scholarly journals Expression of Autographa californica Multiple Nucleopolyhedrovirus Genes in Mammalian Cells and Upregulation of the Host β-Actin Gene

2006 ◽  
Vol 80 (5) ◽  
pp. 2390-2395 ◽  
Author(s):  
Ryosuke Fujita ◽  
Takahiro Matsuyama ◽  
Junya Yamagishi ◽  
Ken Sahara ◽  
Shinichiro Asano ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Mammalian Cells ◽  
De Novo ◽  
Autographa Californica ◽  
Early Gene ◽  
Bhk Cells ◽  
Multiple Nucleopolyhedrovirus ◽  
Viral Genes ◽  
Tata Motif

ABSTRACT The gene expression of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was examined in two types of mammalian cells, human HeLa14 and hamster BHK cells. DNA microarray analysis followed by reverse transcription-PCR identified at least 12 viral genes transcribed in both HeLa14 cells and BHK cells inoculated with AcMNPV. 5′ rapid amplification of cDNA ends was carried out to examine the transcriptional fidelity of these genes in HeLa14 cells. The transcription of ie-1, ie-0 and gp64 was initiated at a baculovirus early gene motif, CAGT, accompanied by a TATA motif. In addition, the same splicing observed for ie-0 mRNA in Sf9 cells occurred in HeLa14 cells. While the transcription initiation sites for pe38 and p6.9 were not located in the CAGT motif, most of them were in a typical eukaryotic RNA polymerase II promoter structure (a conventional TATA motif and/or an initiator). Interestingly, the expression of β-actin was upregulated in the mammalian cells inoculated with AcMNPV. Subsequent experiments using UV-inactivated virus confirmed the upregulation, suggesting that de novo synthesis of viral products is not required for the event. These results indicated that the AcMNPV genome acts as a template for transcription in mammalian cells through the usual infection pathway, though there is no evidence for the functional expression of viral genes at present.

scholarly journals Reduced Expression of the Immediate-Early Protein IE0 Enables Efficient Replication of Autographa californica Multiple Nucleopolyhedrovirus in Poorly Permissive Spodoptera littoralis Cells

2003 ◽  
Vol 77 (1) ◽  
pp. 535-545 ◽  
Author(s):  
Liqun Lu ◽  
Quansheng Du ◽  
Nor Chejanovsky
Keyword(s):  
Steady State ◽  
Spodoptera Littoralis ◽  
Autographa Californica ◽  
Early Gene ◽  
Immediate Early ◽  
Cosmid Library ◽  
Functional Protein ◽  
Early Protein ◽  
Multiple Nucleopolyhedrovirus ◽  
Steady State Levels

ABSTRACT Infection of Spodoptera littoralis SL2 cells with the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) results in apoptosis and low yields of viral progeny, in contrast to infection with S. littoralis nucleopolyhedrovirus (SlNPV). By cotransfecting SL2 cells with AcMNPV genomic DNA and a cosmid library representing the complete SlNPV genome, we were able to rescue AcMNPV replication and to isolate recombinant virus vAcSL2, which replicated efficiently in SL2 cells. Moreover, vAcSL2 showed enhanced infectivity for S. littoralis larvae compared to AcMNPV. The genome of vAcSL2 carried a 519-bp insert fragment that increased the distance between the TATA element and the transcriptional initiation site (CAGT) of immediate-early gene ie0. This finding correlated with low steady-state levels of IE0 and higher steady-state levels of IE1 (the product of the ie1 gene, a major AcMNPV transactivator, and a multifunctional protein) than of IE0. Mutagenesis of the ie0 promoter locus by insertion of the chloramphenical acetyltransferase (cat) gene yielded a new recombinant AcMNPV with replication properties identical to those of vAcSL2. Thus, the analysis indicated that increasing the steady-state levels of IE1 relative to IE0 should enable AcMNPV replication in SL2 cells. This suggestion was confirmed by constructing a recombinant AcMNPV bearing an extra copy of the ie1 gene under the control of the Drosophila hsp70 promoter. These results suggest that IE0 plays a role in the regulation of AcMNPV infection and show, for the first time, that significant improvement in the ability of AcMNPV to replicate in a poorly permissive cell line and organism can be achieved by increasing the expression of the main multiple functional protein, IE1.

scholarly journals Autographa californica Multiple Nucleopolyhedrovirus ORF11 Is Essential for Budded-Virus Production and Occlusion-Derived-Virus Envelopment

2014 ◽  
Vol 89 (1) ◽  
pp. 373-383 ◽  
Author(s):  
Xue Ying Tao ◽  
Jae Young Choi ◽  
Woo Jin Kim ◽  
Saes Byeol An ◽  
Qin Liu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Life Cycle ◽  
Gene Knockout ◽  
Autographa Californica ◽  
Early Gene ◽  
Expression Vectors ◽  
Knockout Mutant ◽  
Transfected Cells ◽  
Multiple Nucleopolyhedrovirus ◽  
Baculovirus Infection

ABSTRACTORF11 (ac11) ofAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role ofac11in the baculovirus life cycle, anac11knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5′ rapid amplification of cDNA ends (RACE) analyses revealed thatac11is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in Ac11KO-transfected Sf9 cells. However, quantitative PCR (qPCR) analysis demonstrated that the deletion ofac11did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of Ac11KO-transfected cells, which demonstrates that the defect in BV production in Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form occlusion-derived viruses (ODVs) and that their subsequent embedding into occlusion bodies (OBs) was also blocked in Ac11KO-transfected cells, demonstrating thatac11is required for ODV envelopment. These results therefore demonstrate thatac11is an early gene that is essential for BV production and ODV envelopment.IMPORTANCEBaculoviruses have been extensively used not only as specific, environmentally benign insecticides but also as helper-independent protein expression vectors. Although the function of baculovirus genes in viral replication has been studied by using gene knockout technology, the functions of more than one-third of viral genes, which include some highly conserved genes, are still unknown. In this study,ac11was proven to play a crucial role in BV production and ODV envelopment. These results will lead to a better understanding of baculovirus infection cycles.

scholarly journals The Rate of c-fos Transcription in Vivo Is Continuously Regulated at the Level of Elongation by Dynamic Stimulus-coupled Recruitment of Positive Transcription Elongation Factor b

2006 ◽  
Vol 282 (7) ◽  
pp. 5075-5084 ◽  
Author(s):  
Stephan Ryser ◽  
Toshitsugu Fujita ◽  
Silvia Tortola ◽  
Isabelle Piuz ◽  
Werner Schlegel
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Thyrotropin Releasing Hormone ◽  
Early Gene ◽  
Regulation Of Transcription ◽  
Dependent Manner ◽  
Tight Coupling ◽  
Releasing Hormone

In mammalian cells, multiple stimuli induce the expression of the immediate early gene c-fos. The specificity of c-fos transcriptional response depends on the activation of signaling protein kinases, transcription factors, and chromatin-modifying complexes but also on a regulated block to elongation in the first intron. Here we show by chromatin immunoprecipitation that finely tuned control of c-fos gene expression by distinct stimuli is associated with a dynamic regulation of transcription elongation and differential phosphorylation of the C-terminal domain of RNA polymerase II. Comparison of two stimuli of c-fos expression in the pituitary cell line GH4C1, namely the thyrotropin-releasing hormone versus depolarizing KCl, shows that both stimuli increase initiation, but only thyrotropin-releasing hormone is efficient to stimulate elongation and thus produce high transcription rates. To control elongation, the elongation factor P-TEFb is recruited to the 5′-end of the gene in a stimuli and time-dependent manner. Transition from initiation to elongation depends also on the dynamic recruitment of the initiation factors TFIIB and TFIIE but not TFIID, which remains constitutively bound on the promoter. It thus appears that tight coupling of signaling input to transcriptional output rate is achieved by c-fos gene-specific mechanisms, which control post-initiation steps rather than pre-initiation complex assembly.

scholarly journals Continuous transcription initiation guarantees robust repair of transcribed genes and regulatory regions in response to DNA damage

2019 ◽  
Author(s):  
Anastasios Liakos ◽  
Dimitris Konstantopoulos ◽  
Matthieu D. Lavigne ◽  
Maria Fousteri
Keyword(s):  
Dna Damage ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
De Novo ◽  
Transcriptional Response ◽  
Genotoxic Stress ◽  
Chromatin Accessibility ◽  
Initiation Rate ◽  
Pol Ii ◽  
Active Genes

ABSTRACTInhibition of RNA synthesis caused by DNA damage-impaired RNA polymerase II (Pol II) elongation is found to conceal a local increase in de novo transcription, slowly progressing from Transcription Start Sites (TSSs) to gene ends. Although associated with accelerated repair of Pol II-encountered lesions and limited mutagenesis, it is still unclear how this mechanism is maintained during recovery from genotoxic stress. Here we uncover a surprising widespread gain in chromatin accessibility and preservation of the active histone mark H3K27ac after UV-irradiation. We show that the concomitant increase in Pol II release from promoter-proximal pause (PPP) sites of most active genes, PROMoter uPstream Transcripts (PROMPTs) and enhancer RNAs (eRNAs) favors unrestrained initiation, as demonstrated by the synthesis of short nascent RNAs, including TSS-associated RNAs (start-RNAs). In accordance, drug-inhibition of the transition into elongation replenished the post-UV reduced levels of pre-initiating pol II at TSSs. Continuous engagement of new Pol II thus ensures maximal transcription-driven DNA repair of active genes and non-coding regulatory loci. Together, our results reveal an unanticipated layer regulating the UV-triggered transcriptional-response and provide physiologically relevant traction to the emerging concept that transcription initiation rate is determined by pol II pause-release dynamics.

scholarly journals Distinct properties of c-myc transcriptional elongation are revealed in Xenopus oocytes and mammalian cells and by template titration, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), and promoter mutagenesis.

1993 ◽  
Vol 13 (9) ◽  
pp. 5647-5658 ◽  
Author(s):  
T Meulia ◽  
A Krumm ◽  
M Groudine
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Premature Termination ◽  
Proximal Region ◽  
Transcriptional Elongation ◽  
Transcription Complexes ◽  
Rna Polymerase Ii Transcription

A block to c-myc transcription elongation has been observed in Xenopus oocytes and mammalian cells. Here, we show that the distribution of RNA polymerase II transcription complexes in the c-myc promoter proximal region in Xenopus oocytes is different from that observed previously in mammalian cells. Thus, there are major differences in the c-myc elongation block observed in the two systems. In addition, as first reported for a Xenopus tubulin gene (K. M. Middleton and G. T. Morgan, Mol. Cell. Biol. 10:727-735, 1990). c-myc template titration experiments reveal the existence of two classes of RNA polymerase II transcription complexes in oocytes: one (at low template concentration) that is capable of reading through downstream sites of premature termination, and another (high template concentration) that does not. We show that these classes of polymerases are distinct from those previously identified by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), which distinguishes transcription complexes on the basis of transcribed distance, rather than on the basis of differential elongation through sites of premature termination. We also show that mutations that affect the efficiency of initiation of transcription from the c-myc P2 promoter can influence premature termination by at least two mechanisms: TATA box mutations function by the titration effect (decrease in transcription initiation results in a relative decrease in premature termination), while an upstream activator (E2F) site functions by contributing to the assembly of polymerase complexes competent to traverse the downstream sites of premature termination.

scholarly journals Nuclear myosin VI regulates the spatial organization of mammalian transcription initiation

2020 ◽  
Author(s):  
Yukti Hari-Gupta ◽  
Natalia Fili ◽  
Ália dos Santos ◽  
Alexander W. Cook ◽  
Rosemarie E. Gough ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Mammalian Cells ◽  
Spatial Organization ◽  
Regulation Of Gene Expression ◽  
Myosin Vi ◽  
Transcription Factories ◽  
Cellular Environment

SUMMARYDuring transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability which underpin the function these transcription factories remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI in the nanoscale organisation of RNAPII. We reveal that myosin VI acts as the molecular anchor that holds RNAPII into transcription factories. Perturbation of myosin VI leads to the disruption of RNAPII localisation, changes in chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of myosin VI in the spatial regulation of gene expression during the rapid response to changes in the cellular environment.

scholarly journals Methylation of RNA polymerase II non-consensus Lysine residues marks early transcription in mammalian cells

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
João D Dias ◽  
Tiago Rito ◽  
Elena Torlai Triglia ◽  
Alexander Kukalev ◽  
Carmelo Ferrai ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Mammalian Cells ◽  
Consensus Sequence ◽  
Gene Promoters ◽  
Post Translational Modification ◽  
Lysine Residues ◽  
Early Transcription ◽  
Gene Expression Levels

Dynamic post-translational modification of RNA polymerase II (RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and processing of nascent RNA. Extensive phosphorylation of serine residues at the largest RNAPII subunit occurs at its structurally-disordered C-terminal domain (CTD), which is composed of multiple heptapeptide repeats with consensus sequence Y1-S2-P3-T4-S5-P6-S7. Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated (K7ac). Here, we describe mono- and di-methylation of CTD Lysine-7 residues (K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation. In contrast, K7ac is associated with RNAPII elongation, Serine-2 phosphorylation and mRNA expression. We identify an unexpected balance between RNAPII K7 methylation and acetylation at gene promoters, which fine-tunes gene expression levels.

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