The Experts below are selected from a list of 291 Experts worldwide ranked by ideXlab platform
Donald H. Gilden - One of the best experts on this subject based on the ideXlab platform.
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Varicella-Zoster Virus Gene 66 Transcription and Translation in Latently Infected Human Ganglia
Journal of virology, 2003Co-Authors: Randall J. Cohrs, Donald H. Gilden, Paul R. Kinchington, Esther Grinfeld, Peter G. E. KennedyAbstract:Latent infection with varicella-zoster Virus (VZV) is characterized by restricted Virus Gene expression and the absence of Virus production. Of the ∼70 predicted VZV Genes, only five (Genes 4, 21, 29, 62, and 63) have been shown by multiple techniques to be transcribed during latency. IE62, the protein product of VZV Gene 62, is the major immediate-early (IE) Virus-encoded transactivator of viral Gene transcription and plays a pivotal role in transactivating viral Genes during lytic infection. The protein kinase (66-pk) encoded by VZV Gene 66 phosphorylates IE62, resulting in cytoplasmic accumulation of IE62 that mitigates nuclear IE62-induced Gene activation. Analysis of latently infected human trigeminal ganglia for 66-pk expression by reverse transcriptase-dependent nested PCR, including DNA sequence analysis, in situ hybridization, and immunohistochemistry, revealed VZV open reading frame 66 to be a previously unrecognized latently expressed Virus Gene and suggests that prevention of IE62 import to the nucleus by VZV 66-pk phosphorylation is one possible mechanism by which VZV latency is maintained.
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Identification of simian varicella Virus Gene 21 promoter region using green fluorescent protein.
Journal of virological methods, 2000Co-Authors: Ravi Mahalingam, Mary Wellish, Tiffany M. White, Donald H. GildenAbstract:Clinical, pathological, immunological and virological features of simian varicella Virus (SVV) infection in primates closely resemble those of varicella zoster Virus (VZV) infection in humans. In ganglia infected latently of humans and monkeys, Gene 21 of VZV and SVV is transcribed, respectively. We determined the nucleotide sequence of the intragenic region between SVV Genes 20 and 21 to identify the putative promoter region for SVV Gene 21. A recombinant clone was prepared in which the Gene encoding green fluorescent protein (GFP) was inserted ten base pairs upstream of the predicted translational start site for SVV Gene 21. SVV-infected monkey kidney cells transfected with the recombinant clone showed the presence of green fluorescence, whereas transfection of these cells with a construct containing the GFP Gene in the opposite orientation, produced no fluorescence. The recombinant clone containing GFP flanked by SVV sequences can be used to prepare a SVV mutant in which the Virus Gene 21 promoter drives GFP. Such a mutant will be useful in analyzing varicella pathoGenesis and latency in experimentally infected animals, studies not possible in humans.
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Varicella-Zoster Virus Gene 21: Transcriptional Start Site and Promoter Region
Journal of virology, 1998Co-Authors: Randall J. Cohrs, Michael Barbour, Donald H. GildenAbstract:Varicella-zoster Virus (VZV) causes chicken pox (varicella), becomes latent in dorsal root ganglia, and reactivates decades later to cause shingles (zoster). During latency, the entire VZV genome is present in a circular form, from which Genes 21, 29, 62, and 63 are transcribed. Immediate-early (IE) VZV Genes 62 and 63 encode regulators of Virus Gene transcription, and VZV Gene 29 encodes a major DNA-binding protein. However, little is known about the function of VZV Gene 21 or the control of its transcription. Using primer extensions, we mapped the start of VZV Gene 21 transcription in VZV-infected cells to a single site located at −79 nucleotides (nt) with respect to the initiation codon. To identify the VZV Gene 21 promoter, the 284-bp region of VZV DNA separating open reading frames (ORFs) 20 and 21 was cloned upstream from the chloramphenicol acetyltransferase Gene. In transient-transfection assays, the VZV Gene 21 promoter was transactivated in VZV-infected, but not uninfected, cells. Further, the protein encoded by ORF 62 (IE62), but not those encoded by VZV ORFs 4, 10, 61, and 63, transactivates the VZV Gene 21 promoter. By use of transient-cotransfection assays in conjunction with 5′ deletions of the VZV Gene 21 promoter, a 40-bp segment was shown to be responsible for the transactivation of the VZV Gene 21 promoter by IE62. This region was located at −96 to −56 nt with respect to the 5′ start of Gene 21 transcription.
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A simian varicella Virus (SVV) homolog to varicella-zoster Virus Gene 21 is expressed in monkey ganglia latently infected with SVV.
Journal of virology, 1996Co-Authors: Penny Clarke, W L Matlock, T Beer, Donald H. GildenAbstract:We have sequenced a simian varicella Virus (SVV) open reading frame (ORF), 3,123 bp in length, whose product has 51% amino acid homology with the sequence encoded by the ORF of varicella-zoster Virus Gene 21. Several regions are highly conserved between the two ORFs, with homologies of approximately 80%. The SVV Gene is transcribed in tissue culture cells productively infected with SVV and in monkey ganglia latently infected with SVV.
Randall J. Cohrs - One of the best experts on this subject based on the ideXlab platform.
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Varicella-Zoster Virus Gene 66 Transcription and Translation in Latently Infected Human Ganglia
Journal of virology, 2003Co-Authors: Randall J. Cohrs, Donald H. Gilden, Paul R. Kinchington, Esther Grinfeld, Peter G. E. KennedyAbstract:Latent infection with varicella-zoster Virus (VZV) is characterized by restricted Virus Gene expression and the absence of Virus production. Of the ∼70 predicted VZV Genes, only five (Genes 4, 21, 29, 62, and 63) have been shown by multiple techniques to be transcribed during latency. IE62, the protein product of VZV Gene 62, is the major immediate-early (IE) Virus-encoded transactivator of viral Gene transcription and plays a pivotal role in transactivating viral Genes during lytic infection. The protein kinase (66-pk) encoded by VZV Gene 66 phosphorylates IE62, resulting in cytoplasmic accumulation of IE62 that mitigates nuclear IE62-induced Gene activation. Analysis of latently infected human trigeminal ganglia for 66-pk expression by reverse transcriptase-dependent nested PCR, including DNA sequence analysis, in situ hybridization, and immunohistochemistry, revealed VZV open reading frame 66 to be a previously unrecognized latently expressed Virus Gene and suggests that prevention of IE62 import to the nucleus by VZV 66-pk phosphorylation is one possible mechanism by which VZV latency is maintained.
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Varicella-Zoster Virus Gene 21: Transcriptional Start Site and Promoter Region
Journal of virology, 1998Co-Authors: Randall J. Cohrs, Michael Barbour, Donald H. GildenAbstract:Varicella-zoster Virus (VZV) causes chicken pox (varicella), becomes latent in dorsal root ganglia, and reactivates decades later to cause shingles (zoster). During latency, the entire VZV genome is present in a circular form, from which Genes 21, 29, 62, and 63 are transcribed. Immediate-early (IE) VZV Genes 62 and 63 encode regulators of Virus Gene transcription, and VZV Gene 29 encodes a major DNA-binding protein. However, little is known about the function of VZV Gene 21 or the control of its transcription. Using primer extensions, we mapped the start of VZV Gene 21 transcription in VZV-infected cells to a single site located at −79 nucleotides (nt) with respect to the initiation codon. To identify the VZV Gene 21 promoter, the 284-bp region of VZV DNA separating open reading frames (ORFs) 20 and 21 was cloned upstream from the chloramphenicol acetyltransferase Gene. In transient-transfection assays, the VZV Gene 21 promoter was transactivated in VZV-infected, but not uninfected, cells. Further, the protein encoded by ORF 62 (IE62), but not those encoded by VZV ORFs 4, 10, 61, and 63, transactivates the VZV Gene 21 promoter. By use of transient-cotransfection assays in conjunction with 5′ deletions of the VZV Gene 21 promoter, a 40-bp segment was shown to be responsible for the transactivation of the VZV Gene 21 promoter by IE62. This region was located at −96 to −56 nt with respect to the 5′ start of Gene 21 transcription.
Bernard Moss - One of the best experts on this subject based on the ideXlab platform.
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Modification of the cascade model for regulation of vaccinia Virus Gene expression: purification of a prereplicative, late-stage-specific transcription factor.
Journal of virology, 1994Co-Authors: G R Kovacs, Ricardo Rosales, J G Keck, Bernard MossAbstract:Abstract In vivo and in vitro studies have provided evidence that vaccinia Virus late Gene transcription factors are intermediate Gene products synthesized exclusively after DNA replication. Here, we describe an additional transcription factor (P3 factor) that stimulates late Gene transcription between 10- and 40-fold but is made in the absence of viral DNA replication. P3 factor activity was not detected either in uninfected cells or in purified virions. A > 1,500-fold purification of P3 factor was achieved by column chromatography of cytoplasmic extracts prepared from cells infected with vaccinia Virus in the presence of a DNA replication inhibitor. P3 factor was stage specific, since it could not substitute for early or intermediate transcription factors. Evidence that late stage-specific transcription factors are made both before and after DNA replication necessitates a modification of the cascade model for vaccinia Virus Gene regulation.
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Cascade Regulation of Vaccinia Virus Gene Expression
Regulation of Gene Expression in Animal Viruses, 1993Co-Authors: Bernard MossAbstract:Vaccinia Virus provides a facile system for combined biochemical and Genetic analyses. The three regulatory classes of vaccinia Virus Genes — early, intermediate and late — have distinctive promoters and stage specific Virus-encoded transcription factors. The expression of vaccinia Virus early Genes occurs soon after infection of the cell and entry of the Virus particle into the cytoplasm. The core associated RNA polymerase, VETF, capping and methylating enzymes, and poly(A) polymerase produce viral transcripts that resemble eukaryotic mRNAs in structure and are translated efficiently. The products of the early mRNAs include RNA polymerase, capping enzyme, and poly(A) polymerase subunits, DNA polymerase and factors for specific transcription of intermediate Genes. The virion DNA cannot serve as a template for intermediate Gene expression, probably because core proteins restrict access by the newly synthesized transcription complex. Following DNA replication, however, intermediate Genes are transcribed. The products of the intermediate Genes include three transactivators of late Gene expression. In the third phase of the cycle, the late Genes are expressed and virion enzymes, early transcription factor and structural proteins are assembled into infectious progeny viral particles to begin the cycle again.
Mani Subramanian - One of the best experts on this subject based on the ideXlab platform.
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Development of a Plant Viral-Vector-Based Gene Expression Assay for the Screening of Yeast Cytochrome P450 Monooxygenases
Assay and Drug Development Technologies, 2003Co-Authors: Kathleen M. Hanley, Long V Nguyen, Faizah Khan, Gregory P Pogue, Fakhrieh Vojdani, Sanjay Panda, Franck Pinot, Vincent B Oriedo, Lada Rasochova, Mani SubramanianAbstract:Development of a Gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant Virus Gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid ω-hydroxylase activity. Yarrowia lipolytica CYP52 Gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant Virus Gene expression vector, and used as a model system for determining heterologous expression. Plants infected with Virus vectors expressing the yeast CYP52 Genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative con...
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Development of a plant viral-vector-based Gene expression assay for the screening of yeast cytochrome p450 monooxygenases.
Assay and drug development technologies, 2003Co-Authors: Kathleen Hanley, Long V Nguyen, Faizah Khan, Gregory P Pogue, Fakhrieh Vojdani, Sanjay Panda, Franck Pinot, Vincent B Oriedo, Lada Rasochova, Mani SubramanianAbstract:Development of a Gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant Virus Gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 Gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant Virus Gene expression vector, and used as a model system for determining heterologous expression. Plants infected with Virus vectors expressing the yeast CYP52 Genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant Virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK Gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of Gene function.
Andrew J. Davison - One of the best experts on this subject based on the ideXlab platform.
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Delineation of a sequence required for nuclear localization of the protein encoded by varicella-zoster Virus Gene 61.
Journal of General Virology, 1994Co-Authors: David Stevenson, Kathryn L. Colman, Andrew J. DavisonAbstract:All characterized alphaherpesViruses encode a protein whose N-terminal region contains a novel zinc-binding motif, the C3HC4 domain. Homology between the different proteins is in General limited to key residues in this domain. In order to identify a separate landmark site in the C3HC4 protein encoded by varicella-zoster Virus Gene 61, namely the region required for nuclear localization, we have analysed a range of mutants in transient expression and immunofluorescence experiments. A basic region (RGAKRR) at residues 387 to 392 was found to be required for nuclear localization, and residues 390 and 391 were critical.
