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)

The expression of a molecular cDNA clone (P1 KIN) of the human RNA-dependent P1/eIF-2 alpha protein kinase (PKR) was examined in transfected monkey cells and in cell-free protein-synthesizing systems. Expression of the wild-type (wt) P1 KIN cDNA, which encodes an active protein kinase, was compared with that of the phosphotransfer catalytic domain II Lys-296-->Arg (K296R) mutant cDNA, which does not encode an active kinase. wt and K296R mutant P1 mRNAs prepared by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of P1 ribosome-associated protein with comparable efficiency in the rabbit reticulocyte system. The K296R mutant P1 protein was also efficiently synthesized in vivo in transfected COS monkey cells. However, synthesis of the wt P1 protein was reduced about 30-fold in transfected COS cells as compared with the K296R mutant P1 protein. Cotransfection of wt P1 KIN cDNA with either K296R mutant P1 KIN cDNA or reovirus S4 cDNA greatly reduced the synthesis of K296R mutant P1 protein and reovirus sigma 3 protein, respectively. Although the wt and K296R mutant P1 KIN plasmid expression vectors replicated with comparable efficiencies in COS cells, the steady-state amount of P1 mRNA was about 3-fold less in COS cells transfected with the wt as compared with the K296R mutant P1 KIN cDNA. These results suggest that RNA-dependent P1 protein kinase expression is autoregulated in vivo in transfected mammalian cells primarily at the level of translation by a mechanism that is likely dependent upon catalytically active P1 kinase.
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PMID:Mechanism of interferon action: autoregulation of RNA-dependent P1/eIF-2 alpha protein kinase (PKR) expression in transfected mammalian cells. 127 95

To further investigate the role of p53 gene inactivation in gastric tumorigenesis, the mutational status of the p53 gene in primary human gastric cancer samples was examined. Reverse transcriptase polymerase chain reaction and subsequent direct sequencing of the p53 gene from gastric cancer samples revealed frequent point mutations of the p53 gene: some of these coincided with those previously identified in gastric cancer cell lines. In addition, both allelic deletion analysis using pYNZ 22 and polymerase chain reaction-restriction fragment length polymorphism analysis demonstrated an allelic deletion of the p53 gene in cancer tissue which contained a point mutation of the p53 gene in the remaining allele. Transfection of the wild-type or mutant p53 genes into gastric cancer cells showed that the wild-type but none of the mutated p53 genes suppressed the colony formation of gastric cancer cells. Furthermore, the incorporation of thymidine into DNA was reduced in cancer cells expressing the wild-type p53 gene. The glutathione S-transferase-wild type p53 fusion protein bound to simian virus 40 large T antigen in COS-1 cell lysate. None of the p53 fusion proteins containing mutations at codons 143, 175, 248, or 273 bound to simian virus 40 large T antigen. By contrast, two different mutant p53 fusion proteins containing mutations specifically observed in gastric cancer bound to simian virus 40 large T antigen. These results indicate that inactivation of the p53 gene through mutations and the allelic deletion may play an important role in gastric tumorigenesis. These mutations may cause a conformational change in the p53 protein resulting in the loss of the suppression by p53 of the growth of gastric cells, partly through disruption of the association of p53 protein with a cellular component.
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PMID:p53 gene mutations in human gastric cancer: wild-type p53 but not mutant p53 suppresses growth of human gastric cancer cells. 132 85

The co-infection or infection-transfection variants of the T7 RNA polymerase/vaccinia vector system were used to express 5-HT1ARs in COS-7, BSC-40 and GH3 cells, with co-infection giving ca. 3-fold higher level than infection-transfection. Binding affinities were similar to those of the endogenous 5-HT1AR, with highest affinities for 5-HT and 8-OH-DPAT. Functional properties were demonstrated by assays of agonist-stimulated GTPase activity and its inhibition by pertussin toxin. Immunoblot assays showed expression of the unglycosylated and glycosylated receptor protein in the membrane and, surprisingly, in the cytosolic fractions.
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PMID:Expression in animal cells of the 5-HT1A receptor by a vaccinia virus vector system. 153 96

Infection of cells with poliovirus results in a rapid inhibition of host RNA and protein synthesis. Concordant with this shutoff, the p220 subunit of the cap-binding protein complex is cleaved, probably indirectly, by the poliovirus proteinase p2A (2Apro). To elucidate the mechanism of action of 2Apro in inhibiting protein synthesis in vivo, we studied the effect of transient expression of 2Apro in COS-1 monkey kidney cells. In cells transfected with a 2Apro expression plasmid, p220 was cleaved and the 2Apro mRNA was reduced 30-fold compared to an identical plasmid containing a translation termination codon within the 2Apro coding region. The reduced expression from the 2Apro vector results from a 4-fold reduction in DNA replication and 22-fold reduction in transcription by RNA polymerase II from the adenovirus major late promoter/SV40 enhancer utilized in this vector. In contrast, no decrease in transcription of the adenovirus virus-associated I RNA gene by RNA polymerase III was observed. The effect of 2Apro expression on cap-dependent mRNA translation was studied by producing a dicistronic beta-globin mRNA harboring the encephalomyocarditis virus leader and 2Apro coding region within the 3' end of the mRNA to mediate cap-independent translation of 2Apro. Expression of this mRNA was also reduced 25-fold compared to an identical plasmid harboring a termination codon within the 2Apro coding region. Translation of the beta-globin marker gene from this mRNA was reduced 3-fold when corrected for mRNA level. These results suggest that p220 cleavage itself is not sufficient for complete inhibition of host translation and that an important effect of 2Apro expression on host protein synthesis is a reduction in RNA polymerase II transcription and to a lesser extent, DNA replication. This reduction could be a primary effect of 2Apro, or a secondary effect caused by the inhibition of translation.
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PMID:The effect of poliovirus proteinase 2Apro expression on cellular metabolism. Inhibition of DNA replication, RNA polymerase II transcription, and translation. 171 90

The effect of Rev on cytoplasmic accumulation of the singly spliced human immunodeficiency virus type 1 (HIV-1) vif, vpr, and env/vpu RNAs was examined by using a quantitative RNA polymerase chain reaction (PCR) analysis following transfection of complete proviral molecular clones into lymphoid cells. Previously published studies using subgenomic env constructs in nonlymphoid cell types concluded that Rev was necessary for cytoplasmic accumulation of high levels of unspliced env RNA and that, by analogy, Rev must be necessary for the cytoplasmic accumulation of all HIV-1 RNAs that contain the Rev-responsive element (RRE). We confirm those results in COS cells. Unexpectedly, in lymphoid cells, we find that although Rev acts somewhat to increase the cytoplasmic level of full-length HIV-1 RNA, Rev has little or no effect on cytoplasmic accumulation of singly spliced HIV-1 RNAs. However, Env protein expression was greatly reduced in the absence of Rev. Analysis of the cytoplasmic RNA revealed that in the absence of Rev or the RRE, the cytoplasmic vif, vpr, and env/vpu 2 RNAs were not associated with polysomes but with a complex of 40S-80S in size. Consequently, efficient expression of the Vif, Vpr, Vpu, and Env proteins from these RNAs is dependent on Rev. These results exclude a mechanism whereby the sole function of Rev is simply to export RNAs from nucleus to cytoplasm. We discuss other models to take into account the dependence on Rev for efficient translation of cytoplasmic HIV-1 RNAs.
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PMID:Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs. 182 22

The E1A region of the adenoviral genome, important for initiation of virus infection and activation of other viral genes, was chosen as a target for engineering antisense RNA (asRNA) to inhibit adenovirus 5 (Ad5) replication in COS-1 cell culture in vitro. The hsp70 promoter, taken from the appropriate heat-shock-protein gene of Drosophila melanogaster, and the VA-1 RNA promoter, derived from the Ad5 gene coding for low-molecular-mass VA-1 RNA and recognized by RNA polymerase III were used as regulatory elements of transcription. The two types of recombinant constructs contained E1A fragments of 710 bp (hsp70 constructs) or 380 or 740 bp (VA-1 RNA constructs) in reverse orientation relative to the promoter position, as well as a transcription termination signal, the SV40 ori, and the gene controlling Geneticin (antibiotic G418) resistance (G418R). After selection of transfected COS-1 cells in the presence of G418, a number of stable G418R cell lines were raised which expressed engineered asRNAs. Plating of Ad5 suspensions of known titre on monolayers of transfected COS-1 cells clearly showed strong inhibition of adenovirus replication by asRNAs: 75% with the hsp70 promoter and 90% with the VA-1 RNA promoter.
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PMID:Inhibition of adenovirus replication by the E1A antisense transcript initiated from hsp70 and VA-1 promoters. 215 50

We have identified an abundant transcript initiated upstream from the canonical cap site of human alpha 1 globin gene in bone marrow cells and in COS-7 cells transfected with an alpha 1 globin gene-containing plasmid. Similar to the major alpha 1 globin transcript, this upstream RNA is present almost exclusively in the cytoplasm of the transfected COS-7 cells. It is also synthesized efficiently in vitro by RNA polymerase II in the nuclear extracts prepared from a Hela cell line and an erythroleukemia cell line, K562. RNAs isolated from these cell lines, however, do not contain this upstream transcript. The putative 5' end of the alpha 1 globin upstream RNA is mapped by primer extension to base -45, which is located in between the CCAAT and TATA boxes. The synthesis of this RNA in vitro and in vivo, and the close proximity of its 5' end to the promoter of the alpha 1 globin gene suggest a common mechanism regulating the transcriptional initiation of both the upstream and the major alpha 1 globin RNAs.
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PMID:Characterization of an unique RNA initiated immediately upstream from human alpha 1 globin gene in vivo and in vitro: polymerase II-dependence, tissue specificity, and subcellular location. 241 22

The gene for rat cholecystokinin (CCK) was isolated from a rat genomic DNA library. The transcription unit spans 7 kilobases and is interrupted by two introns. The initiator methionine codon lies 2 bases into exon 2; therefore, exon 1 is a noncoding exon. The transcription initiation site was determined using avian myeloblastosis reverse transcriptase, a cDNA primer, and mRNA isolated from a rat medullary thyroid carcinoma. A "TATA"-like sequence precedes the transcription initiation site at position -34. The polyadenylation site for the gene was mapped by a nuclease protection assay using a cRNA generated by transcription of the exon 3 region of the CCK gene with SP6 bacteriophage RNA polymerase. The sequence AT-TAAA is found 22 bases 5' to the site determined to be the polyadenylation addition site. Two regions of simple repetitive DNA occur within the CCK lambda clone, one within intron 2 and the other 4 kilobases 3' to the gene. Sequence analysis of the repetitive element 3' distal to the gene revealed two copies of the sequence 5'-(AC)n-3', where n is 22 and 25. A 114-base pair sequence of predominantly repeating purine-pyrimidine nucleotides separates these two d(AC) repeats. Transcriptional control elements were investigated by fusing regions of the CCK gene to the structural gene encoding chloramphenicol acetyltransferase. Promoter activity was determined by transfecting COS-7 cells with plasmids containing the gene fusions, followed by determining chloramphenicol acetyltransferase activity in cellular extracts. The region necessary for expression of the CCK gene fusions in COS-7 cells is within 144 bases 5' to the initiation of transcription.
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PMID:A gene encoding rat cholecystokinin. Isolation, nucleotide sequence, and promoter activity. 298 40

We studied RNA polymerase I transcription in cells transfected with a plasmid, prHuTK, containing the herpes simplex virus tk gene fused to a human rRNA promoter. Primer extension analysis of tk RNA isolated from COS cells transfected with prHuTK reveals that transcription from the RNA polymerase I promoter is highly efficient and initiates at the same position used for the synthesis of endogenous rRNA in HeLa cells. The RNA products derived from prHuTK are distinguishable from normal RNA polymerase II transcripts of tk in that they are not polyadenylated, are extremely unstable, and are found predominantly in the nucleus. Moreover, the transcription observed is resistant to 300 micrograms of alpha-amanitin per ml. These results strongly suggest that prHuTK transcription is under the control of the human rRNA promoter and RNA polymerase I. To further characterize the activity of the human rDNA promoter in vivo, a series of 5' and 3' deletion mutants was tested in this transfection assay. The deletion analysis indicates that a core region of ca. 40 base pairs overlapping the initiation site is critical for transcription. In addition, a region between nucleotides -234 and -131 upstream from the core sequence serves to modulate the efficiency of transcription. Insertion into prHuTK of additional ribosomal nontranscribed spacer DNA or the simian virus 40 enhancer element has no apparent effect on the promoter activity. Surprisingly, RNA polymerase II transcripts synthesized at low levels from two start sites within the core control element of the wild-type RNA polymerase I promoter are activated upon deletion of upstream RNA polymerase I promoter sequences. However, these RNA polymerase II transcripts are not expressed from the endogenous rRNA promoter.
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PMID:Transcription of herpes simplex virus tk sequences under the control of wild-type and mutant human RNA polymerase I promoters. 298 90

The large gene, L, of vesicular stomatitis virus (VSV), which codes for the multifunctional RNA-dependent RNA polymerase, was assembled from five overlapping cDNA clones. The sequence of the 6.4-kilobase gene of the final construct was identical to the consensus sequence reported earlier. The gene was inserted into the simian virus 40 transient expression vector pJC119. Antibodies directed against synthetic peptides corresponding to the amino and carboxyl termini of the L protein were raised in rabbits. Both antibodies specifically immunostained the cytoplasm of COS cells that had been transfected with the vector DNA. The expressed L protein was immunoprecipitated from cell extracts and it was identical in size to the L protein of the virion (241 kilodaltons). Most importantly, COS cells that expressed the recombinant L protein transcribed, replicated, and consequently complemented and rescued temperature-sensitive RNA polymerase mutants of VSV at the nonpermissive temperature. The kinetics of virus release were similar to those of a wild-type VSV infection. We conclude that the recombinant RNA polymerase protein L is indistinguishable in its size and its functions from the VSV polymerase.
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PMID:Expression of a cDNA encoding a functional 241-kilodalton vesicular stomatitis virus RNA polymerase. 299 88


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