Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Non-viral vectors are promising vehicles for gene therapy but delivery of plasmid DNA to post-mitotic cells is challenging as nuclear entry is particularly inefficient. We have developed and evaluated a hybrid mRNA/DNA system designed to bypass the nuclear barrier to transfection and facilitate cytoplasmic gene expression. This system, based on co-delivery of mRNA(A64) encoding for T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid, produced between 10- and 2200-fold higher gene expression in primary dorsal root ganglion neuronal (DRGN) cultures isolated from Sprague-Dawley rats compared to a cytomegalovirus (CMV)-driven plasmid, and 30-fold greater expression than the enhanced T7-based autogene plasmid pR011. Cell-free assays and in vitro transfections highlighted the versatility of this system with small quantities of T7 RNAP mRNA required to mediate expression at levels that were significantly greater than with the T7-driven plasmid alone or supplemented with T7 RNAP protein. We have also characterized a number of parameters, such as mRNA structure, intracellular stability and persistence of each nucleic acid component that represent important factors in determining the transfection efficiency of this hybrid expression system. The results from this study demonstrate that co-delivery of mRNA is a promising strategy to yield increased expression with plasmid DNA, and represents an important step towards improving the capability of non-viral vectors to mediate efficient gene transfer in cell types, such as in DRGN, where the nuclear membrane is a significant barrier to transfection.
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PMID:Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons. 1683 7

The human cytomegalovirus major immediate-early protein IE86 is pivotal for coordinated regulation of viral gene expression throughout infection. A relatively promiscuous transactivator of viral early and late gene transcription, IE86 also acts during infection to negatively regulate its own promoter via direct binding to a 14-bp palindromic IE86-binding site, the cis repression sequence (crs), located between the major immediate-early promoter (MIEP) TATA box and the start of transcription. Although such autoregulation does not involve changes in the binding of basal transcription factors to the MIEP in vitro, it does appear to involve selective inhibition of RNA polymerase II recruitment. However, how this occurs is unclear. We show that autorepression by IE86 at late times of infection correlates with changes in chromatin structure around the MIEP during the course of infection and that this is likely to result from physical and functional interactions between IE86 and chromatin remodeling enzymes normally associated with transcriptional repression of cellular promoters. Firstly, we show that IE86-mediated autorepression is inhibited by histone deacetylase inhibitors. We also show that IE86 interacts, in vitro and in vivo, with the histone deacetylase HDAC1 and histone methyltransferases G9a and Suvar(3-9)H1 and that coexpression of these chromatin remodeling enzymes with IE86 increases autorepression of the MIEP. Finally, we show that mutation of the crs in the context of the virus abrogates the transcriptionally repressive chromatin phenotype normally found around the MIEP at late times of infection, suggesting that negative autoregulation by IE86 results, at least in part, from IE86-mediated changes in chromatin structure of the viral MIEP.
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PMID:Autorepression of the human cytomegalovirus major immediate-early promoter/enhancer at late times of infection is mediated by the recruitment of chromatin remodeling enzymes by IE86. 1700 78

Transcription is a critical component in the expression of genes. Here we describe the design and analysis of a potent core promoter, termed super core promoter 1 (SCP1), which directs high amounts of transcription by RNA polymerase II in metazoans. SCP1 contains four core promoter motifs-the TATA box, initiator (Inr), motif ten element (MTE) and downstream promoter element (DPE)-in a single promoter, and is distinctly stronger than the cytomegalovirus (CMV) IE1 and adenovirus major late (AdML) core promoters both in vitro and in vivo. Each of the four core promoter motifs is needed for full SCP1 activity. SCP1 is bound efficiently by TFIID and exhibits a high propensity to form productive transcription complexes. SCP1 and related super core promoters (SCPs) with multiple core promoter motifs will be useful for the biophysical analysis of TFIID binding to DNA, the biochemical investigation of the transcription process and the enhancement of gene expression in cells.
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PMID:Rational design of a super core promoter that enhances gene expression. 1712 35

Human cytomegalovirus (HCMV) late mRNA expression in megakaryoblast and in turn the pathogenesis of idiopathic thrombocytopenic purpura (ITP) patients with HCMV infection, and effectiveness of ganciclovir were investigated. Colony forming unit-megakaryocytes (CFU-MK) of 46 ITP patients with HCMV infection were incubated from patients' bone marrow mononuclear cells (MNC). Reverse transcriptase-polymerase chain reaction (RT-PCR) was subsequently used for CFU-MK for HCMV-late mRNA detection. Ganciclovir therapy was given to both HCMV-late mRNA positive and negative groups for comparison of therapeutic effectiveness. The results in 19 of 46 CFU-MK culture cells specimens with positive HCMV-DNA by PCR or positive CMV-IgM by enzyme linked immunosorbent assay (ELISA) in the correspondent serum of peripheral blood were positive for HCMV-late mRNA. Sixteen out of 19, patients with positive HCMV-late mRNA CFU-MK had a positive response to ganciclovir. Amongst 27 patients with negative HCMV-late mRNA CFU-MK, only 4 positive responders to ganciclovir therapy were observed. Curative effectiveness of ganciclovir in HCMV-late mRNA positive group was significantly higher than that in HCMV-late mRNA negative group (P<0.01). It was suggested that HCMV could directly infect CFU-MK, which might be one of the mechanisms responsible for HCMV related ITP. The ganciclovir is an effective therapy in resulting in the increases in thrombocyte in the ITP patients whose HCMV- late mRNA was positive in their CFU-MK.
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PMID:Direct infection of colony forming unit-megakaryocyte by human cytomegalovirus contributes the pathogenesis of idiopathic thrombocytopenic purpura. 1721 66

Reverse genetics system is an excellent platform to research the construction and function of viruses. Genome modification, such as gene recombination, mosaicism, and mutation may interfere with replication, assembly and release of viruses. An efficient, convenient and economical method of virus rescue is undoubtedly required for elevating the efficiency of rescuing crippled virus. In this study, we developed a method to rescue infectious bursal disease virus (IBDV) using RNA polymerase II. The genome of IBDV Gt strain, flanked by hammerhead ribozyme and hepatitis delta ribozyme sequences, were cloned downstream of the cytomegalovirus enhancer and the beta chicken actin promoter of the vector pCAGGS. Through direct transfection in various cell lines, IBDV could be rescued efficiently. The RNA polymerase II-based reverse genetics system is efficient, stable, convenient, and fit to various cells. The system not only provides the basis of the gene function research of IBDV, but is also useful for reverse genetics research of other birnaviridae viruses.
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PMID:An improved method for infectious bursal disease virus rescue using RNA polymerase II system. 1738 19

Dendritic cells (DCs) produce type I interferons (IFNs) in greater amounts than other cells, but the mechanisms remain elusive. Here we studied the role of a transcription factor, IRF8, in DC induction of type I IFNs. Upon newcastle disease virus (NDV) infection, bone marrow-derived plasmacytoid and conventional DCs induced IFN transcripts, exhibiting two-phase kinetics. The second, amplifying phase represented an IFN feedback response that accounted for much of IFN protein production. Induction of second phase transcription required IRF8. Mouse cytomegalovirus (MCMV) and Toll-like receptor-mediated IFN induction in DCs also required IRF8. Chromatin immunoprecipitation analysis showed that IRF7, IRF8, and RNA polymerase II were recruited to the IFN promoters upon stimulation. Moreover, sustained RNA polymerase II recruitment to the promoters critically depended on IRF8. Together, these data indicate that IRF8 magnifies the second phase of IFN transcription in DCs by prolonging binding of basic transcription machinery to the IFN promoters, thereby playing a role in innate immunity.
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PMID:The feedback phase of type I interferon induction in dendritic cells requires interferon regulatory factor 8. 1770 15

A hallmark of vertebrate genes is that actively transcribed genes are hypomethylated in critical regulatory sequences. However, the mechanisms that link gene transcription and DNA hypomethylation are unclear. Using a trichostatin A (TSA)-induced replication-independent demethylation assay with HEK 293 cells, we show that RNA transcription is required for DNA demethylation. Histone acetylation precedes but is not sufficient to trigger DNA demethylation. Following histone acetylation, RNA polymerase II (RNAP II) interacts with the methylated promoter. Inhibition of RNAP II transcription with actinomycin D, alpha-amanitin, or CDK7-specific small interfering RNA inhibits DNA demethylation. H3 trimethyl lysine 4 methylation, a marker of actively transcribed genes, was associated with the cytomegalovirus promoter only after demethylation. TSA-induced demethylation of the endogenous cancer testis gene GAGE follows a similar sequence of events and is dependent on RNA transcription as well. These data suggest that DNA demethylation follows rather than precedes early transcription and point towards a novel function for DNA demethylation as a memory of actively transcribed genes.
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PMID:Acetylation-induced transcription is required for active DNA demethylation in methylation-silenced genes. 1770 85

One of the characteristic features of herpesviruses is that most of their genes are intronless. Thus, their replication relies on the selective nuclear export of intronless viral mRNAs, which have to compete with the nuclear export of bulk spliced cellular mRNAs. Therefore, the regulation of nuclear mRNA export is crucial for the replication and pathogenesis of herpesviruses. Besides the thymidine kinase transcript of herpes simplex virus type 1, which contains specific sequences to facilitate the nuclear export of intronless mRNA, other cis-acting RNA elements for nuclear mRNA export have not yet been identified in the rest of herpesviral intronless mRNAs. Instead, emerging studies show that herpesviruses encode viral mRNA export factors, which interact with components of the major cellular mRNA export pathway, the RNA polymerase II transcription complex and specific splicing factors to selectively export intronless herpesviral mRNAs to the cytoplasm in infected cells. These herpesviral mRNA export factors are members of a conserved gene family that can be found in all herpesviruses. The human cytomegalovirus transactivator protein UL69, which has been demonstrated to belong to this conserved protein family, shares common features with its herpesviral homologues but also possesses unique properties that will be discussed in this review.
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PMID:The human cytomegalovirus regulatory protein UL69 and its effect on mRNA export. 1798 67

We describe a simplified RNA polymerase II-based reverse genetics approach that allows for the efficient rescue of high-titer infectious bursal disease virus (IBDV) from cloned cDNAs of genomic segments A and B. Unlike the previously reported RNA polymerase II-based methods, the developed strategy does not necessitate the introduction of a ribozyme sequence at both ends of the genomic cDNA sequences. This was achieved by fusing the 5' terminal sequence of the cDNA of each segment to the transcription start site of the immediate early cytomegalovirus promoter, while a ribozyme sequence was only introduced at the 3' end. Using this strategy, and without complementing with IBDV structural proteins, titers as high as 10(11) tissue culture infectious dose 50 were reproducibly obtained in chicken embryo fibroblast cells immediately upon co-transfection with cDNAs of both segments. We anticipate that this modification could improve reverse genetics for any other RNA virus and may be beneficial for vaccine development and dissection of the viral life cycle.
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PMID:Efficient rescue of infectious bursal disease virus using a simplified RNA polymerase II-based reverse genetics strategy. 1839 69

The use of RNA interference (RNAi) to inhibit gene expression is potentially applicable in the treatment of viral infections such as hepatitis B virus (HBV) persistence. Although efficient HBV gene silencing by short hairpin RNA (shRNA) expressed from RNA polymerase (Pol) III promoters has been reported, constitutive high-level transcription may cause harmful side effects. Here, we report an approach that allows the use of a Pol II promoter to improve transcription regulation of expressed RNAi effecters. Pol II [cytomegalovirus (CMV)] or Pol III (U6) promoter cassettes that transcribe anti-HBV primary microRNA (pri-miR)-122 and pri-miR-31 shuttles were generated. In cultured cells both types of pri-miR-like sequences effected knockdown of markers of viral replication (>80%) and were processed to form intended 21-nucleotide guides. The concentration of CMV-expressed miRs was approximately 85-fold lower than the U6 shRNA-derived guide RNA. When cells were co-transfected with pri-miR expression cassettes, attenuation of independent RNAi-mediated gene silencing was not observed, which is in contrast to the action of U6 shRNA expression cassettes. The efficacy of the anti-HBV pri-miR shuttles in vivo was verified using the murine hydrodynamic injection model. Employing Pol II-expressed pri-miR mimics may be useful in the treatment of HBV infection, and potentially also for generic application in RNAi-based therapy.
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PMID:Expressed anti-HBV primary microRNA shuttles inhibit viral replication efficiently in vitro and in vivo. 1843 60


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