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 antibody-linked polymerase assay is a method which allows one to assign RNA polymerase activity to SDS-denatured polypeptides on nitrocellulose membranes using antibodies which were raised against only partially purified polymerase preparations. Here we show that with this method not only enzyme subunits but also initiation factor(s) can be determined in crude homogenates. Moreover the determination is quantitative. Therefore changes in the amount of individual polymerase subunits and factor(s) can be visualized within different crude homogenates.
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PMID:Quantification of DNA-dependent RNA polymerase subunits and initiation factor(s) by antibody-linked polymerase assays. 329 92

The particles of CPV of silkworm contain double-stranded RNA polymerase and methyltransferase. It was reported in a previous paper that the genome-enzyme complex could be isolated. The genome-enzyme complex shows high enzyme activity of RNA polymerase and methyltransferase in spite of the fact that it consists of only 5 percent of the protein. In order to clarify the protein subunits of the RNA polymerase and methyltransferase, two methods were adopted. The SDS-polyacrylamide gel electrophoretogram showed that the 125I-labeled genome-enzyme complex of CPV contained three protein components in molecular weight of 33 K, 67 K and 142 K daltons respectively and each protein component of them consisted of more than two protein subunits with different isoelectric points in 2-dimensional electrophoretogram. The antibody to the five protein components (P1, P2, P3, P4, P5) was prepared and used to inhibit the enzyme activities of RNA polymerase and methyltransferase. It showed that the RNA polymerase was inhibited by the antibody to proteins P1, P2 and P4, whereas the methyltransferase was mainly inhibited by the antibody to protein P1.
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PMID:The protein subunits of the double-stranded RNA dependent RNA polymerase and methyltransferase of the cytoplasmic polyhedrosis virus of silkworm, Bombyx mori. 329 95

A cDNA encoding the entire amino acid sequence of the matrix (M1) protein of influenza A/WSN/33 virus was cloned, sequenced, and expressed in a vaccinia virus system consisting of the T7 bacteriophage RNA polymerase and a plasmid carrying the M1 gene flanked by T7 polymerase promoter and terminator sequences. The transiently expressed M1 gene product comigrated on SDS-polyacrylamide gels with the endogenous WSN virus M1 protein and was recognized in Western blot analysis by three epitope-specific monoclonal antibodies directed to the M1 protein. The nucleotide sequence and the predicted amino acid sequence of the cloned WSN virus M1 coding region was found to be more than 97% homologous to that of the M1 gene of influenza virus A/PR/8/34 reported by G. Winter and S. Fields (Nucleic Acids Res. 8, 1965-1974, 1980).
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PMID:Transient expression and sequence of the matrix (M1) gene of WSN influenza A virus in a vaccinia vector. 335 9

Purification of RNA polymerase II from chicken myeloblastosis (leukemia) cells to homogeneity and subsequent structural analysis of the purified enzyme revealed that the enzyme contained seven polypeptides with molecular masses ranging from 27 KDa to 220 KDa. Inclusion of protease inhibitors in the buffer system during purification significantly increased the molar ratio of the largest (220 KDa) polypeptide to the second largest (180 KDa) polypeptide. However, proteolytic conversion of the 220 KDa to 180 KDa polypeptide did not inhibit the DNA binding activity of the enzyme. The enzyme, after dissociation into subunits in a SDS-polyacrylamide gel containing urea was blotted onto a nitrocellulose filter. The filter was incubated with 32P-labeled calf thymus DNA and both the 220 KDa and 180 KDa polypeptides of the enzyme bind DNA, suggesting that the DNA-binding site of the enzyme resides on the 180 KDa polypeptide of the largest subunit.
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PMID:The 180 KDa polypeptide contains the DNA-binding domain of RNA polymerase II. 359 59

[35S]Methionine-labeled proteins from total or cytoplasmic extracts of Vero cells infected with African swine fever virus were chromatographed on native and denatured DNA-cellulose and DNA-binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), by DNA binding to Western blots, or by two-dimensional electrophoresis. Thirteen virus-specific DNA-binding proteins were detected by one-dimensional analysis. Major species have molecular mass 44,000 (44K), 38K, 20K, 18K, 14K, 13K, and 12K. The remaining DNA-binding proteins are proteins with molecular mass 130K, 110K, 35K, 33K, 17K, and 14.5K. When viral DNA used in the binding assay the results were very similar but the 13K protein did not bind viral DNA. Seven other minor virus-specific DNA-binding proteins could be detected by two-dimensional analysis. This technique also enabled the assignment of virus-specific proteins. Seven of the virus-specific DNA-binding proteins are structural proteins. Twelve are late proteins, the remaining being early proteins synthesized before viral DNA replication. Most of the virus-specific DNA-binding proteins bind both to double-stranded and to single-stranded DNA. The 110K, 29K, and 18K DNA-binding proteins bind only to single-stranded DNA. Two virus-specific enzymatic activities, DNA polymerase and RNA polymerase, were present in the fractions separated by DNA-cellulose chromatography. The virus-specific single-stranded DNA nuclease did not bind to DNA.
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PMID:DNA-binding proteins specified by African swine fever virus. 368 26

The complete coding sequence for P-450 PBc2 was inserted into a T7-phage promoter system, and a capped cRNA was generated using T7 RNA polymerase. The P-450 PBc2 cRNA was translated in a rabbit reticulocyte lysate. The in vitro translation product was indirectly immunoprecipitated by the monoclonal antibodies 2F5 and 3C3 that recognize P-450 K. SDS-polyacrylamide gel electrophoresis revealed that the translated protein product exhibits the same relative electrophoretic mobility as P-450 K. The N-terminal amino acid sequence was determined to be MDLVVVLGL-LS-LLLLSL- for P-450 K immunopurified from rabbit kidney using the monoclonal antibody 2F5. This sequence agrees with the predicted amino acid sequence derived from the P-450 PBc2 cDNA. These results indicate that P-450 K or a protein closely related to P-450 K is encoded by the plasmid pP-450 PBc2.
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PMID:Evidence that rabbit cytochrome P-450 K is encoded by the plasmid pP-450 PBc2. 380 Oct 23

The specific transcription of a cloned Drosophila melanogaster tRNAVal4 gene and a tRNASer7 gene by extracts from a homologous embryonic cell line showed lag periods of about 30 min before maximum rates were reached. This lag appeared to represent the time to form an active transcription complex. Thus, when extracts were incubated with template DNA for 30 min at 22 degrees C and stored in the cold, the subsequent transcription rate was linear with time and without a lag. After ultracentrifugation of a preincubated reaction mixture on a sucrose step gradient consisting of 20, 30, 40, and 60% shelves, about 40% of the transcription activity in the extract was found in the 40% shelf. This fraction formed almost exclusively RNA I, the unprocessed tRNA gene transcript, and transcription required only addition of ribonucleoside triphosphates. The rate of formation of RNA by the 40% sucrose fraction was linear against time, with no lag, and linear with the quantity of fraction. The yield of activity isolated on the gradient was directly proportional to the quantity of cloned gene in the preincubation mixture. At a limiting concentration of the gene in the preincubation mixture, the turnover number of the isolated complex was approximately 50 transcripts/gene/h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of fractions containing the complex still showed many bands, although the complex activity was greatly purified compared to the extract. From the sedimentation behavior of the isolated active transcription complex and from its stability and transcriptional properties, we conclude that the 40% sucrose fraction contains an active transcription complex containing a cloned tRNA gene, RNA polymerase III, and the accessory protein factors required for transcription.
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PMID:Partial purification of stable transcription complexes with cloned tRNA genes of Drosophila melanogaster. 392 73

Two-dimensional gel electrophoresis (IEF and SDS-PAGE) was used to examine virion polypeptide changes associated with switch-on of transcriptase function in reovirus. Results reveal that switch-on is correlated with altered electrophoretic behavior of a specific minor polypeptide (delta 1) which is present in intermediate subviral particles. A second finding is that each of the molecular weight classes of viral polypeptides exists as a series of subspecies with different isoelectric points. This suggests that extensive posttranslational modification of progeny viral polypeptides occurs during particle morphogenesis. These findings have important theoretical and practical implications.
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PMID:Switch-on of transcriptase function in reovirus: analysis of polypeptide changes using 2-D gels. 406 May 93

1. Rapidly labelled RNA from Escherichia coli K 12 was characterized by hybridization to denatured E. coli DNA on cellulose nitrate membrane filters. The experiments were designed to show that, if sufficient denatured DNA is offered in a single challenge, practically all the rapidly labelled RNA will hybridize. With the technique employed, 75-80% hybridization efficiency could be obtained as a maximum. Even if an excess of DNA sites were offered, this value could not be improved upon in any single challenge of rapidly labelled RNA with denatured E. coli DNA. 2. It was confirmed that the hybridization technique can separate the rapidly labelled RNA into two fractions. One of these (30% of the total) was efficiently hybridized with the low DNA/RNA ratio (10:1, w/w) used in tests. The other fraction (70% of the total) was hybridized to DNA at low efficiencies with the DNA/RNA ratio 10:1, and was hybridized progressively more effectively as the amount of denatured DNA was increased. A practical maximum of 80% hybridization of all the rapidly labelled RNA was first achieved at a DNA/RNA ratio 210:1 (+/-10:1). This fraction was fully representative of the rapidly labelled RNA with regard to kind and relative amount of materials hybridized. 3. In competition experiments, where additions were made of unlabelled RNA prepared from E. coli DNA, DNA-dependent RNA polymerase (EC 2.7.7.6) and nucleoside 5'-triphosphates, the rapidly labelled RNA fraction hybridized at a low (10:1) DNA/RNA ratio was shown to be competitive with a product from genes other than those responsible for ribosomal RNA synthesis and thus was presumably messenger RNA. At higher DNA/rapidly labelled RNA ratios (200:1), competition with added unlabelled E. coli ribosomal RNA (without messenger RNA contaminants) lowered the hybridization of the rapidly labelled RNA from its 80% maximum to 23%. This proportion of rapidly labelled RNA was not competitive with E. coli ribosomal RNA even when the latter was in large excess. The ribosomal RNA would also not compete with the 23% rapidly labelled RNA bound to DNA at low DNA/RNA ratios. It was thus demonstrated that the major part of E. coli rapidly labelled RNA (70%) is ribosomal RNA, presumably a precursor to the RNA in mature ribosomes. 4. These studies have shown that, when earlier workers used low DNA/RNA ratios (about 10:1) in the assay of messenger RNA in bacterial rapidly labelled RNA, a reasonable estimate of this fraction was achieved. Criticisms that individual messenger RNA species may be synthesized from single DNA sites in E. coli at rates that lead to low efficiencies of messenger RNA binding at low DNA/RNA ratios are refuted. In accordance with earlier results, estimations of the messenger RNA content of E. coli in both rapidly labelled and randomly labelled RNA show that this fraction is 1.8-1.9% of the total RNA. This shows that, if any messenger RNA of relatively long life exists in E. coli, it does not contribute a measurable weight to that of rapidly labelled messenger RNA.
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PMID:Characterization of rapidly labelled ribonucleic acid in Escherichia coli by deoxyribonucleic acid-ribonucleic acid hybridization. 488 72

Cardiac hypertrophy in spontaneously hypertensive rats is associated with increased nuclear RNA polymerase activity. In order to explore mechanisms facilitating the interaction of the enzyme with its endogenous template, we compared the structure of nuclear chromatin from myocytes of 20-week-old spontaneously hypertensive rats and normotensive Wistar-Kyoto controls. Enhanced RNA synthesis in hypertensive rats was accompanied by increased susceptibility to digestion by deoxyribonuclease I. Nick translation of nuclei also resulted in higher nucleotide incorporation in hypertensive rats. Salt-extraction abolished the differences in deoxyribonuclease I sensitivity between the two animal groups. Reconstitution with either 0.35 M NaCl-extract or high mobility group (HMG) non-histone proteins restored digestion susceptibility but did not equalize SHR and WKY cells. SDS-polyacrylamide gel electrophoresis of 0.35 M NaCl-extracts and supernatants from deoxyribonuclease I digestion revealed the presence of HMG proteins which were preferentially released in hypertensive rats. There was a small but statistically significant increase in nuclear HMG protein content in hypertensive rats (0.12 +/- 0.02 mg/mg DNA vs. 0.09 +/- 0.02 mg/mg DNA in Wistar-Kyotos, P less than 0.05) but no difference in their electrophoretic appearance. These results indicate that chromatin structure is altered in the hypertrophied myocardium with resultant increase in deoxyribonuclease I susceptibility. This increase appears to be partly dependent on the high-mobility group non-histone proteins.
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PMID:Enhanced myocardial RNA synthesis in spontaneously hypertensive rats possible role of high-mobility group non-histone proteins. 617 42


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