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)

RNA polymerase I transcription has been used for expression of influenza vRNA molecules, with influenza hemagglutinin or other cDNAs precisely inserted between mouse rDNA promoter and terminator sequences. In in vitro studies generation of HA vRNA transcripts in high rates and correct formation of their 5' ends as well as their 3' ends has been achieved for such hybrid DNA templates. For in vivo expression studies, the HA coding region was replaced by chloramphenicol acetyltransferase (CAT), also in vRNA antisense orientation, with both influenza terminal sequences beyond start and stop codons being retained on the resulting transcript. Following transfection with precisely constructed hybrid DNA templates and depending on infection with influenza virus, CAT activity could be demonstrated. Templates resulting in 3' extended vRNA molecules did not give this result. vRNA-CAT molecules were not only recognized by influenza viral RNA polymerase for synthesis of plus strand mRNAs, but also were packaged into progeny virus particles, as shown by CAT activity in infected cells after passaging of virus containing supernatants.
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PMID:RNA polymerase I-mediated expression of influenza viral RNA molecules. 800 59

Hepatitis A virus (HAV) exhibits several characteristics which distinguish it from other picornaviruses, including slow growth in cell culture even after adaptation, and lack of host-cell protein synthesis shut-down. Like other picornaviruses, HAV contains a long 5' nontranslated region (NTR) incorporating an internal ribosomal entry site (IRES), which directs cap-independent translation. We compared HAV IRES-initiated translation with translation initiated by the structurally similar encephalomyocarditis virus (EMCV) IRES, using plasmids in which each of the 5'NTRs is linked in-frame with the chloramphenicol acetyltransferase (CAT) gene. Translation was assessed in an HAV-permissive cell line which constitutively expresses T7 RNA polymerase and transcribes high levels of uncapped RNA from these plasmids following transfection. RNAs containing the EMCV IRES were efficiently translated in these cells, while those containing the HAV IRES were translated very poorly. Analysis of translation of these RNAs in the presence of poliovirus protein 2A, which shuts down cap-dependent translation, demonstrated that their translation was cap independent. Our results suggest that the HAV IRES may function poorly in these cells, and that inefficient translation may contribute to the exceptionally slow replication cycle characteristic of cell culture-adapted HAV.
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PMID:Analysis of hepatitis A virus translation in a T7 polymerase-expressing cell line. 803 60

To characterize in vivo the translational control elements present in the 5' nontranslated region (5'NTR) of hepatitis A virus (HAV) RNA, we created an HAV-permissive monkey kidney cell line (BT7-H) that stably expresses T7 RNA polymerase and carries out cytoplasmic transcription of uncapped RNA from transfected DNA containing the T7 promoter. The presence of an internal ribosomal entry site (IRES) within the 5'NTR of HAV was confirmed by using BT7-H cells transcribing bicistronic RNAs in which the 5'NTR was placed within the intercistronic space, controlling translation of a downstream reporter protein (bacterial chloramphenicol acetyltransferase). However, translation directed by the 5'NTR in these bicistronic transcripts and in monocistronic T7 transcripts in which the HAV 5'NTR was placed upstream of the chloramphenicol acetyltransferase coding sequence was very inefficient compared with the translation of monocistronic transcripts containing either the IRES of encephalomyocarditis (EMC) virus or a short nonpicornavirus 5' nontranslated leader sequence. A large deletion within the HAV IRES (delta 355-532) eliminated IRES activity in bicistronic transcripts. In contrast, larger deletions within the IRES in monocistronic transcripts (delta 1-354, delta 1-532, delta 1-633, and delta 158-633) resulted in 4- to 14-fold increases in translation. In the latter case, this was most probably due to a shift from IRES-directed translation to translation initiation by 5'-end-dependent scanning. Translation of RNAs containing either the EMC virus IRES or the nonpicornavirus leader was significantly enhanced by cotransfection of the reporter constructs with pEP2A, which directs transcription of RNA containing the EMC virus IRES fused to the poliovirus 2Apro coding region. This 2Apro enhancement of cap-independent translation suggests a greater availability of limiting cellular translation factors following 2Apro-mediated cleavage of the p220 subunit of the eukaryotic initiation factor eIF-4F and subsequent shutdown of 5' cap-dependent translation. In contrast, pEP2A cotransfection resulted in severe inhibition of translation directed by the HAV IRES in either monocistronic or bicistronic transcripts. This inhibition was due to competition from the EMC virus IRES present in pEP-2A transcripts, as well as the expression of proteolytically active 2Apro. 2Apro-mediated suppression of HAV translation was not seen with transcripts containing large deletions in the HAV IRES (delta 158-633, delta 1-532, or delta 1-633). These data suggest that the HAV IRES may have a unique requirement for intact p220 or that it may be dependent on active expression of another cellular translation factor which is normally present in severely limiting quantities.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Low efficiency of the 5' nontranslated region of hepatitis A virus RNA in directing cap-independent translation in permissive monkey kidney cells. 803 22

An in vivo system in which expression of a synthetic influenza virus-like chloramphenicol acetyltransferase (CAT) RNA is driven by influenza virus proteins synthesized from cloned cDNAs has been developed. Expression of the four influenza virus core proteins (nucleoprotein, PA, PB1 and PB2) was performed by transfection of four pGEM recombinant plasmids, each containing one of the four viral genes, into cell cultures previously infected with a vaccinia virus recombinant encoding the T7 RNA polymerase (vTF7-3). When a naked negative-sense influenza virus-like CAT RNA was transfected into cells expressing the four influenza virus proteins, CAT activity was detected in the cell extracts, demonstrating that the expressed proteins had RNA-synthesizing activity. In this system, CAT RNA templates containing additional nucleotides at the 3' end were also expressed, resulting in CAT activity. This showed that the influenza virus polymerase can recognize its promoter when located internally on an RNA template. In influenza virus-infected cells however, CAT activity was detected only when the CAT RNA contained the viral promoter at the exact 3' end and was transfected as in vitro assembled ribonucleoprotein. These results are discussed in terms of the different requirements of the two helper systems for expression of an exogenously added RNA.
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PMID:Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system. 804 17

A plasmid containing the reporter gene, chloramphenicol acetyltransferase (CAT), driven by the bacteriophage T7 promoter was co-delivered with purified T7 RNA polymerase by the DC-chol cationic liposomes into human embryonic kidney 293 cells to obtain a transient (2 days) CAT gene expression. To prolong the expression, a T7 autogene which contains the T7 RNA polymerase gene driven by the T7 promoter was included in the transfection complex as a self-amplifying regeneration mechanism for the polymerase. High level CAT gene expression was observed up to 5 days after transfection. This strong and sustained expression system should be useful in gene transfer experiments.
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PMID:A sustained, cytoplasmic transgene expression system delivered by cationic liposomes. 818 68

Proteins entirely expressed from cDNA were used to rescue synthetic RNA genome analogs into infectious defective particles of rabies virus (RV). Synthetic negative-stranded RNAs containing 3'- and 5'-terminal RV sequences and transcriptional signal sequences were transcribed from plasmids transfected into cells expressing T7 RNA polymerase from recombinant vaccinia virus. After simultaneous expression of RV N, P, and L proteins from plasmids containing a T7 RNA polymerase promoter, the synthetic genomes were encapsidated, replicated, and transcribed by the RV polymerase proteins. Insertion of the bacterial chloramphenicol acetyltransferase gene or beta-galactosidase (lacZ) gene between the 3' and 5' termini containing transcriptional signal sequences resulted in transcription of mRNAs and expression of chloramphenicol acetyltransferase and beta-galactosidase, respectively. Upon simultaneous expression of N, P, M, G, and L proteins, virions carrying the foreign genes were assembled and released into the supernatant. The possibility of rescuing cDNA into rabies virions by proteins also expressed entirely from cDNA opens the possibility of studying the functions of each RV protein and analyzing cis-acting signals of the RV genome.
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PMID:Rescue of synthetic genomic RNA analogs of rabies virus by plasmid-encoded proteins. 828 75

Expression of bacteriophage T7 RNA polymerase in mammalian cells can efficiently drive the transcription of a foreign gene controlled by the T7 promoter (Elroy-Stein et al., Proc. Natl. Acad. Sci. USA. 86, 6126-6130, 1989). We have tested the hypothesis that purified T7 RNA polymerase can be co-delivered into mammalian cells together with a reporter gene (chloramphenicol acetyltransferase, CAT) controlled by the T7 promoter (pT7-EMC-CAT) using DC-chol cationic liposomes. Indeed, significant level of CAT activity was observed in human lung adenocarcinoma (A549-1) cells which had been incubated with a complex of T7 RNA polymerase, pT7-EMC-CAT DNA and DC-chol cationic liposomes. The expression was specific in that T3 RNA polymerase could not replace the T7 RNA polymerase, and that co-delivered T7 RNA polymerase did not enhance the expression of a CAT gene controlled by the SV40 early promoter. The system was optimized in terms of enzyme, DNA and liposome concentrations. Time course experiment indicated that the expression of the T7 system was about 8-10 hours sooner than the SV40 system, consistent with the notion that T7 RNA polymerase does not enter into the nucleus and the transcription takes place in the cytoplasm of the transfected cells. The expression of the T7 system was transient; it declined after 30 hours post transfection, probably due to turnover of the phage enzyme in the mammalian cells. The expression system described here should be useful for gene transfer experiments which require a fast but transient expression of a foreign gene. We have also compared our delivery system with a commercial reagent, Lipofectin, which has been used to deliver T3 or T7 RNA polymerase with a reporter plasmid encoding the T3 or T7 promoter.
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PMID:Cytoplasmic expression of a reporter gene by co-delivery of T7 RNA polymerase and T7 promoter sequence with cationic liposomes. 833 95

RNA polymerase I has been used for transcription of influenza hemagglutinin (HA) cDNA precisely linked in the anti-sense configuration to both mouse rDNA promoter and terminator segments. In transcription reactions based on Ehrlich ascites cell nuclear extracts, specific uniform RNA products are synthesized in high rates that are comparable to original rDNA template transcriptions. Primer extension reactions show the 5' ends of these RNA transcripts to be located exactly at position +1, corresponding to the 5' end of negative strand HA viral RNA. RNA 3' ends in a first series of constructs were found extended beyond the accepted location of pre-rRNA 3' ends, in using both hybrid cDNA and original rDNA templates. But upon deletion of six basepairs from the rDNA termination region RNA polymerase I transcription has been adapted to yield correctly terminated influenza viral RNA in vitro. This result has been confirmed in an in vivo experiment via synthesis of an anti-sense viral RNA molecule containing the chloramphenicol acetyltransferase (CAT) gene, which in turn is recognized at its terminal sequence by viral RNA dependent RNA polymerase for plus strand mRNA synthesis and expression of CAT activity.
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PMID:RNA polymerase I catalysed transcription of insert viral cDNA. 836 75

Transcription and replication of hepatitis delta virus (HDV) RNA is thought to be performed by host RNA polymerase II. The mechanism which enables polymerase II to use RNA as a template is unclear. However, since extensive intramolecular complementarity allows HDV RNA to form a rod-shaped structure, it is possible that the mostly double-stranded HDV RNA may resemble double-stranded DNA in structure, and can thus be used by RNA polymerase II as a template. To investigate this possibility, we examined whether the cDNA counterpart of HDV RNA contains a promoter and thus can drive the transcription and replication of HDV RNA. Circularized monomers of HDV cDNA, when transfected into various cell lines, were found to generate both monomeric and dimeric forms of HDV RNA and hepatitis delta antigen at levels comparable to those generated with HDV cDNA multimers under the control of a SV40 late promoter, suggesting that HDV cDNA contains endogenous promoters. Using chloramphenicol acetyltransferase and human growth hormone as reporter genes, the specific promoter activity for the synthesis of antigenomic HDV RNA was localized to a 29-nucleotide region (nucleotides 1650-1679), although an additional 224-nucleotide upstream region was also necessary for maximum activity. Similarly, promoter activity for the synthesis of genomic RNA was localized to a 160-nucleotide region around position 1679 that overlapped with the antigenomic promoter region. Since these regions are in a highly conserved double-stranded region of HDV RNA, they may represent RNA promoters recognized by RNA polymerase II. This result also suggests a convenient method, using circularized monomer HDV cDNA, to study HDV RNA replication.
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PMID:Endogenous promoters can direct the transcription of hepatitis delta virus RNA from a recircularized cDNA template. 837 36

The I-R system of hybrid dysgenesis in Drosophila melanogaster is controlled by a long interspersed nuclear element-like retroposon, the I factor. Transposition of the I factor occurs at a high frequency only in the ovaries of females produced by crossing males of inducer strains that contain functional I factors with females of reactive strains that lack them. In this study, the 5' untranslated region of the I factor was joined to the chloramphenicol acetyltransferase gene, and activity was assayed in transfected D. melanogaster tissue culture cells and transformed flies. The results have identified a promoter that lies within the first 186 pb of the I factor. Deletion analysis shows that nucleotides +1 to +40 are sufficient for high promoter activity and accurate transcription initiation. This region contains sequences that are found in a class of RNA polymerase II promoters that lack both a TATA box and CpG-rich motifs. In transformed flies, high levels of expression from nucleotides +1 to +186 are confined to the ovaries of reactive females, suggesting that the promoter is involved in the tissue and cytotype specificity of transposition.
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PMID:The 5' untranslated region of the I factor, a long interspersed nuclear element-like retrotransposon of Drosophila melanogaster, contains an internal promoter and sequences that regulate expression. 838 Aug 89


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