Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A hybrid recombinant baculovirus-bacteriophage T7 expression system was developed for transient expression in insect cells of plasmids with foreign genes provided with a T7 promoter. The coding sequence for T7 RNA polymerase, with or without a nuclear localization signal, was inserted into the genome of Autographa californica nuclear polyhedrosis virus. Recombinant viruses stably expressed T7 RNA polymerase in insect cells. Upon transfection of infected insect cells with plasmids containing the genes for chloramphenicol acetyltransferase (CAT), the hepatitis B virus precore-, core- or e- antigens under control of the T7 promoter, transient expression of these genes was detected by ELISA. The results obtained indicate that this baculovirus/T7 system provides a simple and widely applicable tool for transient gene expression studies.
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PMID:A hybrid baculovirus-bacteriophage T7 transient expression system. 963 68

The hepatitis B virus (HBV) X protein is essential for viral infectivity, and evidence indicates that it is a strong contributor to HBV-mediated oncogenesis. X has been shown to transactivate a wide variety of RNA polymerase (Pol) II-dependent, as well as RNA Pol III-dependent, promoters. In this study, we have investigated the possibility that X modulates RNA Pol I-dependent rRNA transcription. In both human hepatoma Huh7 and Drosophila Schneider S2 cell lines, X expression stimulated rRNA promoter activity. Extracts prepared from X-expressing cells stably transfected with an X gene also exhibited an increased ability to transcribe the rRNA promoter. The mechanism for X transactivation was examined by determining whether this regulatory event was dependent on Ras activation and increased TATA-binding protein (TBP) levels. Our previous studies have demonstrated that X, and the activation of Ras, produces an increase in the cellular levels of TBP (H.-D. Wang, A. Trivedi, and D. L. Johnson, Mol. Cell. Biol. 17:6838-6846, 1997). Expression of a dominant negative form of Ras blocked the X-mediated induction of the rRNA promoters, whereas expression of a constitutively activated form of Ras mimicked the enhancing effect of X on rRNA promoter activity. When TBP was overexpressed in either Huh7 or S2 cells, a dose-dependent increase in rRNA promoter activity was observed. To analyze whether the increase in TBP was modulating rRNA promoter activity indirectly, by increasing activity of RNA Pol II-dependent promoters, a Drosophila TBP cDNA was constructed with a mutation that eliminated its ability to stimulate RNA Pol II-dependent promoters. Transient expression of wild-type TBP in S2 cells increased the activities of specific RNA Pol I- and Pol II-dependent promoters. Expression of the mutant TBP protein failed to enhance the activity of the RNA Pol II-dependent promoters, yet the protein completely retained its ability to stimulate the rRNA promoter. Furthermore, the addition of recombinant TBP to S2 extracts stimulated rRNA promoter activity in vitro. Together, these results demonstrate that the HBV X protein up-regulates RNA Pol I-dependent promoters via a Ras-activated pathway in two distinct cell lines. The enhanced promoter activity can, at least in part, be attributed to the X- and Ras-mediated increase in cellular TBP, a limiting transcription component.
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PMID:Regulation of RNA polymerase I-dependent promoters by the hepatitis B virus X protein via activated Ras and TATA-binding protein. 981 95

To modulate transcription, regulatory factors communicate with basal transcription factors and/or RNA polymerases in a variety of ways. Previously, it has been reported that RNA polymerase II subunit 5 (RPB5) is one of the targets of hepatitis B virus X protein (HBx) and that both HBx and RPB5 specifically interact with general transcription factor IIB (TFIIB), implying that RPB5 is one of the communicating subunits of RNA polymerase II involved in transcriptional regulation. In this context, we screened for a host protein(s) that interacts with RPB5. By far-Western blot screening, we cloned a novel gene encoding a 508-amino-acid-residue RPB5-binding protein from a HepG2 cDNA library and designated it RPB5-mediating protein (RMP). Expression of RMP mRNA was detected ubiquitously in various tissues. Bacterially expressed recombinant RMP strongly bound RPB5 but neither HBx nor TATA-binding protein in vitro. Endogenous RMP was immunologically detected interacting with assembled RPB5 in RNA polymerase in mammalian cells. The central part of RMP is responsible for RPB5 binding, and the RMP-binding region covers both the TFIIB- and HBx-binding sites of RPB5. Overexpression of RMP, but not mutant RMP lacking the RPB5-binding region, inhibited HBx transactivation of reporters with different HBx-responsive cis elements in transiently transfected cells. The repression by RMP was counteracted by HBx in a dose-dependent manner. Furthermore, RMP has an inhibitory effect on transcriptional activation by VP16 in the absence of HBx. These results suggest that RMP negatively modulates RNA polymerase II function by binding to RPB5 and that HBx counteracts the negative role of RMP on transcription indirectly by interacting with RPB5.
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PMID:RMP, a novel RNA polymerase II subunit 5-interacting protein, counteracts transactivation by hepatitis B virus X protein. 981 40

The hepatitis B virus (HBV) is a coated, incompletely double-stranded DNA virus with some outstanding features. (1) All three coat proteins of HBV contain HBsAg, which is highly immunogenic inducing anti-HBs. These antibodies are protective for HBV outer cells (humoural immunity). Structural viral proteins induce specific T-lymphocytes, which are able to eliminate HBV-infected cells (cytotoxic T-cells; cellular immunity). (2) Intracellular HBV primarily causes little or no damage (non-cytopathogenic), which is an excellent strategy of viral survival. However, viral oligo-peptides of 8-15 amino acids are loaded on host cell MHC-class 1 molecules and are transported to the cell surface. Thus, HBV-specific T-lymphocytes are able to detect infected cells and destroy them, an ingenious defence strategy. However, this cell deletion triggered by inflammation cells may result in acute hepatitis. If HBV is not eliminated, a delicate balance between viral replication and immunodefence prevails which may lead to chronic hepatitis and liver cirrhosis. (3) In chronically infected cells HBV may become partly cytopathogenic--a process still poorly understood--and the viral DNA may integrate into the host cell DNA (through a viral transcriptase). If integration leads to activation of crucial host genes a hepatocellular carcinoma results. These outstanding features are responsible for the highly variable course of HBV infection and its final outcome, e.g. when the load of HBV-infected cells is still low at the time when an efficient immune defence starts, the infection is self-limited and asymptomatic, and immunity results. When there is no immune defence or a defective immune defence (immune tolerance of new-borns or immunosuppressed individuals) the HBV infection very often becomes chronic. In these cases, no acute hepatitis occurs, but hepatocellular carcinoma may result. Treatment with Interferon has become accepted, resulting in up to 30 to 40% of cases in the elimination of the virus. However, treatment is laborious and expensive, and the mechanism of action is still poorly understood (anti-viral and/or immune-modulating).
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PMID:Hepatitis B: virus, pathogenesis and treatment. 991 26

The poly-2'-O-(2,4-dinitrophenyl)-oligoribonucleotide (poly-DNP-RNA) with antisense sequence 5'ggguguauggaaaagccguc-3' was designed to target the sequence 2468-2487 in the polymerase gene of duck hepatitis B virus (DHBV). The stereochemically pure RNA was synthesized by using T7 RNA polymerase with synthetic DNA template and subsequently derivatized with 2,4-dinitrofluorobenzene in mild basic conditions to make the poly-DNP-RNA with an average DNP/base ratio of 0.7. In vitro studies showed that this antisense poly-DNP-RNA can hybridize with sense DNA and has high resistance to RNase A digestion. These poly-DNP-RNA were also found to be potent sequence-independent inhibitors of the reverse transcriptase activity of DHBV DNA-polymerase. For in vivo studies, DHBV-infected ducks were treated with antisense, sense, and random noncomplementary sequence poly-DNP-RNA, respectively. The data showed that the antisense poly-DNP-RNA completely inhibited the duck viremia in all nine ducks that had been treated with a dose of 1 mg/kg (i.v.) per day for 25 days. The viremia did not come back after 10 months recession. In the sense group, three of the four ducks showed no inhibition, and in the random group, both ducks maintained their viremia. After 45 days of treatment with the antisense poly-DNP-RNA, followed by 2 weeks of recession, PCR as well as QC-PCR assay and microscopic examination showed that viral DNA had disappeared in liver and that the histology of the damaged liver (filled with fat granules) had returned to normal.
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PMID:Treatment of duck hepatitis B virus by antisense poly-2'-O-(2,4-dinitrophenyl)-oligoribonucleotides. 991 10

Previous studies have demonstrated the feasibility of implantation of human blood cells or tissues in lethally irradiated mice or rats, radioprotected with SCID mouse bone marrow cells: The Trimera system. In the present study, we describe the development of a mouse Trimera model for human hepatitis B virus (HBV) infection. In this model, viremia is induced by transplantation of ex vivo HBV-infected human liver fragments. Engraftment of the human liver fragments, evaluated by hematoxylin-eosin staining and human serum albumin mRNA expression, was observed in 85% of the transplanted animals 1 month postimplantation. Viremia levels were determined in these mice by measuring serum HBV DNA using polymerase chain reaction (PCR), followed by dot-blot hybridization. HBV DNA is first detected 8 days after liver transplantation. Viremia attains a peak between days 18 and 25 when HBV infection is observed in 85% of the transplanted animals. The HBV-Trimera model was used to evaluate the therapeutic effects of human polyclonal anti-HBs antibodies (Hepatect) and of two reverse-transcriptase inhibitors, lamivudine (3TC) and beta-L-5-fluoro-2',3'-dideoxycytidine (beta-L-5FddC). Treatment of HBV-Trimera mice with these drugs effectively reduced both the percentage of infected animals and the viral load in their sera. Treatment cessation resulted in rebound of viral load, indicating HBV replication upon drug withdrawal. These results show that the HBV-Trimera model represents a novel experimental tool for simulating human HBV infection and evaluating potential anti-HBV therapeutic agents.
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PMID:The hepatitis B virus-trimera mouse: a model for human HBV infection and evaluation of anti-HBV therapeutic agents. 991 35

The hepatitis D virus (HDV) relies on the helper hepatitis B virus (HBV) for the provision of its envelope, which consists of hepatitis B surface antigen (HBsAg). The RNA genome of HDV is a circular rod-like structure due to its extensive intramolecular base-pairing. HDV-RNA has ribozyme activity which includes autocatalytic cleavage and self-ligation properties, essential in virus replication via the rolling circle mechanism. Replication of the RNA is thought to be effected by cellular RNA polymerase II. Hepatitis D antigen (HDAg) is the only protein encoded by HDV-RNA and its long and short forms have a regulatory role in the replication and morphogenesis of the virus. Superinfected HBV carriers who become chronically infected with HDV are at increased risk of developing cirrhosis. Attempts to treat such carriers with interferon have not been particularly successful. In recent years the epidemiology of HDV has changed primarily due to the impact of HBV vaccination in preventing an increase in the pool of susceptible individuals. Copyright 1998 John Wiley & Sons, Ltd.
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PMID:Hepatitis D virus. 1039 91

Accumulating evidence has demonstrated that aberration of the p53 tumour-suppressor gene is one of the pivotal genetic events in hepatocellular carcinogenesis. Recent reports suggest that the product of hepatitis B virus (HBV) interacts with p53 and that the hepatitis C virus (HCV) core protein reduces p53 expression. A novel p73 gene, which is related to p53, has recently been identified and mapped to chromosome 1p36.3, which is a locus of multiple tumour-suppressor genes for many cancers, including hepatocellular carcinoma (HCC) and neuroblastoma. Here, we investigated mRNA expression, allelotype and mutation of p73 in 48 HCCs obtained from untreated patients. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that p73 mRNA was expressed ubiquitously at low levels in all the tumour tissues, as well as in the adjacent normal liver tissues. The frequency of p73 loss of heterozygosity was observed in 20% of HCCs, but PCR-single strand conformation polymorphism (SSCP) analysis showed no mutations in the 48 tumours except for three types of polymorphisms. These results suggest that p73 may play a role in hepatocellular carcinogenesis in a different manner from a Knudson two-hit model. The regulatory mechanism of interaction between p73 and hepatitis viruses remains to be determined.
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PMID:Absence of mutation of the p73 gene localized at chromosome 1p36.3 in hepatocellular carcinoma. 1040 9

The title nucleoside, 4,8-diamino-6-imino-6H-1-beta-d-ribofuranosylimidazo[4,5-e][1,3]-d iazepine, exhibited potent anti-hepatitis B viral activity with minimum toxicity in vitro, and its 5'-triphosphate derivative strongly inhibited the bacteriophage T7 RNA polymerase.
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PMID:Potent anti-hepatitis B viral activity and inhibition of bacteriophage T7 RNA polymerase by a "fat" nucleoside and its 5'-triphosphate derivative: synthetic, biochemical, and biological studies of 4,8-diamino-6-imino-6H-1-beta-D-ribofuranosyl-imidazo[4,5-E] [1,3]diazepine-5'-triphosphate. 1043 89

Prolonged administration of nucleoside analogues for chronic hepatitis B may result in the emergence of hepatitis B viral polymerase mutants. To gain insight into the mechanism involved in the virus's resistance to famciclovir, the amino acid sequences of the terminal protein and reverse-transcriptase (RT) domains of the viral polymerase were determined during therapy among 28 patients. The antiviral response was independent of viral genotypes, and nonresponse to famciclovir was associated with a complex variability of the RT domain. No mutation in the YMDD motif was observed, whereas an L528M mutation was clearly selected by famciclovir treatment in 2 patients, as well as 14 novel mutations in 7 patients. Clone sequence analysis of the RT domains of patients undergoing retreatment with famciclovir and/or lamivudine showed the selection of a preexisting drug-resistant mutant in one case and indicated that sequential antiviral therapy may allow the rapid selection of resistant strains.
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PMID:Evolution of hepatitis B virus polymerase gene sequence during famciclovir therapy for chronic hepatitis B. 1076 59


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