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)

Both pulsed and continuous applications of the RNA polymerase II inhibitor thiolutin cause a dramatic but reversible loss of bioluminescence and its overt rhythmicity in cells of the dinoflagellate Lingulodinium polyedrum (formerly Gonyaulax polyedra). Such cells remain alive, and the rhythm resumes after an interval, the length of which depends on the concentration of thiolutin used. The period and phase of the resumed rhythm were not systematically altered following such treatments, and the effects were not different at different circadian phases. For three different genes, luciferin binding protein (lbp), luciferase (lcf), and glyceraldehyde-3-phosphate dehydrogenase (gapdh), which are circadian-regulated at the level of translation, the amounts of their mRNAs were determined by Northern blots for times up to 12.5 h following the addition of 1.5 microM thiolutin. Consistent with previous reports that their abundances do not change with circadian time, their levels remained high for several hours after thiolutin addition, but then did diminish.
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PMID:Lifetimes of mRNAs for clock-regulated proteins in a dinoflagellate. 1468 Jan 37

RNA interference (RNAi) is a sequence-specific RNA degradation process mediated by short double-stranded RNAs termed small interfering RNAs. Here, we describe the lentivirus-based vector small interfering RNA system expressing. As a pilot study, we generated constructs expressing small hairpin RNA (shRNA) specific for luciferase gene (shLuc) or green fluorescence protein (shGFP) under the control of human H1 RNA polymerase III promoter. The effect of the shRNA was evaluated against HIV-1 infection in a single-round or multiple-round infectious system using an HIV-1 molecular clone carrying the luc or GFP gene. In the single-round infectious system, cells transduced with shLuc by lentiviral vector significantly reduced (approximately 90% reduction) viral gene expression after challenge infection at a multiplicity of infection of 10. These transduced cells continued to resist against at least four sequentially repeated challenge infections. Importantly, this efficient antiviral activity persisted over 35 days in culture. In a multiple-round infectious system using a replication-competent HIV-1 molecular clone carrying the GFP gene, we also observed that a lentiviral vector expressing shGFP could inhibit HIV-1 replication for at least 1 week. The profound effect of lentiviral shRNA was also observed in human primary monocyte-derived macrophages. Thus, shRNA introduced through the lentiviral vector can be useful for efficient and stable gene suppression in human cells including primary non-dividing cells. Moreover, quantitative analysis of viral cDNA synthesis on challenge infection showed that viral genomic RNAs packaged in incoming virus core might not be targeted by shLuc. Instead, the degradation of transcripts from integrated proviral DNAs might be a major cause of the profound reduction in HIV-1 gene expression by shRNA in our system.
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PMID:Expression of small hairpin RNA by lentivirus-based vector confers efficient and stable gene-suppression of HIV-1 on human cells including primary non-dividing cells. 1473 96

Mx proteins belong to the dynamin superfamily of high molecular weight GTPases and interfere with multiplication of a wide variety of viruses. Earlier studies show that nuclear mouse Mx1 and human MxA designed to be localized in the nucleus inhibit the transcription step of the influenza virus genome. Here we set a transient influenza virus transcription system using luciferase as a reporter gene and cells expressing the three RNA polymerase subunits, PB1, PB2 and PA, and NP. We used this reporter assay system and nuclear-localized MxA proteins to get clues for elucidating the anti-influenza virus activity of MxA. Nuclear-localized VP16-MxA and MxA-TAg NLS strongly interfered with the influenza virus transcription. Over-expression of PB2 led to a slight resumption of the transcription inhibition by nuclear MxA, whereas over-expression of PB1 and PA did not affect the MxA activity. Of interest is that the inhibitory activity of the nuclear MxA was markedly neutralized by over-expression of NP. An NP devoid of its C-terminal region, but containing the N-terminal RNA binding domain, also neutralized the VP16-MxA activity in a dose-dependent manner, whereas an NP lacking the N-terminal region did not affect the VP16-MxA activity. Further, not only VP16-MxA but also the wild-type MxA was found to interact with NP in influenza virus-infected cells. This indicates that the nuclear MxA suppresses the influenza virus transcription by interacting with not only PB2 but also NP.
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PMID:Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome. 1475 52

In many developmentally and functionally important higher plant plastid genes, expression depends on a specific nuclear-encoded RNA polymerase (NEP). Molecular mechanisms for NEP-mediated gene expression are poorly understood. We have improved a transient expression assay based on biolistics and the dual-luciferase reporter technique, which facilitated investigations into the regulation of plastid genes in vivo. We scrutinized the 5'-flanking region and the 5'-untranslated region (5'UTR) of accD, a plastid gene encoding a subunit of the prokaryotic-type acetyl-CoA carboxylase which is transcribed exclusively by NEP. The results indicated that two AT-rich sequences, one of them containing two overlapping YRTA-like motifs, were essential for accD expression in vivo. The results also revealed that the length of the 5'UTR rather than a particular sequence element was a determinant for the level of accD expression. Because transcripts accumulated in proportion to reporter enzyme activity and protein levels, and transcript degradation rates were independent of the nature of the 5'UTR, it was unlikely that the 5'UTR acts as a translational enhancer or a stabilizer of the transcripts. Therefore, the length of 5'UTR might be a factor contributing to the efficiency of NEP-dependent transcription in plastids.
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PMID:Possible involvement of the 5'-flanking region and the 5'UTR of plastid accD gene in NEP-dependent transcription. 1498 88

There are several unorthodox features, which distinguish the non-redundant and unique novel matrix metalloproteinase-26 (MMP-26) (an enzyme that has recently evolved and does not exist in rodents but is present in humans) from other members of the MMP superfamily. This report describes our recent efforts to gain a better understanding of the mechanisms which restrict expression of MMP-26 to certain cell/tissue types. We examined transcriptional regulation of the human MMP-26 gene in normal and malignant cells. The AP-1 and Tcf-4 sites of the MMP-26 promoter appear most potent in regulating the expression of the MMP-26-luciferase chimera in HEK293 embryonic kidney and MCF7 breast carcinoma cells. Key regulators of the Wnt pathway (beta-catenin and lymphoid enhancer-binding factor/T-cell factor with which beta-catenin associates) enhanced the transcriptional activity of MMP-26 suggesting that the MMP-26 gene is a likely target of the Wnt pathway. Immunostaining, gene arrays and reverse-transcriptase polymerase chain reaction (RT-PCR) confirm the presence of MMP-26 in normal cells, including the apical epithelial conjunctiva cells of the human eye, as well as in malignant cells of epithelial origin. MMP-26 predominantly accumulates in its proenzyme form in the intracellular milieu of the transfected breast carcinoma MCF7 cells. This study brings us a step forward towards a better understanding of the unconventional role, regulation and functions of epithelial cell MMP-26 in physiological conditions and in neoplasms.
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PMID:Beta-catenin regulates the gene of MMP-26, a novel metalloproteinase expressed both in carcinomas and normal epithelial cells. 1500 46

Silencing of mammalian gene expression by RNA interference (RNAi) technology can be achieved using small interfering RNA (siRNA) or short hairpin RNA (shRNA). However, the relative effectiveness of these two approaches is not known. It is also not clear whether gene-specific shRNA transcribed from an RNA polymerase II (Pol II)-directed promoter in a fusion form can disrupt the targeted gene expression. Here, we report that using both luciferase and antiapoptotic survivin genes as targets, both siRNA and shRNA approaches significantly silenced the targeted gene expression in cancer cells. We further demonstrated that shRNAs transcribed from an RNA Pol II-mediated promoter in a green fluorescent protein (GFP) fusion form at the 3'-untranslated region silenced luciferase and survivin expression as well, suggesting that the extra RNA sequence outside of the shRNA hairpin does not disrupt shRNA function. We also showed that silencing of survivin expression selectively induces apoptosis in transfected cells. Together, we have validated multiple approaches of RNAi technology using both survivin and luciferase genes as targets and demonstrated for the first time that GFP-shRNAs transcribed from an RNA Pol II-mediated promoter could mediate gene silencing, which may lead to new directions for the application of RNAi technology.
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PMID:Silencing of antiapoptotic survivin gene by multiple approaches of RNA interference technology. 1503 60

To efficiently perform collection and functional studies of large-scale cDNAs, we constructed multi-functional cDNA vectors for efficient full-length cDNA cloning, direct sequencing, easy screening, and the expression of cDNA in vitro and in vivo without subcloning the cDNA into other vectors. The constructed vectors, pCNS and pCNS-D2, contain a multi-cloning site for uni-directional full-length cDNA cloning, T7 and Sp6 RNA polymerase promoters for in vitro transcription and translation, and hCMV immediate early promoter and BGH poly(A) to allow expression in mammalian cells. Using these vectors, we constructed full-length enriched cDNA libraries containing 60-75% of the full-length cDNAs using two different oligo-capping methods. The subtracted cDNA libraries could also be constructed by removing of EF1-alpha cDNA, a highly expressed cDNA. In addition, we confirmed the translation of EF1-alpha cDNA in vitro and the expression of luciferase cDNA in mammalian cells. The expression efficiency of luciferase cDNA in different cell lines, such as HeLa, Hep3B, SNU638, and SNU668, showed that pCNS vectors can highly express target genes in different cell types. These results indicated that our multi-purpose vectors, pCNS and pCNS-D2, are useful tools for the construction of full-length cDNA libraries and high-throughput based functional study of cDNAs.
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PMID:Construction of multi-purpose vectors, pCNS and pCNS-D2, are suitable for collection and functional study of large-scale cDNAs. 1510 28

Artificial minigenomes are powerful tools for studying the replication and transcription of negative-strand RNA viruses. Bunyamwera virus (BUN; genus Orthobunyavirus, family Bunyaviridae) is an arbovirus that shows fundamental biological differences when replicating in mammalian versus mosquito cells. To study BUN RNA synthesis in mosquito cells, we developed a bacteriophage T7 RNA polymerase-based minireplicon system similar to that described previously for mammalian cells. An Aedes albopictus C6/36-derived mosquito cell line stably expressing T7 RNA polymerase was established. Viral proteins and artificial minigenomes (containing Renilla luciferase as a reporter) were transcribed and expressed in these cells from transfected T7 promoter-containing plasmids. Transcription of the minigenome required two viral proteins, the nucleocapsid protein N and the RNA-dependent RNA polymerase L, a situation similar to that in mammalian cells. However, unlike the situation in mammalian cells, the viral polymerase was not inhibited by the viral nonstructural protein NSs. We also report that promoter strength is different for vertebrate versus invertebrate cells. The development of this system opens the way for a detailed comparison of bunyavirus replication in cells of disparate phylogeny.
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PMID:A bunyamwera virus minireplicon system in mosquito cells. 1514 Sep 65

Recombinant infectious coxsackievirus B3 (CVB3) particles were generated by packaging of modified viral genomes in which the capsid coding P1-region was replaced by an EGFP-luciferase reporter gene. Efficient packaging of the recombinant genome was achieved by a novel method based on cotransfection of a plasmid encoding the subgenomic viral replicon together with two alternative helper plasmids carrying expression cassettes of the CVB3 capsid proteins, and a T7 RNA polymerase expression plasmid. Transcription of a reporter gene and expression of capsid proteins were achieved in a single step, eliminating the need of a helper virus. Recombinant viral stocks were used to infect human embryonal cardiomyocytes (hCMC) and other cell types, and luciferase activity was measured at different timepoints after infection. Neither progeny virus nor wildtype CVB3 was produced upon infection of target cells, facilitating analyses of infected cells without viral spread. The presence of an IRES sequence upstream of the P1 open reading frame in the helper plasmids was indispensable for the generation of recombinant particles, as no packaging was observed using helper plasmids without this feature. Luciferase data obtained by transfection of reporter plasmids with and without upstream 5'-NTR sequences suggests that the CVB3 IRES facilitates translation in T7 RNA polymerase-dependent gene transcription, both in presence and absence of viral replication.
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PMID:Plasmid-based generation of recombinant coxsackievirus B3 particles carrying capsid gene replacement replicons. 1517 88

7,8-Dihydro-8-oxoguanine (8-oxoG) is the most frequent mutagenic lesion caused by oxidative stress. Eukaryotic cells use a specific DNA glycosylase, OGG1, to excise 8-oxoG from DNA. The mild phenotype of OGG1 null mice has been attributed to the existence of alternative pathways, including Cockayne syndrome B (CSB)-dependent transcription coupled repair (TCR), for removal of 8-oxoG. We have studied repair and transcription activities at 8-oxoG lesions with a reconstituted transcription system (RTS; RNA polymerase II, TBP, TFIIA, TFIIB, TFIIE, TFIIF and TFIIH), as well as in cellular extracts and in vivo. All measurable repair activity at 8-oxoG lesions takes place in the 3'-direction from the lesion, indicating base excision repair (BER) activity and negligible role of nucleotide excision repair (NER). Although 8-oxoG has been shown to be preferentially removed from the transcribed strand, in vitro experiments with purified transcription factors failed to identify a definite block for RNA polymerase II at the lesion. However, a weak block was observed at the lesion during transcription carried out with RTS as well as with cellular extracts. RNA polymerase II was identified at the site of the lesion on obstructed templates. Wild-type cells, as well as cells carrying targeted mutations of genes required for removal of 8-oxoG, were transfected with a luciferase expression vector containing an 8-oxoG lesion. No significant obstruction at 8-oxoG lesions was observed by this in vivo approach. In control experiments transcription elongation was completely blocked by cisplatin.
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PMID:Transcription activities at 8-oxoG lesions in DNA. 1538 Jan 1


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