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Academic literature on the topic 'RNA polymerases'

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Published: 4 June 2021
Last updated: 30 July 2024

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Journal articles on the topic "RNA polymerases"

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Serrano, Alicia, Martín Moret, Isabel Fernández-Parras, Aureliano Bombarely, Francisco Luque, and Francisco Navarro. "RNA Polymerases IV and V Are Involved in Olive Fruit Development." Genes 15, no. 1 (December 19, 2023): 1. http://dx.doi.org/10.3390/genes15010001.

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Transcription is carried out in most eukaryotes by three multimeric complexes (RNA polymerases I, II and III). However, plants contain two additional RNA polymerases (IV and V), which have evolved from RNA polymerase II. RNA polymerases II, IV and V contain both common and specific subunits that may specialise some of their functions. In this study, we conducted a search for the genes that putatively code for the specific subunits of RNA polymerases IV and V, as well as those corresponding to RNA polymerase II in olive trees. Based on the homology with the genes of Arabidopsis thaliana, we identified 13 genes that putatively code for the specific subunits of polymerases IV and V, and 16 genes that code for the corresponding specific subunits of polymerase II in olives. The transcriptomic analysis by RNA-Seq revealed that the expression of the RNA polymerases IV and V genes was induced during the initial stages of fruit development. Given that RNA polymerases IV and V are involved in the transcription of long non-coding RNAs, we investigated their expression and observed relevant changes in the expression of this type of RNAs. Particularly, the expression of the intergenic and intronic long non-coding RNAs tended to increase in the early steps of fruit development, suggesting their potential role in this process. The positive correlation between the expression of RNA polymerases IV and V subunits and the expression of non-coding RNAs supports the hypothesis that RNA polymerases IV and V may play a role in fruit development through the synthesis of this type of RNAs.
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Shareef, Afzaal M., Barbara Ludeke, Paul Jordan, Jerome Deval, and Rachel Fearns. "Comparison of RNA synthesis initiation properties of non-segmented negative strand RNA virus polymerases." PLOS Pathogens 17, no. 12 (December 16, 2021): e1010151. http://dx.doi.org/10.1371/journal.ppat.1010151.

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It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming.
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Crotty, Shane, David Gohara, Devin K. Gilligan, Sveta Karelsky, Craig E. Cameron, and Raul Andino. "Manganese-Dependent Polioviruses Caused by Mutations within the Viral Polymerase." Journal of Virology 77, no. 9 (May 1, 2003): 5378–88. http://dx.doi.org/10.1128/jvi.77.9.5378-5388.2003.

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ABSTRACT Viral RNA-dependent RNA polymerases exhibit great sequence diversity. Only six core amino acids are conserved across all polymerases of positive-strand RNA viruses of eukaryotes. While exploring the function of one of these completely conserved residues, asparagine 297 in the prototypic poliovirus polymerase 3Dpol, we identified three viable mutants with noncanonical amino acids at this conserved position. Although asparagine 297 could be replaced by glycine or alanine in these mutants, the viruses exhibited Mn2+-dependent RNA replication and viral growth. All known RNA polymerases and replicative polymerases of bacterial, eukaryotic, and viral organisms are thought to be magnesium dependent in vivo, and therefore these mutant polioviruses may represent the first viruses with a requirement for an alternative polymerase cation. These results demonstrate the extreme functional flexibility of viral RNA-dependent RNA polymerases. Furthermore, the finding that strictly conserved residues in the nucleotide binding pocket of the polymerase can be altered in a manner that supports virus production suggests that drugs targeting this region of the enzyme will still be susceptible to the problem of drug-resistant escape mutants.
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Egorova, Tatiana, Ekaterina Shuvalova, Sabina Mukba, Alexey Shuvalov, Peter Kolosov, and Elena Alkalaeva. "Method for Rapid Analysis of Mutant RNA Polymerase Activity on Templates Containing Unnatural Nucleotides." International Journal of Molecular Sciences 22, no. 10 (May 14, 2021): 5186. http://dx.doi.org/10.3390/ijms22105186.

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Pairs of unnatural nucleotides are used to expand the genetic code and create artificial DNA or RNA templates. In general, an approach is used to engineer orthogonal systems capable of reading codons comprising artificial nucleotides; however, DNA and RNA polymerases capable of recognizing unnatural nucleotides are required for amplification and transcription of templates. Under favorable conditions, in the presence of modified nucleotide triphosphates, DNA polymerases are able to synthesize unnatural DNA with high efficiency; however, the currently available RNA polymerases reveal high specificity to the natural nucleotides and may not easily recognize the unnatural nucleotides. Due to the absence of simple and rapid methods for testing the activity of mutant RNA polymerases, the development of RNA polymerase recognizing unnatural nucleotides is limited. To fill this gap, we developed a method for rapid analysis of mutant RNA polymerase activity on templates containing unnatural nucleotides. Herein, we optimized a coupled cell-free translation system and tested the ability of three unnatural nucleotides to be transcribed by different T7 RNA polymerase mutants, by demonstrating high sensitivity and simplicity of the developed method. This approach can be applied to various unnatural nucleotides and can be simultaneously scaled up to determine the activity of numerous polymerases on different templates. Due to the simplicity and small amounts of material required, the developed cell-free system provides a highly scalable and versatile tool to study RNA polymerase activity.
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Pan, Junhua, Vikram N. Vakharia, and Yizhi Jane Tao. "The structure of a birnavirus polymerase reveals a distinct active site topology." Proceedings of the National Academy of Sciences 104, no. 18 (April 24, 2007): 7385–90. http://dx.doi.org/10.1073/pnas.0611599104.

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Single-subunit polymerases are universally encoded in both cellular organisms and viruses. Their three-dimensional structures have the shape of a right-hand with the active site located in the palm region, which has a topology similar to that of the RNA recognition motif (RRM) found in many RNA-binding proteins. Considering that polymerases have well conserved structures, it was surprising that the RNA-dependent RNA polymerases from birnaviruses, a group of dsRNA viruses, have their catalytic motifs arranged in a permuted order in sequence. Here we report the 2.5 Å structure of a birnavirus VP1 in which the polymerase palm subdomain adopts a new active site topology that has not been previously observed in other polymerases. In addition, the polymerase motif C of VP1 has the sequence of -ADN-, a highly unusual feature for RNA-dependent polymerases. Through site-directed mutagenesis, we have shown that changing the VP1 motif C from -ADN- to -GDD- results in a mutant with an increased RNA synthesis activity. Our results indicate that the active site topology of VP1 may represent a newly developed branch in polymerase evolution, and that birnaviruses may have acquired the -ADN- mutation to control their growth rate.
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Bettiol, Michael F., Randall T. Irvin, and Paul A. Horgen. "Immunological analyses of selected eukaryotic RNA polymerases II." Canadian Journal of Biochemistry and Cell Biology 63, no. 12 (December 1, 1985): 1217–30. http://dx.doi.org/10.1139/o85-153.

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Polyclonal antibodies to native RNA polymerase II of Achlya ambisexualis and Agaricus bisporus were produced in rabbits and in mice. Monoclonal antibodies were produced against the α-amanitin resistant RNA polymerase II of the mushroom A. bisporus. These antibodies were used in comparative cross-reactivity studies with five purified RNA polymerases II (A. bisporus, A. ambisexualis, Saccharomyces cerevisiae, wheat germ, and calf thymus). A method for quantitatively comparing cross-reactivity was developed utilizing an enzyme-linked immunosorbant assay (ELISA). ELIS A comparisons indicated that the two filamentous fungi cross-reacted effectively with one another and depending upon the preparation reacted less effectively with yeast and wheat germ RNA polymerases II. Cross-reactivity measurements were also made by immunoblotting sodium dodecyl sulfate – polyacrylamide separated RNA polymerases II. The mouse anti-A. bisporus RNA polymerase II immunoglobulin G (IgG) and the monoclonal antibody preparations did not react with high molecular subunits of A. bisporus RNA polymerase II. The sera did, however, cross-react with high molecular weight subunits of A. ambisexualis. Similarily, rabbit anti-A. ambisexualis RNA polymerase II IgG reacted only with low molecular weight subunits of A. bisporus RNA polymerase II, but reacted with high molecular weight subunits of A. ambisexualis and wheat germ. Our results indicate differences in the cross-reactivity of native and denatured RNA polymerases II and suggest differences in the tertiary and quaternary organization of the enzymes examined.
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Cramer, Patrick. "Multisubunit RNA polymerases." Current Opinion in Structural Biology 12, no. 1 (February 2002): 89–97. http://dx.doi.org/10.1016/s0959-440x(02)00294-4.

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Lysenko, E. A., and V. V. Kuznetsov. "Plastid RNA Polymerases." Molecular Biology 39, no. 5 (September 2005): 661–74. http://dx.doi.org/10.1007/s11008-005-0081-1.

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Blair, D. G. R. "Eukaryotic RNA polymerases." Comparative Biochemistry and Physiology Part B: Comparative Biochemistry 89, no. 4 (January 1988): 647–70. http://dx.doi.org/10.1016/0305-0491(88)90306-9.

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Peramachi Palanivelu. "Identification of Polymerase and Proofreading Exonuclease Domains in the DNA Polymerases IA, IB and Nuclear-Encoded RNA Polymerase of the Plant Chloroplasts." World Journal of Advanced Research and Reviews 17, no. 3 (March 30, 2023): 706–27. http://dx.doi.org/10.30574/wjarr.2023.17.3.0455.

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Chloroplast plays a crucial role in all photosynthetic plants and converts the light energy to chemical energy. It is a semi-autonomous organelle and is mostly controlled by its own genome and partly by the nuclear imports. To replicate its own genome, it uses two DNA polymerases, viz. polymerases IA and IB. DNA polymerase IA showed 72.45% identity to polymerase IB, but only 35.35% identity to the E. coli DNA polymerase I. Multiple sequence alignment (MSA) analysis have shown that the DNA polymerases IA and IB and the E. coli DNA polymerase I possess almost identical active sites for polymerization and proofreading (PR) functions, suggesting their possible common evolutionary origin. The nuclear-encoded RNA polymerase (NEP) is imported from the nucleus and involves in the transcription of all the four subunits of the chloroplast RNA polymerase. The polymerase catalytic core of the DNA polymerases IA, IB and the NEP are remarkably conserved and is in close agreement with other DNA/RNA polymerases reported already, and possess a typical template-binding pair (-YG-), a basic catalytic amino acid (K) to initiate catalysis and a basic nucleotide selection amino acid R at -4 from K. The DNA polymerases IA and IB are very similar to prokaryotic DNA polymerases, except in possessing a zinc-binding motif (ZBM) in them, like the eukaryotic replicases. Interestingly, the PR exonucleases of all three polymerases belong to the DEDD-superfamily of exonucleases. The DNA polymerases IA and IB belong to the DEDD(Y)-subfamily, whereas the NEP belongs to the DEDD(H)-subfamily.
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Dissertations / Theses on the topic "RNA polymerases"

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Salgado, Maria Paula Santos Cordeiro. "Structural studies of RNA-dependent RNA polymerases." Thesis, Open University, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.430559.

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Forrest, David Andrew. "Novel types of RNA polymerases." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/2808.

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Transcription, the first step of gene expression, is accomplished in all domain of life by the multisubunit RNA polymerase (RNAP). Accordingly, the RNAP is an ancient enzyme that is highly conserved in all cellular organisms. However, in-depth bioinformatics has led to the identification of proteins distantly related to the multisubunit RNAP, such as YonO and ORF6 RNAP. Whilst being putative single subunit RNAPs, YonO and ORF6 RNAP are completely unrelated to T7 RNAP. YonO, encoded by the Bacillus subtilis SPβ prophage, has incredibly little similarity to RNAP. Conversely, ORF6 RNAP belonging to the Kluyveromyces lactis killer system contains half of the conserved RNAP active centre. YonO and ORF6 RNAP are potentially novel RNAPs and their characterisation will give new insights into mechanisms of transcription as well as the biology of the organisms which they belong to. Despite lacking most of the conserved RNAP active centre, we have shown YonO is a very efficient DNA dependant RNAP. Striking, unlike all of its multisubunit relatives, YonO is able to initiate transcription on double stranded DNA without accessory factors (such as σ factors in bacteria). Furthermore, our work suggests YonO is expressed during induction of SPβ and functions to transcribe the SPβ late genes. This potentially makes YonO the first bacteriophage single subunit RNAP that resembles the multisubunit RNAP. On the other hand, ORF6 RNAP was very poor at polymerisation, with or without its putative accessory subunit. This suggests additional, currently unknown proteins are required for RNAP activity and potentially a new molecular mechanism of RNA polymerisation. YonO homologues exist in a wide range of bacteria including fermicutes and cyanobacteria. YonO represents a new class of RNAP and our discoveries potentially open up a new area of research as well as being potentially useful for biotechnology and synthetic biology. In contrast, transcription by ORF6 RNAP is more complex than previously thought, with alternative lines of investigation required to identify additional factors that contribute to ORF6 RNAP activity.
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Wright, Sam Mathew. "Structural and biophysical studies of RNA-dependent RNA polymerases." Thesis, University of Oxford, 2010. http://ora.ox.ac.uk/objects/uuid:d5c2a16d-e1e2-4c22-aca5-70f72aa96853.

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RNA-dependent RNA polymerases (RdRps) play a vital role in the life cycle of RNA viruses, being responsible for genome replication and mRNA transcription. In this thesis viral RdRps (vRdRps) of dsRNA bacteriophage phi6 (phi6 RdRp) and Severe Acute Respiratory Syndrome (SARS) coronavirus [non structural protein 12 (NSP-12)] are studied. For SARS polymerase NSP-12, a library-based screening method known as ESPRIT (Expression of Soluble Protein by Random Incremental Truncation) was employed in an attempt to isolate domains of NSP-12 that express solubly in Escherichia coli (E. coli) and are thereby suitable for structural studies. This experiment identified for the first time in a systematic fashion, conditions under which the SARS polymerase could be solubly expressed at small scale and allowed mapping of domain boundaries. Further experiments explored different approaches for increasing expression levels of tractable fragments at large scale. Bacteriophage phi6 RdRp is one of the best studied vRdRps. It initiates RNA synthesis using a de novo mechanism without the need for a primer. Although formation of the de novo initiation complex has been well studied, little is known about the mechanism for the transition from initiation to elongation (i.e. extension of an initiated dinucleotide daughter strand). In the phi6 RdRp initiation complex the C-terminal domain (CTD) blocks the exit path of the newly synthesised dsRNA which must be displaced for the addition of the third nucleotide. The crystal structure of a C-terminally truncated phi6 RdRp (P2T1) reveals the strong non-covalent interactions between the CTD and the main body of the polymerase that must be overcome for the elongation reaction to proceed. Comparing new crystal structures of complexes of both wild-type (WT) and a mutant RdRp (E634 to Q, which removes a salt-bridge between the CTD and main body of the polymerase) with various oligonucleotides (linear and hairpin), nucleoside triphosphates (NTPs) and divalent cations, alongside their biophysical and biochemical properties, provides an insight into the precise molecular details of the transition reaction. Thermal denaturation experiments reveal that Mn2+ acquired from the cell and bound at the phi6 RdRp non-catalytic ion site sufficiently weakens the polymerase structure to facilitate the displacement of the CTD. Our crystallographic and biochemical data also indicate that Mn2+ is released during this displacement and must be replaced for the elongation to proceed. Our data explain the role of the non-catalytic divalent cation in vRdRps and pinpoint the Mn2+-dependent step in viral replication. In addition, by inserting a dysfunctional Mg2+ at the non-catalytic ion site for both WT and E634Q RdRps we captured structures with two NTPs bound within the active site in the absence of Watson-Crick base pairing with template and could map movements of divalent cations during preinitiation through to initiation. Oligonucleotides present on the surface of phi6 RdRp allowed mapping of key residues involved in template entry and unwinding of dsRNA; these preinitiation stages have not been observed previously. Considering the high structural homology of phi6 RdRp with other vRdRps, particularly from (+)ssRNA hepatitis C virus (HCV), insights into the mechanistic and structural details of phi6 RdRp are thought to be relevant to the general understanding of vRdRps.
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Chan, Annie Yee-Man. "Interactions between the influenza virus RNA polymerase and cellular RNA polymerase II." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670083.

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Niedbala, Angela Rochelle. "Kinetic studies of transcription initiation by wild type T7 RNA polymerase, his-tagged wild type T7 RNA polymerase and GP1-Lys222 T7 RNA polymerase." Thesis, Georgia Institute of Technology, 1995. http://hdl.handle.net/1853/27288.

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Nottebaum, Sven. "In vitro assembly of recombinant archaeal RNA polymerases." Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.443827.

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Ma, Hok-tsun, and 馬學俊. "RNA-Dependent RNA polymerase activity of the infectious bursal diseasevirus viral protein 1." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B30408192.

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Curti, Elena. "Structure function studies of selected RNA and DNA polymerases." Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414158.

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Yin, Chang. "Evolution of phage-type RNA polymerases in higher plants." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16270.

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In mono- und eudikotylen Pflanzen kodiert eine Genfamilie (RpoT, RNA-Polymerase des T3/T7-Typs) mitochondriale und plastidäre RNA-Polymerasen (RNAP), die den ungeraden T-Phagen-Polymerasen ähneln. RpoT-Gene von Angiospermen sind gut charakterisiert, während aus tiefer abzweigenden Pflanzenspecies bisher lediglich die Gene aus dem Moos Physcomitrella beschrieben wurden. Um einen Beitrag zur Aufklärung der molekularen Evolution der RpoT-Polymerasen im Pflanzenreich zu liefern und um Erkenntnisse über die potentielle Bedeutung von multiplen Phagen-Typ (RNAP) in Pflanzen zu gewinnen, wurden die RpoT-Gene aus dem Lycophyten Selaginella moellendorffii und aus dem basalen Angiosperm Nuphar advena identifiziert und charakterisiert. Selaginella moellendorffii (Moosfarn)-Trace-Sequenzdaten mit hoher Ähnlichkeit zu RpoT-Sequenzen von Angiospermen wurden benutzt, um das full-length SmRpoT-Gen und die entsprechende cDNA zu isolieren. Die SmRpoT-mRNA ist 3542 nt lang und weist einen offenen Leserahmen von 3006 nt auf, der für ein putatives Protein aus 1002 Aminosäuren mit einer molekularen Masse von 113 kDa kodiert. Das SmRpoT-Gen besteht aus 19 Exons und 18 Introns, die in ihren Positionen mit denen aus den Angiosperm- und Physcomitrella-Genen konserviert sind. Mittels Southernblot-Analyse wurde nachgewiesen, dass S. moellendorffii ein single-copy RpoT-Gen kodiert. Für das N-terminale Transitpeptid von SmRpoT konnte gezeigt werden, dass es bei transienter Expression in Arabidopsis- und Selaginella-Protoplasten den Transport von GFP (green fluorescent protein) exclusiv in Mitochondrien vermittelt. In N. advena wurden mittels Screening einer BAC-Bibliothek drei RpoT-Gene identifiziert. Sowohl die genomischen als auch die cDNA-Sequenzen wurden aufgeklärt. Die NaRpoT-mRNAs kodieren putative Polypeptide von 996, 990 und 985 Aminosären. Alle drei Gene besitzen 19 Exons und 18 Introns, die in ihren Positionen mit denen der RpoT-Gene aus Selaginella und allen anderen Landpflanzen konserviert sind. Die kodierten Proteine weisen auf Aminosäureebene einen hohen Konservierungsgrad auf, einschließlich aller essentiellen Regionen und Aminosäurereste, die für die T7-RNAP bekannt sind. Die N-terminalen Transitpeptide zweier der kodierten RNAP, NaRpoTm1 und NaRpoTm2, vermittelten den Import von GFP exclusiv in Mitochondrien, während die dritte Polymerase, NaRpoTp, in Chloroplasten importiert wurde. Interessanterweise muß die Translation der NaRpoTp-mRNA an einem CUG-Codon initiiert werden, um ein funktionelles Protein mit plastidärem Transitpeptid zu erhalten. Die N. advena RpoTp-RNAP ist somit neben AGAMOUS aus Arabidopsis und der RpoTp-RNAP aus Nicotiana, ein weiteres Beispiel für jene selten vorkommenden pflanzlichen mRNAs, deren Translation exclusiv an nicht-AUG-Codons initiiert wird. Die Rekonstruktion von phylogenetischen Bäumen resultierte in unterschiedlichen Positionen für die Selaginella- und Nuphar-Polymerasen: Im Gegensatz zu der RpoT-Polymerase aus S. moellendorffii und denen aus Physcomitrella, die in den phylogenetischen Analysen Schwesterpositionen zu allen anderen Phagentyp-RNAP der Angiospermen einnehmen, clusterten die Nuphar-RpoTs zusammen mit den deutlich separierten mitochondrialen (NaRpoTm1 und NaRpoTm2) und plastidären (NaRpoTp) Polymerasen. Selaginella kodiert eine einzige mitochondriale RNAP, während Nuphar zwei mitochondriale und eine plastidäre RNAP besitzt. Die Identifizierung einer Plastiden-lokalisierten Phagentyp-RNAP in diesem basalen Eudikotylen, die ortholog zu allen anderen RpoT-Enzymen der Blütenpflanzen ist, läßt darauf schließen, daß die Acquisition einer nukleär kodierten plastidären RNAP, die noch in den Lycopoden fehlt, nach der Trennung der Leucopoden von allen anderen Tracheophyten erfolgte. Eine “dual-targeting” RNAP (mitochondrial und plastidär lokalisiert), wie sie in Eudikotylen, nicht jedoch in Monokotylen vorkommt, wurde weder in Selaginella noch in Nuphar nachgewiesen, vermutlich ist sie ein evolutionäres Novum von eudikotylen Pflanzen wie Arabidopsis.
In mono- and eudicot plants, a small nuclear gene family (RpoT, RNA polymerase of the T3/T7 type) encodes mitochondrial as well as chloroplast RNA polymerases homologous to the T-odd bacteriophage enzymes. RpoT genes from angiosperms are well characterized, whereas data from deeper branching plant species until recently were limited to the moss Physcomitrella. To elucidate the molecular evolution of the RpoT polymerases in the plant kingdom and to get more insight into the potential importance of having more than one phage-type RNA polymerase (RNAP) available, we identified and characterized RpoT genes in the lycophyte Selaginella moellendorffii and the basal eudicot Nuphar advena. Selaginella moellendorffii (spikemoss) sequence trace data encoding a polypeptide highly similar to angiosperm and moss phage-type organelle RNA polymerases were used to isolate a BAC clone containing the full-length gene SmRpoT as well as the corresponding cDNA. The SmRpoT mRNA comprises 3452 nt with an open reading frame of 3,006 nt, encoding a putative protein of 1,002 amino acids with a molecular mass of 113 kDa. The SmRpoT gene comprises 19 exons and 18 introns, conserved in their position with those of the angiosperm and Physcomitrella RpoT genes. Using Southern blot analysis, it was shown that S. moellendorffii encodes a single RpoT gene. The N-terminal transit peptide of SmRpoT was shown to confer targeting of green fluorescent protein (GFP) exclusively to mitochondria after transient expression in Arabidopsis and Selaginella protoplasts. In Nuphar advena three RpoT genes were identified by BAC library screening. Both genomic gene sequences and full-length cDNAs were determined. The NaRpoT mRNAs specify putative polypeptides of 996, 990 and 985 amino acids, respectively. All three genes comprise 19 exons and 18 introns, conserved in their positions with those from S. moellendorffii and the RpoT genes of other land plants. The encoded proteins show a high degree of conservation at the amino acid sequence level, including all functional crucial regions and residues known from the phage T7 RNAP. The N-terminal transit peptides of two of the encoded polymerases, NaRpoTm1 and NaRpoTm2, conferred targeting of GFP exclusively to mitochondria, whereas the third polymerase, NaRpoTp, was targeted to chloroplasts. Remarkably, translation of NaRpoTp mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis and the Nicotiana RpoTp polymerase, N. advena RpoTp provides another example for a plant mRNA that is exclusively translated from a non-AUG codon. Reconstruction of phylogenetic trees revealed different positions of the RpoTs from the lycophyte Selaginella and the basal eudicot Nuphar. In contrast to the RpoTs of S. moellendorffii and those of the moss Physcomitrella, which are according to the phylogenetic analyses in sister positions to all other phage-type polymerases of angiosperms, the Nuphar RpoTs clustered with the well separated clades of mitochondrial (NaRpoTm1 and NaRpoTm2) and plastid (NaRpoTp) polymerases. Selaginella encodes a single mitochondrial RNAP, whereas Nuphar harbors two mitochondrial and one plastid phage-type polymerases. Identification of a plastid localized phage-type RNAP in this basal eudicot, orthologous to all other RpoTp enzymes of flowering plants, suggests that the acquisition of a nuclear encoded plastid RNA polymerase, not present in lycopods, took place after the split of lycopods from all other tracheophytes. A dual-targeted mitochondrial and plastid RNA polymerase (RpoTmp), as present in eudicots but not monocots, was not detected in Nuphar or Selaginella suggesting that its occurrence is an evolutionary novelty of eudicotyledoneous plants like Arabidopsis.
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Vasale, Jessica J. "Roles of Cellular RNA-Dependent RNA Polymerases in Endogenous Small RNA Pathways in Caenorhabditis elegans: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/481.

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The RNA interference (RNAi) pathway in Caenorhabditis elegans is a two-step, small RNA-mediated silencing pathway. Unlike in other organisms, Dicer processing of double-stranded RNA into small interfering (si) RNAs is not sufficient in worms to induce gene silencing. The activity of cellular RNA-dependent RNA polymerase (RdRP) is necessary to synthesize a secondary pool of siRNAs, which interact with a unique class of Argonaute proteins to form the functional effector complexes that mediate silencing. The aims of this thesis were to: 1) characterize the role of RdRP family members in endogenous small RNA biogenesis; 2) identify the Argonaute proteins that interact with RdRP-dependent small RNAs; and 3) investigate the biological function of RdRP-dependent small RNA pathways in C. elegans. In this thesis, I describe genetic, deep sequencing, and molecular studies, which identify 22G-RNAs as the most abundant class of endogenous small RNA in C. elegans. The 22G-RNAs resemble RdRP-dependent secondary siRNAs produced during exogenous RNAi, in that they possess a triphosphorylated 5’ guanine residue and exhibit a remarkable strand bias at target loci. Indeed, I show that 22G-RNAs are dependent on the activity of the RdRPs RRF-1 and EGO-1 and function in multiple distinct endogenous small RNA pathways. Interestingly, I have found that RRF-1 and EGO-1 function redundantly in the germline to generate 22G-RNAs that are dependent on and interact with members of an expanded family of worm-specific Argonaute (WAGO) proteins. The WAGO/22G-RNA pathway appears to be a transcriptome surveillance pathway that silences coding genes, pseudogenes, transposons, and non-annotated, or cryptic, transcripts. In contrast, I have found that EGO-1 alone is required for the biogenesis of a distinct class of 22G-RNAs that interact with the Argonaute CSR-1. Surprisingly, the CSR-1/22G-RNA pathway does not appear to silence its targets transcripts. Instead, the CSR-1/22G-RNA pathway is essential for the proper assembly of holocentric kinetochores and chromosome segregation. Lastly, I show that a third endogenous small RNA pathway, the ERI pathway, is a two-step silencing pathway that requires the sequential activity of distinct RdRPs and Argonautes. In the first step of this pathway, the RdRP, RRF- 3, is required for the biogenesis of 26G-RNAs that associate with the Argonaute, ERGO-1. In the second step, RRF-1 and EGO-1 generate 22G-RNAs that associate with the WAGO Argonautes. This work demonstrates how several C. elegans small RNAs pathways utilize RdRPs to generate abundant populations of small RNAs. These distinct categories of small RNAs function together with specific Argonaute proteins to affect gene expression, to play essential roles in development, and in the maintenance of genome and transcriptome integrity.
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Books on the topic "RNA polymerases"

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Landick, Robert, Terence Strick, and Jue Wang, eds. RNA Polymerases as Molecular Motors. 2nd ed. Cambridge: Royal Society of Chemistry, 2021. http://dx.doi.org/10.1039/9781839160561.

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Buc, Henri, and Terence Strick, eds. RNA Polymerases as Molecular Motors. Cambridge: Royal Society of Chemistry, 2009. http://dx.doi.org/10.1039/9781847559982.

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Henri, Buc, and Strick Terence, eds. RNA polymerases as molecular motors. Cambridge, UK: RSC Pub., 2009.

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L, Adhya S., and Garges Susan, eds. RNA polymerases and associated factors. San Diego, CA: Elsevier Academic Press, 2003.

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White, Robert J. RNA polymerase III transcription. Austin: R.G. Landes, 1994.

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J, White Robert. RNA polymerase III transcription. 2nd ed. Austin, TX: Landes Bioscience, 1998.

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Lal, Adhya Sankar, ed. RNA polymerase and associated factors. San Diego, Calif: Academic Press, 1996.

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1943-, Paule Marvin R., ed. Transcription of ribosomal RNA genes by eukaryotic RNA polymerase I. Berlin: Springer, 1998.

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1943-, Erlich Henry A., ed. PCR technology: Principles and applications for DNA amplification. New York: Stockton Press, 1989.

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Wisconsin--Madison), Steenbock Symposium (16th 1986 University of. RNA polymerase and the regulation of transcription: Proceedings of the Sixteenth Steenbock Symposium held July 13th through July 17th, 1986, at the University of Wisconsin--Madison, U.S.A. New York: Elsevier, 1987.

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Book chapters on the topic "RNA polymerases"

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Merkl, Philipp E., Christopher Schächner, Michael Pilsl, Katrin Schwank, Catharina Schmid, Gernot Längst, Philipp Milkereit, Joachim Griesenbeck, and Herbert Tschochner. "Specialization of RNA Polymerase I in Comparison to Other Nuclear RNA Polymerases of Saccharomyces cerevisiae." In Ribosome Biogenesis, 63–70. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2501-9_4.

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AbstractIn archaea and bacteria the major classes of RNAs are synthesized by one DNA-dependent RNA polymerase (RNAP). In contrast, most eukaryotes have three highly specialized RNAPs to transcribe the nuclear genome. RNAP I synthesizes almost exclusively ribosomal (r)RNA, RNAP II synthesizes mRNA as well as many noncoding RNAs involved in RNA processing or RNA silencing pathways and RNAP III synthesizes mainly tRNA and 5S rRNA. This review discusses functional differences of the three nuclear core RNAPs in the yeast S. cerevisiae with a particular focus on RNAP I transcription of nucleolar ribosomal (r)DNA chromatin.
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Basu, Ritwika S., and Katsuhiko S. Murakami. "Bacteriophage RNA Polymerases." In Nucleic Acid Polymerases, 237–50. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-39796-7_10.

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Boehr, David D., Jamie J. Arnold, Ibrahim M. Moustafa, and Craig E. Cameron. "Structure, Dynamics, and Fidelity of RNA-Dependent RNA Polymerases." In Nucleic Acid Polymerases, 309–33. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-39796-7_14.

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Bushnell, David A., and Roger D. Kornberg. "Eukaryotic RNA Polymerase II." In Nucleic Acid Polymerases, 277–87. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-39796-7_12.

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Ferrer-Orta, Cristina, and Nuria Verdaguer. "RNA Virus Polymerases." In Viral Genome Replication, 383–401. Boston, MA: Springer US, 2009. http://dx.doi.org/10.1007/b135974_18.

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Bautz, Ekkehard K. F., and Gabriele Petersen. "Eukaryotic RNA Polymerases." In Molecular Biology of Chromosome Function, 157–79. New York, NY: Springer New York, 1989. http://dx.doi.org/10.1007/978-1-4612-3652-8_7.

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Yin, Y. Whitney. "Mitochondrial DNA and RNA Polymerases." In Nucleic Acid Polymerases, 251–75. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-39796-7_11.

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Ream, Thomas S., Jeremy R. Haag, and Craig S. Pikaard. "Plant Multisubunit RNA Polymerases IV and V." In Nucleic Acid Polymerases, 289–308. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-39796-7_13.

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Kireeva, Maria L., Mikhail Kashlev, and Zachary F. Burton. "RNA Polymerases and Transcription." In Encyclopedia of Biophysics, 1–9. Berlin, Heidelberg: Springer Berlin Heidelberg, 2018. http://dx.doi.org/10.1007/978-3-642-35943-9_443-1.

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Kireeva, Maria L., Mikhail Kashlev, and Zachary F. Burton. "RNA Polymerases and Transcription." In Encyclopedia of Biophysics, 2264–73. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-16712-6_443.

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Conference papers on the topic "RNA polymerases"

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Singatulina, A. S., M. V. Sukhanova, and O. I. Lavrik. "FACTOR HPF1 REGULATES THE ACTIVITY OF POLY(ADP-RIBOSE)POLYMERASES 1 AND 2 AND THE FORMATION OF POLY(ADP-RIBOSE)-CONTAINING COMPARTMENTS WITH THE PARTICIPATION OF THE RNA-BINDING PROTEIN FUS." In X Международная конференция молодых ученых: биоинформатиков, биотехнологов, биофизиков, вирусологов и молекулярных биологов — 2023. Novosibirsk State University, 2023. http://dx.doi.org/10.25205/978-5-4437-1526-1-281.

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Poly(ADP-ribose) polymerases 1 and 2 (PARP1/2) synthesize poly(ADP-ribose) (PAR) by covalently modifying a number of proteins involved in DNA/RNA metabolism, including themselves. PARP1/2 are key regulators of DNA repair via autopoly(ADP-ribosyl)ation at the site of DNA damage. The study of factors that modulate PARP1/2 activity in response to genotoxic stress is an important task in modern biology.
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Timkin, P. D., and A. A. Penzin. "An experimental approach for diagnosing cercosporosis using RPA+CRISPR/Cas12a." In II All-Russian (national) scientific conference with international participation "Russian Science, Innovation, Education". Krasnoyarsk Science and Technology City Hall, 2023. http://dx.doi.org/10.47813/rosnio-ii.2023.8.263-266.

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The article demonstrates the prospects for using a new experimental approach for the detection of the fungus Cercospora Sojina Hara. The main principle underlying the presented method is a combination of two technologies: RPA (Recombinase polymerase amplification) and CRISPR/Cas12a. RPA - is an alternative to classical PCR, with features in the form of a faster reaction rate and its passage under isothermal conditions. Using RPA technology will reduce amplification to 15-30 minutes. Amplified genomic DNA can be detected fluorescently labeled with CRISPR/Cas12a. The difficulties of this method lie in the selection of specific primers and the selection of spacers for the guide RNA within the amplicon. As a result of the work carried out, using the primer3 plugin on the Unipro Ugene platform, it was possible to select a specific pair of primers that would make it possible to identify this particular phytopathogen. The genome spacer was identified in the ChopChop web toolkit. The resulting primer pair and spacer having complementarity exclusively to the CP036215 locus in Cercospora Sojina H.
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Lee, Sangyool, Sun Hyun Chang, Taeyoung Um, Geupil Jang, Ju-Kon Kim, and Yang Do Choi. "Characterization of the Plastid RNA Polymerase Complex." In The 3rd World Congress on New Technologies. Avestia Publishing, 2017. http://dx.doi.org/10.11159/icbb17.115.

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Gong, Ruyi, Nicolas Acosta, Patrick Su, Luay Almassalha, and Vadim Backman. "Analysis of Multiplexed Single-Molecule Localization Microscopy of Chromatin and Transcription." In Microscopy Histopathology and Analytics. Washington, D.C.: Optica Publishing Group, 2024. http://dx.doi.org/10.1364/microscopy.2024.mm5a.2.

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We developed a protocol on staining and imaging of three-color single-molecule localization microscopy of constitutive heterochromatin (H3K9me3), euchromatin (H3K27ac) and RNA polymerase II and proposed analysis methods to show their functional coupling.
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Benslimane, Fatiha M., Hebah Al Khatib, Dana Albatesh, Ola Al-Jamal, Sonia Boughattas, Asmaa A. Althani, and Hadi M. Yassine. "Nanopore Sequencing SARS-CoV-2 Genome in Qatar." In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2020. http://dx.doi.org/10.29117/quarfe.2020.0289.

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Background: The current pandemic, COVID-19, is cause by an RNA Coronavirus that was recently identified as SARS-CoV-2. RNA viruses tend to have a high mutation rate; the rate is around a million times greater than that of their hosts. The mutagenic potential of the virus depends on many factors, including the fidelity of nucleic acid-replicating viral enzymes, such as SARSCoV-2 RNA dependent RNA polymerase (RdRp). The rate of mutation drives viral evolution and genome variability, consequently allowing viruses to escape the immunity of the host and develop resistance to drugs. Therefore, the characterization of SARS-CoV-2 variants might lead to implement better therapeutics treatments, vaccines design and identify new diagnostics approaches. Aim: The aim of this study was to establish a fast sequencing method to identify SARS-CoV-2 mutations in Qatar. This will help to assess if there are new viral variants that are spreading in country. Methods: RNA was isolated from samples collected from Qatar COVID-19 positive patients. The Artic Network V3 primer scheme and Oxford Nanopore ligation sequencing kit were used to prepare the sequencing libraries. Libraries were loaded on to R9.4.1 flow cells and ran on a GridION. Bioinformatics analysis was done following the Artic Network SARA-CoV-2 bioinformatics tools. Results: Genome coverage of sequenced samples was >80% and the depth was average at 200x. The coverage was highly dependable on sample viral load; samples of CT value lower than 30 resulted in better sequence coverage. The sequenced genomes were deposited in GISAID and were mainly clustering with genomes deposited from the UK. Sequences were compared to Illumina and sanger sequences and they showed compatible results. Conclusion: The use of ONT to sequence SARA-CoV-2 is a quick, affordable, and reliable technique to determine viral mutation. Using this technique, the first sequences from Qatar were deposited in to GISAID. Up to date, 700 genomes have been sequenced from Qatari samples.
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Grierson, Patrick, Kate Lillard, Gregory Behbehani, Kelly Combs, Saumitri Bhattacharyya, Acharya Samir, and Joanna Groden. "Abstract PR3: The BLM helicase facilitates RNA polymerase l-mediated ribosomal RNA transcription." In Abstracts: Second AACR International Conference on Frontiers in Basic Cancer Research--Sep 14-18, 2011; San Francisco, CA. American Association for Cancer Research, 2011. http://dx.doi.org/10.1158/1538-7445.fbcr11-pr3.

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Harrod, K. S., H. T. Pacl, J. L. Tipper, S. Ahmad, A. Ahmad, G. Holder, C. Petit, T. Green, A. J. C. Steyn, and M. Might. "Water Soluble Tocopherol Derivatives Inhibit the SARS-CoV-2 RNA-Dependent RNA Polymerase." In American Thoracic Society 2022 International Conference, May 13-18, 2022 - San Francisco, CA. American Thoracic Society, 2022. http://dx.doi.org/10.1164/ajrccm-conference.2022.205.1_meetingabstracts.a3610.

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Stoicescu, Ramona, Razvan-Alexandru Stoicescu, Codrin Gheorghe, Adina Honcea, and Iulian Bratu. "CONSIDERATIONS ON SARS-COV-2 DIAGNOSIS IN THE LABORATORY OF UNIVERSITY EMERGENCY CLINICAL HOSPITAL OF CONSTANTA." In GEOLINKS Conference Proceedings. Saima Consult Ltd, 2021. http://dx.doi.org/10.32008/geolinks2021/b1/v3/07.

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Coronaviruses are members of the Coronaviridae family. They are enveloped, non-segmented, positive-sense, single-stranded RNA viruses. Their genome size is about 30 kilobases (kb) which consist, at the 5’ end, of non-structural open reading frames (ORFs: ORF1a, ORF 1b) which code for 16 non structural proteins, and at the 3’ end the genes which code for four structural proteins including membrane (M), envelope (E), spike (S), and nucleocapsid (N) proteins. Due to the rapid spread of COVID-19, a reliable detection method is needed for patient diagnosis especially in the early stages of the disease. WHO has recommended nucleic acid amplification tests such as real-time reverse transcription-polymerase chain reaction (RT-PCR). The assay detects three SARS-CoV-2 RNA targets: the envelope (E) gene, the nucleocapsid (N) gene and a region of the open reading frame (ORF1) of the RNA-dependent RNA polymerase (RdRp) gene from SARS-CoV-2 virus isolate Wuhan-Hu-1. Our study was made in the first 3 months of the year 2021 using the real-time RT PCR results obtained in the Cellular Biology ward of the University Emergency Clinical Hospital. In our lab we are testing the inpatients from the hospital wards (Neurology, Pediatrics, Surgery, Internal medicine, ICU, Cardiology, etc.); we are also testing the outpatients from Dialysis and Oncology, 2 days prior to their therapy; we also test the health care personnel. The number of tests we performed was: in January 1456, with 399 positive results (27.4%), 33 deaths; in February 1273 tests, 221 positive (17.36%), 16 deaths; in March 1471 tests, 373 positive (25.36%), 37 deceased.
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Yang, Shanmin, Luqiang Huang, Chun Chen, Mei Zhang, Zhenhuan Zhang, Deping Han, Jinsheng Hong, et al. "Abstract 3849: Triptolide reduces activity of RNA polymerase II." In Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL. American Association for Cancer Research, 2012. http://dx.doi.org/10.1158/1538-7445.am2012-3849.

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Muda, Nora, Abdul Rahman Othman, Nazalan Najimudin, and Zeti Azura Mohamed Hussein. "The Phylogenetic Tree of RNA Polymerase Constructed Using MOM Method." In 2009 International Conference of Soft Computing and Pattern Recognition. IEEE, 2009. http://dx.doi.org/10.1109/socpar.2009.99.

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Reports on the topic "RNA polymerases"

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Gal-On, Amit, Shou-Wei Ding, Victor P. Gaba, and Harry S. Paris. role of RNA-dependent RNA polymerase 1 in plant virus defense. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597919.bard.

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Objectives: Our BARD proposal on the impact of RNA-dependent RNA polymerase 1 (RDR1) in plant defense against viruses was divided into four original objectives. 1. To examine whether a high level of dsRNA expression can stimulate RDR1 transcription independent of salicylic acid (SA) concentration. 2. To determine whether the high or low level of RDR1 transcript accumulation observed in virus resistant and susceptible cultivars is associated with viral resistance and susceptibility. 3. To define the biogenesis and function of RDR1-dependent endogenous siRNAs. 4. To understand why Cucumber mosaic virus (CMV) can overcome RDR1-dependent resistance. The objectives were slightly changed due to the unique finding that cucumber has four different RDR1 genes. Background to the topic: RDR1 is a key plant defense against viruses. RDR1 is induced by virus infection and produces viral and plant dsRNAs which are processed by DICERs to siRNAs. siRNAs guide specific viral and plant RNA cleavage or serve as primers for secondary amplification of viral-dsRNA by RDR. The proposal is based on our preliminary results that a. the association of siRNA and RDR1 accumulation with multiple virus resistance, and b. that virus infection induced the RDR1-dependent production of a new class of endogenous siRNAs. However, the precise mechanisms underlying RDR1 induction and siRNA biogenesis due to virus infection remain to be discovered in plants. Major conclusions, solutions and achievements: We found that in the cucurbit family (cucumber, melon, squash, watermelon) there are 3-4 RDR1 genes not documented in other plant families. This important finding required a change in the emphasis of our objectives. We characterized 4 RDR1s in cucumber and 3 in melon. We demonstrated that in cucumber RDR1b is apparently a new broad spectrum virus resistance gene, independent of SA. In melon RDR1b is truncated, and therefore is assumed to be the reason that melon is highly susceptible to many viruses. RDR1c is dramatically induced due to DNA and RNA virus infection, and inhibition of RDR1c expression led to increased virus accumulation which suggested its important on gene silencing/defense mechanism. We show that induction of antiviral RNAi in Arabidopsis is associated with production of a genetically distinct class of virus-activated siRNAs (vasiRNAs) by RNA dependent RNA polymerase-1 targeting hundreds of host genes for RNA silencing by Argonaute-2. Production of vasiRNAs is induced by viruses from two different super groups of RNA virus families, targeted for inhibition by CMV, and correlated with virus resistance independently of viral siRNAs. We propose that antiviral RNAi activate broad-spectrum antiviral activity via widespread silencing of host genes directed by vasiRNAs, in addition to specific antiviral defense Implications both scientific and agricultural: The RDR1b (resistance) gene can now be used as a transcription marker for broad virus resistance. The discovery of vasiRNAs expands the repertoire of siRNAs and suggests that the siRNA-processing activity of Dicer proteins may play a more important role in the regulation of plant and animal gene expression than is currently known. We assume that precise screening of the vasiRNA host targets will lead in the near future for identification of plant genes associate with virus diseases and perhaps other pathogens.
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Gnatt, Averall. Structural Determination of A Transcribing RNA Polymerase II Complex. Fort Belvoir, VA: Defense Technical Information Center, March 1999. http://dx.doi.org/10.21236/ada367567.

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Laurence, Jeffrey. Antibody to the RNA-Dependent DNA Polymerase of HTLV-III: Characterization and Clinical Associations. Fort Belvoir, VA: Defense Technical Information Center, March 1990. http://dx.doi.org/10.21236/ada231466.

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Laurence, Jeffrey. Antibody to the RNA-Dependent DNA Polymerase of HTLV-III: characterization and Clinical Associations. Fort Belvoir, VA: Defense Technical Information Center, July 1988. http://dx.doi.org/10.21236/ada227404.

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Laurence, Jeffrey. Antibody to the RNA-Dependent DNA Polymerase of HTLV-III: characterization and Clinical Associations. Fort Belvoir, VA: Defense Technical Information Center, December 1988. http://dx.doi.org/10.21236/ada227519.

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Vakharia, Vikram, Shoshana Arad, Yonathan Zohar, Yacob Weinstein, Shamila Yusuff, and Arun Ammayappan. Development of Fish Edible Vaccines on the Yeast and Redmicroalgae Platforms. United States Department of Agriculture, February 2013. http://dx.doi.org/10.32747/2013.7699839.bard.

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Betanodaviruses are causative agents of viral nervous necrosis (VNN), a devastating disease of cultured marine fish worldwide. Betanodavirus (BTN) genome is composed of two single-stranded, positive-sense RNA molecules. The larger genomic segment, RNA1 (3.1 kb), encodes the RNA-dependent RNA polymerase, while the smaller genomic segment, RNA 2 (1.4kb), encodes the coat protein. This structural protein is the host-protective antigen of VNN which assembles to form virus-like particles (VLPs). BTNs are classified into four genotypes, designated red-spotted grouper nervous necrosis virus (RGNNV), barfin flounder nervous necrosis virus (BFNNV), tiger puffer nervous necrosis virus (TPNNV), and striped jack nervous necrosis virus (SJNNV), based on phylogenetic analysis of the coat protein sequences. RGNNV type is quite important as it has a broad host-range, infecting warm-water fish species. At present, there is no commercial vaccine available to prevent VNN in fish. The general goal of this research was to develop oral fish vaccines in yeast and red microalgae (Porphyridium sp.) against the RGNNV genotype. To achieve this, we planned to clone and sequence the coat protein gene of RGNNV, express the coat protein gene of RGNNV in yeast and red microalgae and evaluate the immune response in fish fed with recombinantVLPs antigens produced in yeast and algae. The collaboration between the Israeli group and the US group, having wide experience in red microalgae biochemistry, molecular genetics and large-scale cultivation, and the development of viral vaccines and eukaryotic protein expression systems, respectively, was synergistic to produce a vaccine for fish that would be cost-effective and efficacious against the betanodavirus infection.
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Stern, David, and Gadi Schuster. Manipulating Chloroplast Gene Expression: A Genetic and Mechanistic Analysis of Processes that Control RNA Stability. United States Department of Agriculture, June 2004. http://dx.doi.org/10.32747/2004.7586541.bard.

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New potential for engineering chloroplasts to express novel traits has stimulated research into relevant techniques and genetic processes, including plastid transformation and gene regulation. This BARD-funded research dealt with the mechanisms that influence chloroplast RNA accumulation, and thus gene expression. Previous work on cpRNA catabolism has elucidated a pathway initiated by endonucleolytic cleavage, followed by polyadenylation and exonucleolytic degradation. A major player in this process is the nucleus-encoded exoribo-nuclease/polymerase polynucleotide phosphorylase (PNPase). Biochemical characterization of PNPase has revealed a modular structure that controls its RNA synthesis and degradation activities, which in turn are responsive to the phosphate (P) concentration. During the funding period, new insights emerged into the molecular mechanism of RNA metabolism in the chloroplast and cyanobacteria, suggesting strategies for improving agriculturally-important plants or plants with novel introduced traits.
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Gafny, Ron, A. L. N. Rao, and Edna Tanne. Etiology of the Rugose Wood Disease of Grapevine and Molecular Study of the Associated Trichoviruses. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575269.bard.

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Rugose wood is a complex disease of grapevines, characterized by modification of the woody cylinder of affected vines. The control of rugose wood is based on the production of healthy propagation material. Detection of rugose wood in grapevines is difficult and expensive: budwood from tested plants is grafted onto sensitive Vitis indicators and the appearance of symptoms is monitored for 3 years. The etiology of rugose wood is complex and has not yet been elucidated. Several elongated clostero-like viruses are consistently found in affected vines; one of them, grapevine virus A (GVA), is closely associated with Kober stem grooving, a component of the rugose wood complex. GVA has a single-stranded RNA genome of 7349 nucleotides, excluding a polyA tail at the 3' terminus. The GVA genome includes five open reading frames (ORFs 1-5). ORF 4, which encodes for the coat protein of GVA, is the only ORF for which the function was determined experimentally. The original objectives of this research were: 1- To produce antisera to the structural and non-structural proteins of GVA and GVB and to use these antibodies to establish an effective detection method. 2- Develop full length infectious cDNA clones of GVA and GVB. 3- Study the roll of GVA and GVB in the etiology of the grapevine rugose wood disease. 4- Determine the function of Trichovirus (now called Vitivirus) encoded genes in the virus life cycle. Each of the ORFs 2, 3, 4 and 5 genes of GVA were cloned and expressed in E. coli and used to produce antisera. Both the CP (ORF 4) and the putative MP (ORF 3) were detected with their corresponding antisera in-GVA infected N. benthamiana and grapevine. The MP was first detected at an early stage of the infection, 6-12 h after inoculation, and the CP 2-3 days after inoculation. The MP could be detected in GVA-infected grapevines that tested negative for CP, both with CP antiserum and with a commercially available ELISA kit. Antisera to ORF 2 and 5 encoded proteins could react with the recombinant proteins but failed to detect both proteins in GVA infected plants. A full-length cDNA clone of grapevine virus A (GVA) was constructed downstream from the bacteriophage T7 RNA polymerase promoter. Capped in vitro transcribed RNA was infectious in N. benthamiana and N. clevelandii plants. Symptoms induced by the RNA transcripts or by the parental virus were indistinguishable. The infectivity of the in vitro-transcribed RNA was confirmed by serological detection of the virus coat and movement proteins and by observation of virions by electron microscopy. The full-length clone was modified to include a gus reporter gene and gus activity was detected in inoculated and systemic leaves of infected plants. Studies of GVA mutants suggests that the coat protein (ORF 4) is essential for cell to cell movement, the putative movement protein (ORF 3) indeed functions as a movement protein and that ORF 2 is not required for virus replication, cell to cell or systemic movement. Attempts to infect grapevines by in-vitro transcripts, by inoculation of cDNA construct in which the virus is derived by the CaMV 35S promoter or by approach grafting with infected N. benthamiana, have so far failed. Studies of the subcellular distribution of GFP fusion with each of ORF 2, 3 and 4 encoded protein showed that the CP fusion protein accumulated as a soluble cytoplasmatic protein. The ORF 2 fusion protein accumulated in cytoplasmatic aggregates. The MP-GFP fusion protein accumulated in a large number of small aggregates in the cytoplasm and could not move from cell to cell. However, in conditions that allowed movement of the fusion protein from cell to cell (expression by a PVX vector or in young immature leaves) the protein did not form cytoplasmatic aggregates but accumulated in the plasmodesmata.
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Jordan, Ramon L., Abed Gera, Hei-Ti Hsu, Andre Franck, and Gad Loebenstein. Detection and Diagnosis of Virus Diseases of Pelargonium. United States Department of Agriculture, July 1994. http://dx.doi.org/10.32747/1994.7568793.bard.

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Pelargonium (Geranium) is the number one pot plant in many areas of the United States and Europe. Israel and the U.S. send to Europe rooted cuttings, foundation stocks and finished plants to supply a certain share of the market. Geraniums are propagated mainly vegetatively from cuttings. Consequently, viral diseases have been and remain a major threat to the production and quality of the crop. Among the viruses isolated from naturally infected geraniums, 11 are not specific to Pelargonium and occur in other crops while 6 other viruses seem to be limited to geranium. However, several of these viruses are not sufficiently characterized to conclude that they are distinct agents and their nomenclature and taxonomy are confusing. The ability to separate, distinguish and detect the different viruses in geranium will overcome obstacles te developing effective detection and certification schemes. Our focus was to further characterize some of these viruses and develop better methods for their detection and control. These viruses include: isolates of pelargonium line pattern virus (PLPV), pelargonium ringspot virus (PelRSV), pelargonium flower break virus (PFBV), pelargonium leaf curl (PLCV), and tomato ringspot virus (TomRSV). Twelve hybridoma cell lines secreting monoclonal antibodies specific to a geranium isolate of TomRSV were produced. These antibodies are currently being characterized and will be tested for the ability to detect TomRSV in infected geraniums. The biological, biochemical and serological properties of four isometric viruses - PLPV, PelRSV, and PFBV (and a PelRSV-like isolate from Italy called GR57) isolated from geraniums exhibiting line and ring pattern or flower break symptoms - and an isolate ol elderbeny latent virus (ELV; which the literature indicates is the same as PelRSV) have been determined Cloned cDNA copies of the genomic RNAs of these viruses were sequenced and the sizes and locations of predicted viral proteins deduced. A portion of the putative replicase genes was also sequenced from cloned RT-PCR fragments. We have shown that, when compared to the published biochemical and serological properties, and sequences and genome organizations of other small isometric plant viruses, all of these viruses should each be considered new, distinct members of the Carmovirus group of the family Tombusviridae. Hybridization assays using recombinant DNA probes also demonstrated that PLPV, PelRSV, and ELV produce only one subgenomic RNA in infected plants. This unusual property of the gene expression of these three viruses suggests that they are unique among the Carmoviruses. The development of new technologies for the detection of these viruses in geranium was also demonstrated. Hybridization probes developed to PFBV (radioactively-labeled cRNA riboprobes) and to PLPV (non-radioactive digoxigenin-labeled cDNAs) were generally shown to be no more sensitive for the detection of virus in infected plants than the standard ELISA serology-based assays. However, a reverse transcriptase-polymerase chain reaction assay was shown to be over 1000 times more sensitive in detecting PFBV in leaf extracts of infected geranium than was ELISA. This research has lead to a better understanding of the identity of the viruses infecting pelargonium and to the development of new tools that can be used in an improved scheme of providing virus-indexed pelargonium plants. The sequence information, and the serological and cloned DNA probes generated from this work, will allow the application of these new tools for virus detection, which will be useful in domestic and international indexing programs which are essential for the production of virus-free germplasm both for domestic markets and the international exchange of plant material.
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10

Grumet, Rebecca, and Benjamin Raccah. Identification of Potyviral Domains Controlling Systemic Infection, Host Range and Aphid Transmission. United States Department of Agriculture, July 2000. http://dx.doi.org/10.32747/2000.7695842.bard.

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Potyviruses form one of the largest and most economically important groups of plant viruses. Individual potyviruses and their isolates vary in symptom expression, host range, and ability to overcome host resistance genes. Understanding factors influencing these biological characteristics is of agricultural importance for epidemiology and deployment of resistance strategies. Cucurbit crops are subject to severe losses by several potyviruses including the highly aggressive and variable zucchini yellow mosaic virus (ZYMV). In this project we sought to investigate protein domains in ZYMV that influence systemic infection and host range. Particular emphasis was on coat protein (CP), because of known functions in both cell to cell and long distance movement, and helper component-protease (HC-Pro), which has been implicated to play a role in symptom development and long distance movement. These two genes are also essential for aphid mediated transmission, and domains that influence disease development may also influence transmissibility. The objectives of the approved BARD project were to test roles of specific domains in the CP and HC-Pro by making sequence alterations or switches between different isolates and viruses, and testing for infectivity, host range, and aphid transmissibility. These objectives were largely achieved as described below. Finally, we also initiated new research to identify host factors interacting with potyviral proteins and demonstrated interaction between the ZYMV RNA dependent RNA polymerase and host poly-(A)-binding protein (Wang et al., in press). The focus of the CP studies (MSU) was to investigate the role of the highly variable amino terminus (NT) in host range determination and systemic infection. Hybrid ZYMV infectious clones were produced by substituting the CP-NT of ZYMV with either the CP-NT from watermelon mosaic virus (overlapping, but broader host range) or tobacco etch virus (TEV) (non- overlapping host range) (Grumet et al., 2000; Ullah ct al., in prep). Although both hybrid viruses initially established systemic infection, indicating that even the non-cucurbit adapted TEV CP-NT could facilitate long distance transport in cucurbits, after approximately 4-6, the plants inoculated with the TEV-CPNT hybrid exhibited a distinct recovery of reduced symptoms, virus titer, and virus specific protection against secondary infection. These results suggest that the plant recognizes the presence of the TEV CP-NT, which has not been adapted to infection of cucurbits, and initiates defense responses. The CP-NT also appears to play a role in naturally occurring resistance conferred by the zym locus in the cucumber line 'Dina-1'. Patterns of virus accumulation indicated that expression of resistance is developmentally controlled and is due to a block in virus movement. Switches between the core and NT domains of ZYMV-NAA (does not cause veinal chlorosis on 'Dina-1'), and ZYMV-Ct (causes veinal chlorosis), indicated that the resistance response likely involves interaction with the CP-NT (Ullah and Grumet, submitted). At the Volcani Center the main thrust was to identify domains in the HC-Pro that affect symptom expression or aphid transmissibility. From the data reported in the first and second year report and in the attached publications (Peng et al. 1998; Kadouri et al. 1998; Raccah et al. 2000: it was shown that: 1. The mutation from PTK to PAK resulted in milder symptoms of the virus on squash, 2. Two mutations, PAK and ATK, resulted in total loss of helper activity, 3. It was established for the first time that the PTK domain is involved in binding of the HC-Pro to the potyvirus particle, and 4. Some of these experiments required greater amount of HC-Pro, therefore a simpler and more efficient purification method was developed based on Ni2+ resin.
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