, Joseph J.; Xie, Xiaoliang S.

    2016-01-01

    Genomic integrity is compromised by DNA polymerase replication errors, which occur in a sequence-dependent manner across the genome. Accurate and complete quantification of a DNA polymerase's error spectrum is challenging because errors are rare and difficult to detect. We report a high-throughput sequencing assay to map in vitro DNA replication errors at the single-molecule level. Unlike previous methods, our assay is able to rapidly detect a large number of polymerase errors at base resolution over any template substrate without quantification bias. To overcome the high error rate of high-throughput sequencing, our assay uses a barcoding strategy in which each replication product is tagged with a unique nucleotide sequence before amplification. This allows multiple sequencing reads of the same product to be compared so that sequencing errors can be found and removed. We demonstrate the ability of our assay to characterize the average error rate, error hotspots and lesion bypass fidelity of several DNA polymerases. PMID:27185891

  8. [The contamination under polymerase chain reaction studies: problems and solutions].

    PubMed

    Titov, V N; Ameliushkina, V A; Rozhkova, T A

    2015-01-01

    The study was carried out to determine risk factors of false positive and false negative results under polymerase chain reaction-analysis of clinical material. The samples with high viral load can be the source of false positive results. The contamination with nucleic acids can occur at any section of polymerase chain reaction analysis. The study data permitted to establish that the most sensitive stage is isolation and purification of nucleic acids especially under manual mode of operation. The detection of positive signal in most samples of one setting indicates total contamination. The cases when only several samples are polluted are special challenge. The presence of sample with high concentration of viral nucleic acid and several samples with low concentration in one setting means necessity of repeated analysis beginning with stage of isolation of nucleic acid. The analysis of curves of accumulation of products of amplification, their forms and positioning on chart is the obligatory stage of polymerase chain reaction study in real time regimen. These actions permit to exclude the readouts of false negative testing results to departments. The study conclusions are equipotent for polymerase chain reaction testing of any nucleic acid targets.

  9. Detection of Listeria monocytogenes by using the polymerase chain reaction

    SciTech Connect

    Bessesen, M.T.; Luo, Q.; Blaser, M.J.; Ellison, R.T. III.; Rotbart. H.A. )

    1990-09-01

    A method was developed for detection of Listeria monocytogens by polymerase chain reaction amplification followed by agarose gel electrophoresis or dot blot analysis with {sup 32}P-labeled internal probe. The technique identified 95 of 95 L. monocytogenes strains, 0 of 12 Listeria strains of other species, and 0 of 12 non-Listeria strains.

  10. Polymerase Chain Reaction for Detection of Systemic Plant Pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter outlines the advances and application of the polymerase chain reaction (PCR) since its development in 1984 and its enhancements and applications to detection of viruses, viroids and phytoplasma in pome and stone fruits. PCR is probably the most rapidly and widely adopted technology eve...

  11. Tankyrase Polymerization Is Controlled by Its Sterile Alpha Motif and Poly(ADP-Ribose) Polymerase Domains

    PubMed Central

    De Rycker, Manu; Price, Carolyn M.

    2004-01-01

    Tankyrases are novel poly(ADP-ribose) polymerases that have SAM and ankyrin protein-interaction domains. They are found at telomeres, centrosomes, nuclear pores, and Golgi vesicles and have been shown to participate in telomere length regulation. Their other function(s) are unknown, and it has been difficult to envision a common role at such diverse cellular locations. We have shown that tankyrase 1 polymerizes through its sterile alpha motif (SAM) domain to assemble large protein complexes. In vitro polymerization is reversible and still allows interaction with ankyrin-domain binding proteins. Polymerization can also occur in vivo, with SAM-dependent association of overexpressed tankyrase leading to formation of large tankyrase-containing vesicles, disruption of Golgi structure, and inhibition of apical secretion. Finally, tankyrase polymers are dissociated efficiently by poly(ADP-ribosy)lation. This disassembly is prevented by mutation of the PARP domain. Our findings indicate that tankyrase 1 has the unique capacity to promote both assembly and disassembly of large protein complexes. Thus, tankyrases appear to be master scaffolding proteins that regulate the formation of dynamic protein networks at different cellular locations. This implies a common scaffolding function for tankyrases at each location, with specific tankyrase interaction partners conferring location-specific roles to each network, e.g., telomere compaction or regulation of vesicle trafficking. PMID:15509784

  12. Mutation tendency of mutator Plasmodium berghei with proofreading-deficient DNA polymerase δ

    PubMed Central

    Honma, Hajime; Niikura, Mamoru; Kobayashi, Fumie; Horii, Toshihiro; Mita, Toshihiro; Endo, Hiroyoshi; Hirai, Makoto

    2016-01-01

    In this study, we investigated the mutation tendency of a mutator rodent malaria parasite, Plasmodium berghei, with proofreading-deficient DNA polymerase δ. Wild-type and mutator parasites were maintained in mice for over 24 weeks, and the genome-wide accumulated mutations were determined by high-throughput sequencing. The mutator P. berghei had a significant preference for C/G to A/T substitutions; thus, its genome had a trend towards a higher AT content. The mutation rate was influenced by the sequence context, and mutations were markedly elevated at TCT. Some genes mutated repeatedly in replicate passage lines. In particular, knockout mutations of the AP2-G gene were frequent, which conferred strong growth advantages on parasites during the blood stage but at the cost of losing the ability to form gametocytes. This is the first report to demonstrate a biased mutation tendency in malaria parasites, and its results help to promote our basic understanding of Plasmodium genetics. PMID:27845384

  13. Single-molecule measurements of replisome composition and function reveal the mechanism of polymerase exchange

    NASA Astrophysics Data System (ADS)

    Loparo, Joseph

    2011-03-01

    A complete understanding of the molecular mechanisms underlying the functioning of large, multiprotein complexes requires experimental tools capable of simultaneously visualizing molecular architecture and enzymatic activity in real time. I will describe a novel single-molecule assay that combines the flow-stretching of individual DNA molecules to measure the activity of the DNA-replication machinery with the visualization of fluorescently labeled DNA polymerases at the replication fork. By correlating polymerase stoichiometry with DNA synthesis of T7 bacteriophage replisomes, we are able to quantitatively describe the mechanism of polymerase exchange. We find that even at relatively modest polymerase concentration (2 nM), soluble polymerases are recruited to an actively synthesizing replisome, dramatically increasing local polymerase concentration. These excess polymerases remain passively associated with the replisome through electrostatic interactions with the T7 helicase for 50 seconds until a stochastic and transient dissociation of the synthesizing polymerase from the primer-template allows for a polymerase exchange event to occur.

  14. Polymerase chain reaction system using magnetic beads for analyzing a sample that includes nucleic acid

    DOEpatents

    Nasarabadi, Shanavaz [Livermore, CA

    2011-01-11

    A polymerase chain reaction system for analyzing a sample containing nucleic acid includes providing magnetic beads; providing a flow channel having a polymerase chain reaction chamber, a pre polymerase chain reaction magnet position adjacent the polymerase chain reaction chamber, and a post pre polymerase magnet position adjacent the polymerase chain reaction chamber. The nucleic acid is bound to the magnetic beads. The magnetic beads with the nucleic acid flow to the pre polymerase chain reaction magnet position in the flow channel. The magnetic beads and the nucleic acid are washed with ethanol. The nucleic acid in the polymerase chain reaction chamber is amplified. The magnetic beads and the nucleic acid are separated into a waste stream containing the magnetic beads and a post polymerase chain reaction mix containing the nucleic acid. The reaction mix containing the nucleic acid flows to an analysis unit in the channel for analysis.

  15. Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus.

    PubMed

    Bates, H J; Farjah, M; Osman, T A; Buck, K W

    1995-06-01

    A template-bound RNA polymerase was isolated from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus (RCNMV) by differential centrifugation, solubilization with dodecyl beta-D-maltopyranoside, and chromatography on columns of Sephacryl S-400 and Q-Sepharose. Analysis of the purified polymerase by SDS-polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting, showed that it contained virus-encoded proteins of molecular masses 27 kDa and 88 kDa together with several minor proteins possibly of host origin. After removal of endogenous RNA with micrococcal nuclease, the polymerase became template-dependent. It was also template-specific, being able to utilize as templates RNA of two strains of RCNMV, but not RNAs of three viruses in different taxonomic groups, namely cucumber mosaic cucumovirus, tomato bushy stunt tombusvirus and tomato mosaic tobamovirus. The products of RNA polymerase reactions were double-stranded RNAs corresponding to RCNMV RNAs 1 and 2. The ability of the template-dependent RNA polymerase to synthesize RNA was completely inhibited by antibodies to a peptide containing the GDD motif, whereas the activity of the template-bound enzyme was unaffected by these antibodies.

  16. Proceedings: Summer Conference for College Teachers on Applied Mathematics, University of Missouri-Rolla, 1971.

    ERIC Educational Resources Information Center

    Committee on the Undergraduate Program in Mathematics, Berkeley, CA.

    Proceedings from four sessions of the Summer Conference for College Teachers on Applied Mathematics are presented. The four sessions were: (1) Applications of Elementary Calculus, (2) Applications of Linear Algebra, (3) Applications of Elementary Differential Equations, and (4) Applications of Probability and Statistics. Nine lectures were given…

  17. Mammary epithelial morphogenesis and early breast cancer. Evidence of involvement of basal components of the RNA Polymerase I transcription machinery.

    PubMed

    Rossetti, Stefano; Wierzbicki, Andrzej J; Sacchi, Nicoletta

    2016-09-16

    Upregulation of RNA Polymerase (Pol I)-mediated transcription of rRNA and increased ribogenesis are hallmarks of breast cancer. According to several datasets, including The Cancer Genome Atlas (TCGA), amplification/upregulation of genes encoding for basal components of the Pol I transcriptional machinery is frequent at different breast cancer stages. Here we show that knock down of the RNA polymerase I-specific transcription initiation factor RRN3 (TIF-IA) in breast cancer cells is sufficient to reduce rRNA synthesis and inhibit cell proliferation, and second that stable ectopic expression of RRN3 in human mammary epithelial (HME1) cells, by increasing rRNA transcription, confers increased sensitivity to the anti-proliferative effects of a selective Pol I inhibitor. Further, RRN3-overexpressing HME1 cells, when grown in in vitro 3-dimensional (3D) culture, develop into morphologically aberrant acinar structures lacking a lumen and filled with proliferative cells, thus acquiring a morphology resembling in situ ductal breast cancer lesions (DCIS). Consequently, interference with RRN3 control of Pol I transcription seems capable of both compromising mammary epithelial morphogenetic processes at early breast cancer stages, and driving breast cancer progression by fostering proliferation.

  18. Ethics in the computer age. Conference proceedings

    SciTech Connect

    Kizza, J.M.

    1994-12-31

    These proceedings contain the papers presented at the Ethics in the Computer Age conference held in Gatlinburg, Tennessee, November 11-13, 1994. The conference was sponsored by ACM SIGCAS (Computers and Society) to which I am very grateful. The Ethics in the Computer Age conference sequence started in 1991 with the first conference at the campus of the University of Tennessee at Chattanooga. The second was help at the same location a year later. These two conferences were limited to only invited speakers, but their success was overwhelming. This is the third in the sequence and the first truly international one. Plans are already under way for the fourth in 1996.

  19. Multisubunit RNA Polymerases IV and V: Purveyors of Non-Coding RNA for Plant Gene Silencing

    SciTech Connect

    Haag, Jeremy R.; Pikaard, Craig S.

    2011-08-01

    In all eukaryotes, nuclear DNA-dependent RNA polymerases I, II and III synthesize the myriad RNAs that are essential for life. Remarkably, plants have evolved two additional multisubunit RNA polymerases, RNA polymerases IV and V, which orchestrate non-coding RNA-mediated gene silencing processes affecting development, transposon taming, antiviral defence and allelic crosstalk. Biochemical details concerning the templates and products of RNA polymerases IV and V are lacking. However, their subunit compositions reveal that they evolved as specialized forms of RNA polymerase II, which provides the unique opportunity to study the functional diversification of a eukaryotic RNA polymerase family.

  20. Modified pseudomonas oleovorans phaC1 nucleic acids encoding bispecific polyhydroxyalkanoate polymerase

    DOEpatents

    Srienc, Friedrich; Jackson, John K.; Somers, David A.

    2000-01-01

    A genetically engineered Pseudomonas oleovorans phaC1 polyhydroxyalkanoate (PHA) polymerase having tailored substrate specificity is provided. The modified PHA polymerase is preferably a "bispecific" PHA polymerase capable of copolymerizing a short chain length monomer and a medium chain length monomer is provided. Methods for making the modified PHA polymerase and for making nucleic acids encoding the modified PHA polymerase are also disclosed, as are methods of producing PHA using the modified PHA polymerase. The invention further includes methods to assay for altered substrate specificity.

  1. Cairo conference affirms CEDPA priorities.

    PubMed

    1995-01-01

    The International Conference on Population and Development (ICPD) that was held in Cairo during September adopted a 20-year Programme of Action endorsing the empowerment of women as the foundation of sustainable development. 178 countries and more than a 1000 nongovernmental organizations (NGOs), including the Centre for Development and Population Activities (CEDPA), from 100 countries attended the conference and the parallel NGO forum. The final document sets out specific steps for achievement of universal access to a full range of voluntary, quality family planning and reproductive health services for women and men; provision of services for the special needs of adolescents; closure of the gender gap in education; and empowerment of women via education, health care, and economic options. The CEDPA network of alumnae from 30 countries had worked over the 3 years prior to the conference for the inclusion of women's priorities in policies and to achieve consensus among the government and NGO caucuses. 14 alumnae, including Peggy Curlin (CEDPA President and US delegate), were appointed to their countries' delegations and directly influenced the Programme of Action. The NGO Forum provided a place to exchange experiences and expertise; CEDPA mounted an exhibit, "Empowering Women." The network's theme was "Access, Choice, and Participation." With support from the United Nations Population Fund, CEDPA developed a manual, "After Cairo: A Handbook on Advocacy for Women Leaders," which has been distributed at training sessions and workshops and was translated into French (with support from the US Agency for International Development in Mali) for distribution at the Dakar conference in November in preparation for the World Conference on Women. CEDPA and The Global Committee for Cairo honored the secretary-general of the conference, Dr. Nafis Sadik, for her leadership of the ICPD and UNFPA, and Aziza Hussein, co-chair of the NGO steering committee, at a luncheon; Dr. Sadik

  2. Population conference: consensus and conflict.

    PubMed

    Willson, P D

    1984-01-01

    The United Nations-sponsored International Conference on Population held in Mexico City was both a rejection and an affirmation of a new policy of the Reagan administration. The policy denies international family planning funds to nongovernmental organizations that perform or actively promote abortion as a family planning method in other nations. A compromise statement was accepted urging governments to take appropriate measures to discourage abortion as a family planning method and when possible to provide for the humane treatment and counseling of women ho resorted to abortion. The statement on abortion was 1 of 88 reccomendations approved by the conference. The commitment expressed in the 10-year-old World Population Plan of Action to the rights and responsiblity to all people as reaffirmed. The conference also endorsed family life education and sex education as well as suitable family planning, information and services for adolescents, with due consideration given to the role, rights and obligations of parents. Increased support for international population and family planning programs was urged and World Bank President, Clausen, urged a 4-fold increase in international funding by the year 2000. Most of the conference's recommendations re devoted to the broad range of population policy issues, including morbidity and mortality, international and internal migration, the relationship between population and economic development and the status of women. The purpose of the recommendations is to increase the momentum of international support. The Mexico City conference was characterized by a remarkable degree of consensus about population policies with respect to integration with economic development, the need to respect individual rights and the recognition that all nations have sovereign rights to develop and implement their own population policies. Conflict and controversy arose in the areas of the arms race and the Middle East. The US position on abortion funding

  3. Editorial for the Third Pangu Stroke Conference.

    PubMed

    Chen, Gang; Chen, Jun; Ji, Xunming; Xi, Guohua; Zhang, John

    2015-10-01

    The Pangu Stroke Conference has been held annually in China since 2012 and is based on the successful templates of the Princeton Stroke Conference in the United States and the Marburg Conference on Cerebral Ischemia in Germany. All participants in the Pangu Stroke Conference are expert stroke clinicians or stroke basic science researchers of Chinese origin. This conference promotes collaboration between clinicians and basic science researchers and between stroke researchers in mainland China and other parts of the world. The Pangu Stroke Conference fosters translational stroke research, discussions of stroke research milestones, and proposals for future directions. Some of the keynote presentations in the third Pangu Stroke Conference are included in this special issue.

  4. Identification of co-occurring Branchinecta fairy shrimp species from encysted embryos using multiplex polymerase chain reaction

    USGS Publications Warehouse

    Vandergast, A.G.; Wood, D.A.; Simovich, M.; Bohonak, A.J.

    2009-01-01

    Morphological identification of many fairy shrimp species is difficult because distinguishing characters are restricted to adults. We developed two multiplex polymerase chain reaction assays that differentiate among three Branchinecta fairy shrimp with distributional overlap in southern California vernal pools. Two of the species are federally listed as threatened. Molecular identification of Branchinecta from cysts allows for species surveys to be conducted during the dry season, expanding the timeframe for population assessment and providing a less intrusive method of sampling sensitive vernal pool habitats. ?? Published 2009. This article is a US Government work and is in the public domain in the USA.

  5. Crustal differentiation

    NASA Astrophysics Data System (ADS)

    Melekhova, E.; Blundy, J.

    2012-12-01

    Few erupted arc magmas are sufficiently primitive to be in equilibrium with mantle wedge peridotite, meaning a significant volume of arc crust must comprise plutonic cumulates formed during differentiation of primitive basalts. This cumulate material is typically not available for petrological study. A notable exception is the Lesser Antilles arc, which is renowned for the exceptional abundance and variety of cumulate xenoliths. Additionally, several Lesser Antilles islands erupt primitive basaltic magmas that are close to being in mantle equilibrium. The abundance of plutonic cumulate xenolith and presence of primitive basalts make the Lesser Antilles an ideal natural laboratory for understanding crust-building processes. Here we evaluate the chemical consequences of basalt differentiation in the mid to lower crust and uppermost mantle (10 to 30 km) by means of experiments on a primitive basalt from St. Vincent. The results were combined with compositional and textural observation of plutonic cumulate xenoliths from the island. Our goal was to constrain the conditions under which basalt differentiation can generate the observed chemical diversity of erupted magmas at St. Vincent and the compositions of minerals in cumulate xenoliths. Our experimental results show that it is possible to produce a wide compositional range of melts by differentiation at different depths and water contents from the same primitive source. The melts provide a close match to the full range of erupted lavas on the island. The cumulate assemblages, however, have a consistently lower pressure origin (6-10 km). They are formed by crystallisation of ascending melts generated in the deep crust. Phencocrysts in the lavas are distinct from those in cumulates, notably in the absence of amphibole. The phenocrysts demonstrably grew in response to crystallisation at very shallow depth, probably in sub-volcanic magma chambers. Thus St. Vincent shows clear evidence for polybaric crustal

  6. Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases

    PubMed Central

    Masuda, Yuji; Kanao, Rie; Kaji, Kentaro; Ohmori, Haruo; Hanaoka, Fumio; Masutani, Chikahide

    2015-01-01

    Translesion DNA synthesis (TLS) by the Y-family DNA polymerases Polη, Polι and Polκ, mediated via interaction with proliferating cell nuclear antigen (PCNA), is a crucial pathway that protects human cells against DNA damage. We report that Polη has three PCNA-interacting protein (PIP) boxes (PIP1, 2, 3) that contribute differentially to two distinct functions, stimulation of DNA synthesis and promotion of PCNA ubiquitination. The latter function is strongly associated with formation of nuclear Polη foci, which co-localize with PCNA. We also show that Polκ has two functionally distinct PIP boxes, like Polη, whereas Polι has a single PIP box involved in stimulation of DNA synthesis. All three polymerases were additionally stimulated by mono-ubiquitinated PCNA in vitro. The three PIP boxes and a ubiquitin-binding zinc-finger of Polη exert redundant and additive effects in vivo via distinct molecular mechanisms. These findings provide an integrated picture of the orchestration of TLS polymerases. PMID:26170230

  7. Eighth International Conference on Paleoceanography

    NASA Astrophysics Data System (ADS)

    Grousset, Francis; Peterson, Larry; Delaney, Peggie; Elderfield, Harry; Emeis, Kay; Haug, Gerald; Stocker, Thomas; Wang, Pinxian

    Every three years since 1983, the paleoceanographic community has come together at a different venue to share new data and discoveries at the International Conference on Paleoceanography (ICP). For the recent ICP-8, France was the host country for a conference focused on the theme of “An Ocean View of Global Change.” The Environnements et Paleoenvironnement Oceanique (EPOC) paleoceanography group of the University Bordeaux I acted as the local organizing committee.Scientific presentations at ICP-8 addressed the latest discoveries in paleoceanography and highlighted both emerging and as-yet-unsolved questions on global climate change. Thirty-five speakers, invited by the ICP-8 Science Committee, gave overview talks during morning sessions organized around five major scientific themes. These themes were Cenozoic-Mesozoic Oceans; Carbonate and Silica Systems of the Pleistocene Ocean; Biogeochemical Cycles of the Past; High-Frequency Climate Variability; and Interhemispheric Ocean-Continent-Climate Linkages.

  8. STS-102 Prelaunch Press Conference

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Joel Wells, NASA Public Affairs, introduces Ron Dittemore, NASA Shuttle Program Manager, Tommy Holloway, NASA International Space Station Program Manager, Dave King, NASA Director of Shuttle Processing, and Captain Clif Stargardt, US Air Force Meteorologist, in this STS-102 prelaunch press conference. The men give an overview of the prelaunch processing for the Discovery Orbiter (such as the PRSD loading) and give a weather forecast for launch. They then answer questions from the press.

  9. Apollo 11 preflight press conference

    NASA Technical Reports Server (NTRS)

    1969-01-01

    The three prime crewmen of the Apollo 11 lunar landing mission participate in a pre-flight press conference in the bldg 1 auditorium on July 5, 1969. Left to right, are Astronauts Neil A. Armstrong, commander; Edwin E. Aldrin Jr., lunar module pilot; and Michael Collins, command module pilot. The box-like enclosure surrounding the three astronauts was part of elaborate precautions in effect to reduce the possibility of exposing the crewmen to infectious disease in the preflight period.

  10. 1981 Image II Conference Proceedings.

    DTIC Science & Technology

    1981-11-01

    reverse side if nece.a rid ldentify b block number) area of interest computer simulation flight simulation behavioral visual research data base...visual cue requirements. visual data base development for terrain flight simulation. transformation realism, and strategies to optimize CIG image content... data base design and structure. The Image Generation/Display Conference was conceived in 1977 at the Opera- tions Training Division (formerly the

  11. Fifth international fungus spore conference

    SciTech Connect

    Timberlake, W.E.

    1993-04-01

    This folio contains the proceedings of the Fifth International Fungal Spore Conference held August 17-21, 1991 at the Unicoi State Park at Helen, Georgia. The volume contains abstracts of each oral presentation as well as a collection of abstracts describing the poster sessions. Presentations were organized around the themes (1) Induction of Sporulation, (2) Nuclear Division, (3) Spore Formation, (4) Spore Release and Dispersal, and (4) Spore Germination.

  12. Fast transcription rates of RNA polymerase II in human cells

    PubMed Central

    Maiuri, Paolo; Knezevich, Anna; De Marco, Alex; Mazza, Davide; Kula, Anna; McNally, Jim G; Marcello, Alessandro

    2011-01-01

    Averaged estimates of RNA polymerase II (RNAPII) elongation rates in mammalian cells have been shown to range between 1.3 and 4.3 kb min−1. In this work, nascent RNAs from an integrated human immunodeficiency virus type 1-derived vector were detectable at the single living cell level by fluorescent RNA tagging. At steady state, a constant number of RNAs was measured corresponding to a minimal density of polymerases with negligible fluctuations over time. Recovery of fluorescence after photobleaching was complete within seconds, indicating a high rate of RNA biogenesis. The calculated transcription rate above 50 kb min−1 points towards a wide dynamic range of RNAPII velocities in living cells. PMID:22015688

  13. Functional state of rat liver RNA polymerase IA and IB.

    PubMed

    Zoncheddu, A; Accomando, R; Pertica, M; Orunesu, M

    1979-01-01

    Phosphocellulose chromatography has been employed to characterize RNA polymerase I present in two different functional states in rat liver cells. The actively transcribing enzyme solubilized from nuclei appears to belong both to the IA and IB classes, whereas the non-transcribing enzyme present in the cytoplasmic fraction has been found to belong only to the IA class. Indirect and direct evidence indicates, however, that in isolated nuclei only the IB form is to be regarded as the physiological form of the enzyme, the IA form arising as a procedural artefact during the extraction process. It may, therefore, be concluded that rat liver IA and IB RNA polymerase are to be strictly regarded as the non-transcribing and transcribing form of the enzyme, respectively.

  14. A movie of the RNA polymerase nucleotide addition cycle.

    PubMed

    Brueckner, Florian; Ortiz, Julio; Cramer, Patrick

    2009-06-01

    During gene transcription, RNA polymerase (Pol) passes through repetitive cycles of adding a nucleotide to the growing mRNA chain. Here we obtained a movie of the nucleotide addition cycle by combining structural information on different functional states of the Pol II elongation complex (EC). The movie illustrates the two-step loading of the nucleoside triphosphate (NTP) substrate, closure of the active site for catalytic nucleotide incorporation, and the presumed two-step translocation of DNA and RNA, which is accompanied by coordinated conformational changes in the polymerase bridge helix and trigger loop. The movie facilitates teaching and a mechanistic analysis of transcription and can be downloaded from http://www.lmb.uni-muenchen.de/cramer/pr-materials.

  15. Structure-Function Relationships Among RNA-Dependent RNA Polymerases

    PubMed Central

    Ng, Kenneth K.-S.; Arnold, Jamie J.; Cameron, Craig E.

    2008-01-01

    RNA-dependent RNA polymerases (RdRPs) play key roles in viral transcription and genome replication, as well as epigenetic and post-transcriptional control of cellular gene expression. In this article, we review the crystallographic, biochemical, and molecular genetic data available for viral RdRPs that have led to a detailed description of substrate and cofactor binding, fidelity of nucleotide selection and incorporation, and catalysis. It is likely that the cellular RdRPs will share some of the basic structural and mechanistic principles gleaned from studies of viral RdRPs. Therefore, studies of the viral RdRP establish a framework for the study of cellular RdRPs, an important yet understudied class of nucleic acid polymerases. PMID:18268843

  16. Substrate recognition by norovirus polymerase: microsecond molecular dynamics study

    NASA Astrophysics Data System (ADS)

    Maláč, Kamil; Barvík, Ivan

    2013-04-01

    Molecular dynamics simulations of complexes between Norwalk virus RNA dependent RNA polymerase and its natural CTP and 2dCTP (both containing the O5'-C5'-C4'-O4' sequence of atoms bridging the triphosphate and sugar moiety) or modified coCTP ( C5' -O5'-C4'-O4'), cocCTP ( C5' -O5'-C4'- C4'') substrates were produced by means of CUDA programmable graphical processing units and the ACEMD software package. It enabled us to gain microsecond MD trajectories clearly showing that similar nucleoside triphosphates can bind surprisingly differently into the active site of the Norwalk virus RNA dependent RNA polymerase. It corresponds to their different modes of action (CTP—substrate, 2dCTP—poor substrate, coCTP—chain terminator, cocCTP—inhibitor). Moreover, extremely rare events—as repetitive pervasion of Arg182 into a potentially reaction promoting arrangement—were captured.

  17. The Mitochondrial DNA Polymerase in Health and Disease

    PubMed Central

    Copeland, William C.

    2014-01-01

    Since mutations in mitochondrial DNA (MtDNA) have been shown to be a cause of many mitochondrial diseases as well as aging, it is important to understand the origin of these mutations and how replication proteins modulate this process. DNA polymerase γ (pol γ) is the polymease that is responsible for replication and repair of mtDNA. Pol γ has three main roles in mtDNA maintanence and mutagenesis. As the only known DNA polymerase in mitochondria, pol γ is required for all replication and repair functions and is the main source of errors produced in our mtDNA. Pol γ is also sensitive to a host of antiviral nucleoside analogs used to treat HIV-1 infections, which can cause an induced mitochondrial toxicity. Finally, the gene for pol γ, POLG, is a genetic locus for several mitochondrial disease with over 150 genetic mutations currently identified. PMID:20012584

  18. Detection of Entamoeba histolytica by Recombinase Polymerase Amplification.

    PubMed

    Nair, Gayatri; Rebolledo, Mauricio; White, A Clinton; Crannell, Zachary; Richards-Kortum, R Rebecca; Pinilla, A Elizabeth; Ramírez, Juan David; López, M Consuelo; Castellanos-Gonzalez, Alejandro

    2015-09-01

    Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions.

  19. Characterization of the DNA polymerase gene of human herpesvirus 6.

    PubMed Central

    Teo, I A; Griffin, B E; Jones, M D

    1991-01-01

    The construction of a recombinant bacteriophage lambda library containing overlapping clones covering 155 kbp of the 161-kbp genome of the Ugandan U1102 isolate of human herpesvirus 6 (HHV-6) is described. The use of degenerate-primer polymerase chain reaction allowed the isolation of a DNA probe for the DNA polymerase gene of HHV-6, which was subsequently used to isolate and position the pol gene on the physical map of the viral genome. A 4.4-kbp EcoRI DNA restriction fragment containing the pol gene was isolated and sequenced. The open reading frames flanking the pol gene code for the HHV-6 glycoprotein B gene and the human cytomegalovirus UL53 homolog. This arrangement is different from that seen in the alpha and gamma herpesvirus families, lending further support to the notion that HHV-6 is a member of the beta herpesvirus group. Images PMID:1651403

  20. Detection of Entamoeba histolytica by Recombinase Polymerase Amplification

    PubMed Central

    Nair, Gayatri; Rebolledo, Mauricio; White, A. Clinton; Crannell, Zachary; Richards-Kortum, R. Rebecca; Pinilla, A. Elizabeth; Ramírez, Juan David; López, M. Consuelo; Castellanos-Gonzalez, Alejandro

    2015-01-01

    Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions. PMID:26123960