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Edward G Niles - One of the best experts on this subject based on the ideXlab platform.
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Determinants of vaccinia virus Early Gene transcription termination.
Virology, 2008Co-Authors: Sarah C. Piacente, Benjamin K. Dickerman, Mohamed Ragaa Mohamed, Linda Christen, Edward G NilesAbstract:Vaccinia virus Early Gene transcription requires the vaccinia termination factor, VTF, nucleoside triphosphate phosphohydrolase I, NPH I, ATP, the virion RNA polymerase, and the motif, UUUUUNU, in the nascent RNA, found within 30 to 50 bases from the poly A addition site, in vivo. In this study, the relationships among the vaccinia Early Gene transcription termination efficiency, termination motif specificity, and the elongation rate were investigated. A low transcription elongation rate maximizes termination efficiency and minimizes specificity for the UUUUUNU motif. Positioning the termination motif over a 63 base area upstream from the RNA polymerase allowed efficient transcript release, demonstrating a remarkable plasticity in the transcription termination complex. Efficient transcript release was observed during ongoing transcription, independent of VTF or UUUUUNU, but requiring both NPH I and either ATP or dATP. This argues for a two step model: the specifying step, requiring both VTF and UUUUUNU, and the energy-dependent step employing NPH I and ATP. Evaluation of NPH I mutants for the ability to stimulate transcription elongation demonstrated that ATPase activity and a stable interaction between NPH I and the Rap94 subunit of the viral RNA polymerase are required. These observations demonstrate that NPH I is a component of the elongating RNA polymerase, which is catalytically active during transcription elongation.
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Effect of UTP sugar and base modifications on vaccinia virus Early Gene transcription
Virology, 2006Co-Authors: Mohamed Ragaa Mohamed, Sarah C. Piacente, Benjamin K. Dickerman, Edward G NilesAbstract:Prior efforts demonstrated that RNA oligonucleotides containing the transcription termination signal UUUUUNU stimulate premature termination of vaccinia virus Early Gene transcription, in vitro. This observation suggests that viral transcription termination may be an attractive target for the development of anti-poxvirus agents. Since short RNA molecules are readily susceptible to nuclease digestion, their use would require stabilizing modifications. In order to evaluate the effect of both ribose and uracil modifications of the U5NU signal on Early Gene transcription termination, UTP derivatives harboring modifications to the uracil base, the 2′ position of the ribose sugar and the phosphodiester bond were examined in an in vitro vaccinia virus Early Gene transcription termination system. Incorporation of 4-S-U, 5-methyl-U, 2-S-U, pseudo U and 2′-F-dU into the nascent transcript inhibited transcription termination. 6-aza-U, 2′-amino-U, 2′-azido-U and 2′-O methyl-U inhibited transcription elongation resulting in the accumulation of short transcripts. The majority of the short transcripts remained in the ternary complex and could be chased into full-length transcripts. Initially, derivatives of all uridines in the termination signal were tested. Partial modification of the termination signal reduced termination activity, as well. Introduction of 2′-O methyl ribose to the first three uridines of the U9 termination signal reduced the ability of U9 containing oligonucleotides to stimulate in vitro transcription termination, in trans. Further modifications eliminated this activity. Thus, viral Early Gene transcription termination demonstrates a rigorous requirement for a U5NU signal that is unable to tolerate modification to the base or sugar. Additionally, VTF was shown to enhance transcription elongation through the T9 sequence in the template. These results suggest that VTF may play a subtle role in Early Gene transcription elongation in addition to its known function in mRNA cap formation, Early Gene transcription termination and intermediate Gene transcription initiation.
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UUUUUNU oligonucleotide stimulation of vaccinia virus Early Gene transcription termination, in trans.
Journal of Biological Chemistry, 2003Co-Authors: Mohamed Ragaa Mohamed, Edward G NilesAbstract:Abstract Vaccinia virus Early Gene transcription termination requires the vaccinia termination factor (VTF), NPH I, a single stranded DNA-dependent ATPase, the virion form of RNA polymerase containing the Rap 94 subunit, and the signal UUUUUNU, which resides in the nascent mRNA, located 30 to 50 bases upstream from the poly(A) addition site. Evidence indicates that a required termination factor acts through binding to the UUUUUNU signal. To further investigate the function of UUUUUNU, the ability of UUUUUNU containing oligonucleotides to inhibit transcription termination was tested. A 22-mer RNA oligonucleotide containing a central U9 sequence exhibited sequence and concentration-dependent stimulation of premature transcription termination and transcript release, in trans. Activation of premature termination required VTF, NPH I, Rap 94, and ATP, demonstrating that the normal termination machinery was employed. Premature termination was not stimulated by RNA harboring a mutant UUUUUNU, demonstrating specificity. These data are consistent with a model in which a required termination factor is converted from an inactive to an active form by binding to a UUUUUNU containing oligonucleotide. The active termination factor then interacts with the ternary complex stimulating transcription termination through the normal mechanism, independent of the nascent mRNA sequence.
Mohamed Ragaa Mohamed - One of the best experts on this subject based on the ideXlab platform.
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Determinants of vaccinia virus Early Gene transcription termination.
Virology, 2008Co-Authors: Sarah C. Piacente, Benjamin K. Dickerman, Mohamed Ragaa Mohamed, Linda Christen, Edward G NilesAbstract:Vaccinia virus Early Gene transcription requires the vaccinia termination factor, VTF, nucleoside triphosphate phosphohydrolase I, NPH I, ATP, the virion RNA polymerase, and the motif, UUUUUNU, in the nascent RNA, found within 30 to 50 bases from the poly A addition site, in vivo. In this study, the relationships among the vaccinia Early Gene transcription termination efficiency, termination motif specificity, and the elongation rate were investigated. A low transcription elongation rate maximizes termination efficiency and minimizes specificity for the UUUUUNU motif. Positioning the termination motif over a 63 base area upstream from the RNA polymerase allowed efficient transcript release, demonstrating a remarkable plasticity in the transcription termination complex. Efficient transcript release was observed during ongoing transcription, independent of VTF or UUUUUNU, but requiring both NPH I and either ATP or dATP. This argues for a two step model: the specifying step, requiring both VTF and UUUUUNU, and the energy-dependent step employing NPH I and ATP. Evaluation of NPH I mutants for the ability to stimulate transcription elongation demonstrated that ATPase activity and a stable interaction between NPH I and the Rap94 subunit of the viral RNA polymerase are required. These observations demonstrate that NPH I is a component of the elongating RNA polymerase, which is catalytically active during transcription elongation.
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Effect of UTP sugar and base modifications on vaccinia virus Early Gene transcription
Virology, 2006Co-Authors: Mohamed Ragaa Mohamed, Sarah C. Piacente, Benjamin K. Dickerman, Edward G NilesAbstract:Prior efforts demonstrated that RNA oligonucleotides containing the transcription termination signal UUUUUNU stimulate premature termination of vaccinia virus Early Gene transcription, in vitro. This observation suggests that viral transcription termination may be an attractive target for the development of anti-poxvirus agents. Since short RNA molecules are readily susceptible to nuclease digestion, their use would require stabilizing modifications. In order to evaluate the effect of both ribose and uracil modifications of the U5NU signal on Early Gene transcription termination, UTP derivatives harboring modifications to the uracil base, the 2′ position of the ribose sugar and the phosphodiester bond were examined in an in vitro vaccinia virus Early Gene transcription termination system. Incorporation of 4-S-U, 5-methyl-U, 2-S-U, pseudo U and 2′-F-dU into the nascent transcript inhibited transcription termination. 6-aza-U, 2′-amino-U, 2′-azido-U and 2′-O methyl-U inhibited transcription elongation resulting in the accumulation of short transcripts. The majority of the short transcripts remained in the ternary complex and could be chased into full-length transcripts. Initially, derivatives of all uridines in the termination signal were tested. Partial modification of the termination signal reduced termination activity, as well. Introduction of 2′-O methyl ribose to the first three uridines of the U9 termination signal reduced the ability of U9 containing oligonucleotides to stimulate in vitro transcription termination, in trans. Further modifications eliminated this activity. Thus, viral Early Gene transcription termination demonstrates a rigorous requirement for a U5NU signal that is unable to tolerate modification to the base or sugar. Additionally, VTF was shown to enhance transcription elongation through the T9 sequence in the template. These results suggest that VTF may play a subtle role in Early Gene transcription elongation in addition to its known function in mRNA cap formation, Early Gene transcription termination and intermediate Gene transcription initiation.
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UUUUUNU oligonucleotide stimulation of vaccinia virus Early Gene transcription termination, in trans.
Journal of Biological Chemistry, 2003Co-Authors: Mohamed Ragaa Mohamed, Edward G NilesAbstract:Abstract Vaccinia virus Early Gene transcription termination requires the vaccinia termination factor (VTF), NPH I, a single stranded DNA-dependent ATPase, the virion form of RNA polymerase containing the Rap 94 subunit, and the signal UUUUUNU, which resides in the nascent mRNA, located 30 to 50 bases upstream from the poly(A) addition site. Evidence indicates that a required termination factor acts through binding to the UUUUUNU signal. To further investigate the function of UUUUUNU, the ability of UUUUUNU containing oligonucleotides to inhibit transcription termination was tested. A 22-mer RNA oligonucleotide containing a central U9 sequence exhibited sequence and concentration-dependent stimulation of premature transcription termination and transcript release, in trans. Activation of premature termination required VTF, NPH I, Rap 94, and ATP, demonstrating that the normal termination machinery was employed. Premature termination was not stimulated by RNA harboring a mutant UUUUUNU, demonstrating specificity. These data are consistent with a model in which a required termination factor is converted from an inactive to an active form by binding to a UUUUUNU containing oligonucleotide. The active termination factor then interacts with the ternary complex stimulating transcription termination through the normal mechanism, independent of the nascent mRNA sequence.
Mike Dragunow - One of the best experts on this subject based on the ideXlab platform.
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Immediate Early Gene transcription and synaptic modulation.
Journal of neuroscience research, 1999Co-Authors: M. Walton, Wickliffe C. Abraham, P. Lawlor, C. Henderson, Sara E. Mason-parker, David K. Bilkey, Mike DragunowAbstract:Long-term changes in Gene expression appear to be critical to the formation of memory, but little is known about its stimulus- transcription coupling. Numerous studies in the last decade, by focusing on unraveling this signal transduction pathway, have investigated the potential role of the immediate-Early Genes in this process. The krox family of immediate-Early Gene proteins are of particular interest because they may be involved in stabilizing the synaptic modifications that underlie hippocampal long-term potentiation (LTP). A potential upstream mediator of krox induction is cyclic AMP-responsive element binding protein (CREB), a posttranslationally activated transcription factor that has been implicated in numerous memory paradigms. In this study we investigated whether the activation of CREB by phosphorylation may have a role in the development of rat perforant- path-stimulated LTP and associated dentate granule cell krox-24 mRNA expression. Contrary to what was expected, we failed to show any difference in the levels of phosphorylated CREB after LTP or following endogenous synaptic facilitation stimulated by novelty. Using these same model systems we also investigated the protein levels of brain- derived neurotrophic factor (BDNF), another immediate-Early Gene that is induced following a durable form of LTP. However, BDNF protein was not induced within the hippocampus after LTP and was transiently decreased following novel environmental stimulation. J. Neurosci. Res. 58:96–106, 1999. © 1999 Wiley-Liss, Inc.
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MK-801 Does Not Attenuate Immediate-Early Gene Expression Following an Amygdala Afterdischarge
Experimental neurology, 1994Co-Authors: Paul E. Hughes, Kerin Singleton, Mike DragunowAbstract:Abstract It has been suggested that the increased transient expression of immediate-Early Gene transcription factors seen in nerve cells following an afterdischarge may initiate longer lasting or permanent changes in Gene expression which underly the development of kindling. Since the development of kindling is sensitive to pharmacological blockade of the N -methyl- d -aspartate receptor, we tested whether the increased expression of the immediate-Early Genes c-fos, jun-B, c-jun, krox-20 , and krox-24 following a kindling afterdischarge was also sensitive to N -methyl- d -aspartate receptor blockade by MK-801. In this report we demonstrate that all five immediate-Early Genes are induced by an amygdala afterdischarge. N -methyl- d -aspartate receptor blockade by a dose of MK-801 that significantly retards the development of amygdala kindling failed to attenuate immediate-Early Gene expression. These results suggest that although expression of these five immediate-Early Genes occurs after an amygdala afterdischarge their expression is not involved in the N -methyl- d -aspartate receptor-mediated component of amygdala kindling.
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Correlations between immediate Early Gene induction and the persistence of long-term potentiation
Neuroscience, 1993Co-Authors: Wickliffe C. Abraham, S.e. Mason, Jerome Demmer, Joanna M. Williams, C.l. Richardson, P. Lawlor, Warren P. Tate, Mike DragunowAbstract:Abstract The duration of long-term potentiation in the dentate gyrus of awake rats was examined following systematic manipulation of the number of stimulus trains delivered. This was correlated with the induction of immediate Early Genes in separate groups of animals given identical stimulus regimes. Following 10 trains of stimulation, long-term potentiation decayed with a time constant of up to several days (long-term potentiation 2), and this correlated with the appearance of an increase in the messenger RNA and protein levels of zif/268. Increasing the number of stimulus trains resulted in a greater probability of eliciting long-term potentiation with a time constant of several weeks (long-term potentiation 3), as well as increasing the induction of zif/268, c-Jun, Jun-B, Jun-D and Fos-related proteins. When 10 trains were delivered repeatedly on up to five consecutive days, only the zif/268 protein levels showed associated changes. These data provide support for the hypothesis that long-term potentiation 3 involves mechanisms additional to those for long-term potentiation 2. One possible mechanism is altered Gene expression, initiated by immediate Early Gene transcription factors such as zif/268 and possibly homo- or heterodimers of Fos and Jun family members, that then contributes to the stabilization or maintenance of long-term potentiation 3.
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Brain-derived neurotrophic factor is induced as an immediate Early Gene following N-methyl-d-aspartate receptor activation
Neuroscience, 1993Co-Authors: Paul E. Hughes, Erica J. Beilharz, Peter D. Gluckman, Mike DragunowAbstract:Recent studies show that focal brain injury, cerebral ischaemia, hypoglycaemia and seizures increase the expression of c-fos and brain-derived neurotrophic factor in brain. Here we report that hippocampal focal brain injury transiently induces the immediate Early Genes c-fos, jun-B, c-jun and krox-24 (zif-268) messenger RNA and protein and brain-derived neurotrophic factor messenger RNA in rat dentate gyrus neurons, an effect that was blocked by the N-methyl-D-aspartate receptor antagonist MK-801. Prior administration of the protein synthesis inhibitor cycloheximide super-induced immediate Early Gene messenger RNA, abolished immediate Early Gene protein induction, but had no effect on injury-mediated induction of brain-derived neurotrophic factor messenger RNA. Thus, while N-methyl-D-aspartate receptor activation results in the induction of both immediate Early Genes and brain-derived neurotrophic factor messenger RNA, de novo synthesis of immediate Early Gene proteins is not critical for the increased expression of brain-derived neurotrophic factor messenger RNA seen in brain after focal injury. These results suggest that brain-derived neurotrophic factor is induced after injury as an immediate Early Gene.
Zhixin Yin - One of the best experts on this subject based on the ideXlab platform.
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shrimp nf κb binds to the immediate Early Gene ie1 promoter of white spot syndrome virus and upregulates its activity
Virology, 2010Co-Authors: Xiande Huang, Li Zhao, Haiqing Zhang, Xiaoting Jia, Yihong Chen, Peihui Wang, Shaoping Weng, Zhixin YinAbstract:The immediate-Early Gene ie1 carried by white spot syndrome virus (WSSV) exhibits very strong promoter activity and expresses highly throughout the infection cycle. Here we identified a NF-κB binding motif in the ie1 promoter region. Electrophoretic mobility shift assays indicated that the recombinant Rel homology domain (RHD) of shrimp NF-κB homolog LvRelish bound to the putative NF-κB site in the ie1 promoter. A transactivity assay of the WSSV ie1 promoter in Drosophila Schneider 2 cells demonstrated that LvRelish could increase ie1 promoter activity. These results show that shrimp NF-κB homolog LvRelish transactivates WSSV ie1 Gene expression and contributes to its high promoter activity. Further transactivation assays showed that WSSV IE1 protein expression upregulated the promoter activities of WSSV ie1 Gene and antimicrobial peptide Genes regulated by the NF-κB system. We suggested that WSSV may annex the shrimp NF-κB system, which it uses to enhance the expression of viral immediate-Early Genes.
Sarah C. Piacente - One of the best experts on this subject based on the ideXlab platform.
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Determinants of vaccinia virus Early Gene transcription termination.
Virology, 2008Co-Authors: Sarah C. Piacente, Benjamin K. Dickerman, Mohamed Ragaa Mohamed, Linda Christen, Edward G NilesAbstract:Vaccinia virus Early Gene transcription requires the vaccinia termination factor, VTF, nucleoside triphosphate phosphohydrolase I, NPH I, ATP, the virion RNA polymerase, and the motif, UUUUUNU, in the nascent RNA, found within 30 to 50 bases from the poly A addition site, in vivo. In this study, the relationships among the vaccinia Early Gene transcription termination efficiency, termination motif specificity, and the elongation rate were investigated. A low transcription elongation rate maximizes termination efficiency and minimizes specificity for the UUUUUNU motif. Positioning the termination motif over a 63 base area upstream from the RNA polymerase allowed efficient transcript release, demonstrating a remarkable plasticity in the transcription termination complex. Efficient transcript release was observed during ongoing transcription, independent of VTF or UUUUUNU, but requiring both NPH I and either ATP or dATP. This argues for a two step model: the specifying step, requiring both VTF and UUUUUNU, and the energy-dependent step employing NPH I and ATP. Evaluation of NPH I mutants for the ability to stimulate transcription elongation demonstrated that ATPase activity and a stable interaction between NPH I and the Rap94 subunit of the viral RNA polymerase are required. These observations demonstrate that NPH I is a component of the elongating RNA polymerase, which is catalytically active during transcription elongation.
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Effect of UTP sugar and base modifications on vaccinia virus Early Gene transcription
Virology, 2006Co-Authors: Mohamed Ragaa Mohamed, Sarah C. Piacente, Benjamin K. Dickerman, Edward G NilesAbstract:Prior efforts demonstrated that RNA oligonucleotides containing the transcription termination signal UUUUUNU stimulate premature termination of vaccinia virus Early Gene transcription, in vitro. This observation suggests that viral transcription termination may be an attractive target for the development of anti-poxvirus agents. Since short RNA molecules are readily susceptible to nuclease digestion, their use would require stabilizing modifications. In order to evaluate the effect of both ribose and uracil modifications of the U5NU signal on Early Gene transcription termination, UTP derivatives harboring modifications to the uracil base, the 2′ position of the ribose sugar and the phosphodiester bond were examined in an in vitro vaccinia virus Early Gene transcription termination system. Incorporation of 4-S-U, 5-methyl-U, 2-S-U, pseudo U and 2′-F-dU into the nascent transcript inhibited transcription termination. 6-aza-U, 2′-amino-U, 2′-azido-U and 2′-O methyl-U inhibited transcription elongation resulting in the accumulation of short transcripts. The majority of the short transcripts remained in the ternary complex and could be chased into full-length transcripts. Initially, derivatives of all uridines in the termination signal were tested. Partial modification of the termination signal reduced termination activity, as well. Introduction of 2′-O methyl ribose to the first three uridines of the U9 termination signal reduced the ability of U9 containing oligonucleotides to stimulate in vitro transcription termination, in trans. Further modifications eliminated this activity. Thus, viral Early Gene transcription termination demonstrates a rigorous requirement for a U5NU signal that is unable to tolerate modification to the base or sugar. Additionally, VTF was shown to enhance transcription elongation through the T9 sequence in the template. These results suggest that VTF may play a subtle role in Early Gene transcription elongation in addition to its known function in mRNA cap formation, Early Gene transcription termination and intermediate Gene transcription initiation.
