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 transcription of chromatin from adenovrius 2 transformed rat cells by murine plasmacytoma RNA polymerases I, II and III has been studied. Both the total RNA synthesis and transcription of the integrated adenovirus 2 genes by RNA polymerase II represent de novo DNA transcription as assessed by their sensitivity to actinomycin D. It is shown that each RNA polymerase class has characteristic ionic strength activation profiles and metal ion requirements. RNA polymerase II transcribes the integrated adenovirus 2 genes in chromatin at a frequency 25- to 50-fold higher than their sequences are represented in the genome. In contrast, no detectable viral RNA is synthesized when deproteinized DNA is transcribed. In the presence of Mn2+, all three RNA polymerases (I, II and III) transcribe the integrated viral genes at approximately the same relative frequencey. However, the Mg2+ as divalent cation, the proportion of the total RNA which represents viral gene transcripts is increased 3- to 4-fold with RNA polymerase II, while it remains unchanged for RNA polymerases I or III.
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PMID:Transcription of viral genes in chromatin from adenovirus 2 transformed cells by exogenous eukaryotic RNA polymerases. 49 52

Iodination of alpha-amanitin at the 7-position in the 6-hydroxy-2-sulfoxytryptophan moiety is effected with 1 equiv of iodine monochloride in methanol. The isolated product shows a lambdamax in methanol at 301 nm, compared with 305 nm for the parent alpha-amanitin; in methanolic 0.01 M NaOH the lambdamax are 330 and 332 nm for the product and parent, respectively. Spectrophotometric titration of the phenolic hydroxyl shows a decrease in pKa from 9.72 (alpha-amanitin) to 7.94 (7 iodo-alpha-amanitin). Appropriate spectrophotometric examination therefore distinguishes between parent and product. Proton magnetic resonance shows two aromatic protons (v4H = 7.57; V5H = 6.90 ppm; j4,5 = 9) in the 7-iodo-alpha-amanitin and three aromatic protons (v4H = 7.64; V5H = 6.78; V7H = 6.94 ppm; j4,5 = 9; J5,7 = 2) in alpha amanitin thus establishing the extent and position of iodine substitution. The 7-iodo-alpha-amanitin effectively inhibits RNA polymerase activity with half-maximal inhibition at 2 X 10(-9) M and 10(-4) M for the sea urchin RNA polymerases II and III, respectively. Addition of [125I]-7-iodo-alpha-amanitin (200 Ci/mmol) to crude extracts from sea urchin blastula, MOPC 315 plasmacytoma, and adult Oregon R Drosophila melanogaster followed by resolution on DEAE-Sephadex demonstrates that the radioactive ligand binds stably and specifically with RNA polymerase II in each of these extracts.
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PMID:Biochemistry of the amatoxins: preparation and characterization of a stably iodinated alpha-amanitin. 62 38

Mouse plasmacytoma (MOPC) 460 cells contain two chromatographic forms of RNA polymerase III (IIIA and IIIB) in addition to the major class I and II RNA polymerases. Nuclei isolated from these cells actively synthesize RNA. Among the discrete transcription products observed are the 5S and 4.5S RNAs and additional low molecular weight RNA species (approximately 5.8S, 6.3S, and 6.6S in size). The 4.5S RNAs appear to be tRNA precursors since they can be converted in vitro to 4S RNAs. Studies with alpha-amanitin have shown that the synthesis of these discrete RNA species, and other uncharacterized transcripts somewhat larger in size, is mediated by an endogenous RNA polymerase III activity(ies). Nuclear RNA synthesis is stimulated by exogenous purified RNA polymerases. Exogenous MOPC class III RNA polymerases stimulate the synthesis of each of the distinct low molecular weight species (including 5S and 4.5S RNAs) about 3-6 fold. The hybridization of nuclear transcripts to purified 5S genes (5S DNA) confirms that exogenous class III RNA polymerases stimulate (approximately 4 fold) the synthesis of ribosomal 5S RNA. The 5S RNA genes in nuclei are transcribed asymmetrically by both the endogenous and the exogenous class III enzymes. Exogenous RNA polymerase III from Xenopus laevis ovaries stimulates 4.5S and 5S RNA synthesis in MOPC nuclei as effectively as do the MOPC class III RNA polymerases. However, exogenous MOPC class I and II RNA polymerases do not stimulate 4.5S and 5S RNA synthesis, suggesting that this effect is specific for the structurally similar class III RNA polymerases.
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PMID:Transcription of specific genes in isolated nuclei by exogenous RNA polymerases. 84

Class III RNA polymerases purified from the murine plasmacytoma MOPC 315 and from Xenopus laevis ovaries were compared. The subunit structures of the chromatographically distinct murine enzymes IIIA and IIIB were indistinguishable and were remarkably similar to that of the amphibian enzyme III. The plasmacytoma class III RNA polymerases were also compared with purified plasmacytoma RNA polymerases I and II. Sedimentation studies indicated that RNA polymerase III si significantly larger than RNA polymerase II, which is slightly larger than RNA polymerase I. Structural analyses showed that the molecular weights of the large subunits present in the class III enzymes (138,000 and 155,000) differ from those of the class II enzymes (140,000 and either 170,000, 205,000, or 240,000) and from those of the class I enzymes (117,000 and 195,000). Some low-molecular-weight subunits are also unique to each enzyme class. These results clearly distinguish the class I, II, and III enzymes on a structural basis. In addition, polypeptides of molecular weight 29,000 and 19,000 were found in all enzyme classes, a polypeptide of molecular weight 52,000 was found only in class I and III enzymes, and a polypeptide of molecular weight 41,000 was found only in class II and III enzymes. These findings are discussed in terms of the function and regulation of the RNA polymerases.
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PMID:Distinct molecular structures of nuclear class I, II, and III DNA-dependent RNA polymerases. 105 9

DNA-dependent RNA polymerase II was purified from the mouse plasmacytoma, MOPC 315. Soluble enzyme was obtained from a nucleoplasmic fraction and subjected to chromatography on phosphocellulose, DEAE-cellulose, and DEAE-Sephadex ion exchange resins and was subjected to sedimentation in sucrose density gradients. A chromatographically homogeneous enzyme was obtained which was purified about 25,000-fold relative to whole cell extracts and which had a specific activity (on native DNA) similar to those reported for other purified eukaryotic class II RNA polymerase preparations. Analysis of purified RNA polymerase II by polyacrylamide gel electrophoresis under nondenaturing conditions revealed three protein bands, designated II-O, II-A, and II-B in order of electrophoretic mobility. The subunit compositions of these nondenatured bands were subsequently analyzed by electrophoresis under denaturing conditions. Each enzyme II form contained subunits with molecular weights of 140,000 (II-c), 41,000 (II-d), 30,000 (II-e), 25,000 (II-f), 22,000 (II-g), 20,000 (II-h), and 16,000 (II-i). Molar ratios were unity for all subunits except subunit II-h which had a molar ratio of 2. Each enzyme form was distinguished by its highest molecular weight subunit. II-O contained subunit II-o (molecular weight 240,000), II-A contained subunit II-a (molecular weight 205,000), and II-B contained subunit II-b (molecular weight 170,000). Total molecular weights for II-O, II-A, and II-B were calculated as 554,000, 519,000, and 484,000, respectively. In addition, the number of RNA polymerase II molecules per MOPC 315 tumor cell was calculated to be about 5 times 10-4.
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PMID:Purification and subunit structure of deoxyribonucleic acid-dependent ribonucleic acid polymerase II from the mouse plasmacytoma, MOPC 315. 116 91

To investigate the chromatin surrounding an active gene, we have determined the distribution of RNA polymerase molecules, the intactness of nucleosomal structure, and the subnuclear compartmentalization along 15 kilobase pairs (kb) of the mouse kappa immunoglobulin locus of MPC-11 plasmacytoma cells. Hybridization of in vitro nuclear transcripts to probes specific for the template strand reveals that transcription terminates within the region between 1.1 and 2.3 kb downstream from the poly(A) addition site. Ten different short sequences (8-13 base pairs) reside within 460 base pairs of this termination region that exhibit homology with sequences found in the termination regions of mouse beta-globin and chicken ovalbumin genes. Transcription of the nontemplate strand occurs on either side of this termination region. We find that both within the transcription unit and 6.5 kb downstream of the termination region of the kappa gene, the canonical nucleosomal structure is perturbed, the chromatin exhibits pronounced insolubility, and the nucleosomes liberated by micrococcal nuclease appear to lack histone H1. The insolubility is characterized by interactions that are disrupted by 0.3 to 0.6 M NaCl treatment. We conclude that the active chromatin phenotype spreads a considerable distance along the kappa locus, well beyond the region of transcription termination.
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PMID:Transcription termination and chromatin structure of the active immunoglobulin kappa gene locus. 308 10

Transcription of immunoglobulin kappa genes is regulated by enhancer and promoter elements, both of which function in a tissue-specific fashion. We have studied the interaction of these elements by transfecting plasmacytoma cells with genes that have tandem kappa promoters located next to a single kappa enhancer and assaying these genes for transient or stable transcription. We find that the promoters located proximal and distal to the enhancer function identically whether they are separated by 440 bp or by 2.7 kb or whether they are located 1.7 or 7.7 kb away from the enhancer. Our results indicate that the immunoglobulin kappa enhancer does not operate as a bidirectional entry site for RNA polymerase or for other factors associated with the transcription complex. Rather, they suggest that the enhancer exerts its influence uniformly over large distances and independently of the presence of intervening promoters.
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PMID:Tandem kappa immunoglobulin promoters are equally active in the presence of the kappa enhancer: implications for models of enhancer function. 308 28

A variety of cell lines representing different maturation stages of the B lymphocyte were used to analyse developmental changes in the transcriptional pattern through the mu-delta locus and the relationship between mu mRNA accumulation and transcriptional activity. As anticipated from earlier studies, we observed that RNA polymerase loading in the region between the mu m cleavage/poly A addition site and the delta 1 exon is markedly decreased in IgM secreting cells compared to cells bearing surface IgM or surface IgM and IgD. In several IgM secreting hybridomas, transcriptional termination mainly occurred downstream of the first mu m exon. Thus, the predominance of mu s-terminated transcripts in these cells would appear to be principally determined by RNA processing events, most likely by more efficient cleavage at the mu s poly A site and/or less efficient splicing of the C mu and mu m exons. In two plasmacytoma lines, polymerase unloading between the mu s and mu m sites also contributed significantly to the high mu s mRNA phenotype. Our results further indicate that posttranscriptional regulation is largely responsible for the greatly increased accumulation of mu mRNA in the IgM secretors. Interestingly, the sterile-mu RNA components do not seem to be subject to this posttranscriptional regulation.
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PMID:Transcriptional and posttranscriptional control of immunoglobulin mRNA production during B lymphocyte development. 309 May 17

A procedure has been developed for the production of MAb against weakly immunogenic subunits of a multisubunit enzyme. This procedure takes into account the problems of insufficient antigen, single epitope immunodominance and the difficulty of mapping non-sequential determinants. Small quantities of mammalian RNA polymerase II subunits were purified by SDS-polyacrylamide gel electrophoresis and were used to immunize splenocytes in vitro. After fusion with plasmacytoma cells, the hybrid cells were cloned and screened by ELISA utilizing native RNA polymerase II. This procedure is biased towards the production of MAb directed against sequential epitopes accessible on the native enzyme. Monoclonal antibodies, produced by in vitro immunization, were shown to be useful in protein transblot analyses, to inhibit enzyme activity in vitro and to have binding affinities comparable with MAbs produced by in vivo immunization.
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PMID:Production of monoclonal antibody against electrophoretically purified RNA polymerase II subunits using in vitro immunization. 321 73

The effect of encephalomyocarditis virus infection of MOPC 460 mouse plasmacytoma cells on host RNA synthesis and RNA polymerases was investigated. Consistent with work performed in other virus host systems, rates of RNA synthesis appeared to be inhibited in infected cells, whereas RNA degradation appeared normal. These results were further extended with isolated nuclei, in which distinct RNA polymerase activities could be studied under conditions where problems with RNA turnover and endogenous nucleotide pool sizes were insignificant. Endogenous nuclear RNA polymerase II activity was inhibited early postinfection and at 1 to 2 h prior to endogenous RNA polymerase I plus III activity. However, the solubilized enzymes were fully active with exogenous DNA as template. In fact, the levels of RNA polymerases I, II, and III, isolated from infected cells and nuclei, were indistinguishable from levels in uninfected cells and nuclei at each stage of their partial purification procedure. The chromatographic properties of the enzymes on DEAE-Sephadex were also unaltered. Furthermore, the RNA synthetic activity of these isolated enyzmes, or of nuclei isolated from uninfected cells, was resistant to extracts of nuclei or of cytoplasmic fractions from infected cells. These results are discussed in terms of a possible inhibition of RNA synthesis in vivo at the level of transcription initiation.
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PMID:Encephalomyocarditis virus infection of mouse plasmacytoma cells. II. Effect on host RNA synthesis and RNA polymerases. 436 75


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