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)

DNA-dependent RNA polymerase was solubilized from nuclei of Ehrlich ascites carcinoma (EAC) cells by sonic disruption in the presence of 0.3 M (NH4)2 SO4, and the multiple RNA polymerases were separated by chromatography on DEAE-Sephadex A-25. Elution with a nine-step gradient of (NH4)2 SO4 yielded five peaks of activity designated RNA polymerases Ia, Ib, IIa, IIb, and III, of which IIb was the most prominent. Linear-gradient elusion also yielded five peaks of the same designation, but Ia and Ib, as well as IIa and IIb, were not well separated. IIa and IIb were inhibited completely by 0.1 mug alpha-amanitin/ml, whereas the other forms were not. EAC RNA polymerases Ia, Ib, IIa, and IIb possessed Mg2+ ion, Mn2+ ion, and (NH4)2 SO4 optima, molecular weights, and thermal sensitivities similar to those reported for other mammalian DNA-dependent RNA polymerases. As measured by relative ribonucleoside monophosphate incorporation, with native calf thymus DNA template, EAC RNA polymerases Ia and Ib synthesized ribosomal RNA-like products, whereas forms IIa, IIb, and the parent enzyme mixture synthesized compounds that were more similar to DNA. No species specificity was found for DNA templates, and denatured DNA was consistently preferred to the native template by RNA polymerases IIa and IIb; the two kinds of template were about equally efficient for RNA polymerases Ia and Ib. Although EAC RNA polymerases Ia, IIa, and IIb were inhibited by daunomycin, form IIa was preferentially affected. 3',5'-Cyclic AMP, 3',5'-Cyclic GMP, and gibberellic acid, implicated as RNA polymerase regulators in other systems, were generally ineffective. The levels of nuclear RNA polymerase activities, per mg DNA, of 3 mouse ascites tumors (EAC, 6C3HED lymphosarcoma, and TA3 adenocarcinoma) were compared with those from 3 normal mouse tissues (kidney, liver, and spleen). On the average, the tumor cell nuclei contained (per mg of DNA) 8.9, 1.5, 2.7, 20.0, and 3.8 times the activities of RNA polymerases Ia, Ib, IIa, IIb, and III, respectively, as the normal cells, but the difference was significantly only for IIb.
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PMID:DNA-dependent RNA polymerases of Ehrlich carcinoma, other murine ascites tumors, and murine normal tissues. 117 42

An improved method for the purification of the alpha-amanitin-sensitive deoxyribonucleic acid dependent ribonucleic acid polymerase [ribonucleosidetriphosphate:RNA-nucleotidyltransferase, EC 2.7.7.6-A1 (RNA polymerase II or RNA polymerase B) from wheat germ is presented. The method involves homogenization of wheat germ in a buffer of moderate ionic strength, precipitation of RNA polymerase with Polymin P (a polyethylenimine), elution of RNA polymerase from the Polymin P precipitate, ammonium sulfate precipitation, and chromatography on DEAE-cellulose and phosphocellulose. RNA polymerase II is purified over 4000-fold with a 60% recovery, resulting in a yield of 25-30 mg of RNA polymerase from 1 kg of starting material.
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PMID:A new method for the large-scale purification of wheat germ DNA-dependent RNA polymerase II. 118 7

Guinea-pig and mouse liver chromatin responds to the partial hepatectomy by an increase in binding of a basic dye acridine orange (AO) and by a decrease of its stability to heat in thermal denaturation test in situ. Degree of the changes in AO chromatin binding is identical in the cells of different ploidy and proportional to their DNA content. Treatment of the preparations by 0.6 M NaCl solutions under conditions bringing about the selective removal of histone H1 from the cells produces in vitro changes in DNA properties taking place in cells in vivo in the course of their activation. The treatment of cells with 0.35 M NaCl solution results in the disappearance of changes occurring in the chromatin of activated cells whereas the properties of control cells remain unchanged. The data obtained are interpreted as a result of the removal of some non-histone regulatory proteins from the chromatin of activated cells that is accompanied by changes in the character of DNA-histone interaction. At the time of maximum increase of AO binding a significant intensification of endogenous RNA polymerase activity was found, the incorporation of [3H] UTP in the nucleolus being higher than that in the extranucleolar part of the nucleus. High ionic strength in the incubation medium (0.4 M (NH4)2SO4) results in drastic increase of radioactive label in the nucleus and in the disappearance of differences between activated and non-activated chromatin. It is concluded that the intensification of RNA synthesis under the influence of proliferative stimulus is more likely dependent on the additional opening of DNA-matrix than on the direct activation of the enzyme.
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PMID:[Early changes in liver chromatin in response to partial hepatectomy]. 121 68

The limiting factor in RNA synthesis by isolated kidney nuclei is RNA nucleotidyltransferase at high salt concentrations but at low salt concentrations template availability becomes limiting. alpha-Amanitin inhibits 85% of the activity at high salt concentrations but only 20-50% of the activity at low salt concentrations. Exogenous DNA is utilized at low salt concentrations [up to 0-1M (NH4)2SO4] but not at high salt concentrations. The effect of increasing salt concentration is mainly to cause an increase in the length of chains synthesized. Initiation rates are not increased by high salt concentrations. The apparent Km for UTP is 8-10 muM at high salt concentrations, indicating that assays performed at low UTP concentrations are likely to give inaccurate results. The activation energy for the reaction at low salt concentration is less than that for the reaction at high salt concentration. The RNA synthesizing capacity of kidney nuclei is dependent on the method of isolation, and preparation by a modification of the Chauveau method (Chauveau et al. 1956) yields the most active nuclei.
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PMID:Characterization of the RNA synthesizing activity of isolated kidney nuclei. 122 83

Spermine stimulates activities of higherly purified rat liver nuclear RNA olymerases I, II and III 3 to 4 fold. Inclusion of (NH4)2SO4 at concentrations required for maximal enzyme activities does not significantly enhance the degree of stimulation of polymerase activities by spermine, but maintains the stimulatory levels of enzymes over a broader range of spermine concentrations. The stimulatory effect of spermine at a concentration of 1 mM is a useful method for the elevation of activities of all RNA polymerases and thus provides a means to measure these enzymes when extracted from small quantities of tissues or cells. Based on the differential stimulation of the polymerases by spermine, a higher concentration of spermine (5 mM) can be selected to inhibit RNA polymerase I specifically.
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PMID:Stimulation of RNA polymerases I, II and III from rat liver by spermine, and specific inhibition of RNA polymerase I by higher spermine concentrations. 124 14

Poly (A) polymerase activity from cytoplasm and nuclei of 12-16-day-old mouse embryos has been partially purified by (NH4)2SO4 fractionation, DEAE-cellulose, phosphocellulose and tRNA-Sepharose affinity chromatography, and their properties have been compared. The nuclear and cytoplasmic enzymes exhibit similar chromatographic elution profiles, and similar biochemical and physical properties. Poly(A) polymerase has an absolute requirement for a divalent cation, ATP and an oligo- or polyribonucleotide primer. With tRNA, the divalent salt concentrations for optimum enzyme activity are 1 mM MnCl2 or 10 mM MgCl2. The enzyme activity with MnCl2 is 10-15-fold higher than that with MgCl2. The molecular weight of the native enzyme is about 65 000 and its sedimentation coefficient is around 4.5 S. The average chain length synthesized by the enzyme is between 10 and 13 nucleotides. The inhibitors of RNA polymerase do not affect poly (A) polymerase activity; however, some synthetic rifamycin SV derivatives are potent inhibitors of this enzyme.
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PMID:Polyadenylate polymerase from cytoplasm and nuclei of N.I.H.-Swiss mouse embryos. 124 20

The class III DNA dependent RNA polymerases (nucleoside triphosphate:RNA nucleotidyltransferase EC 2.7.7.6 from HeLa cells have been solubilized and characterized as to function and properties. Two chromatographically distinct forms of enzyme III, designated polymerases IIIA and IIIB, can be resolved when cell extracts are chromatographed on DEAE-Sephadex columns. Enzymes IIIA and IIIB exhibit nearly identical catalytic properties such as divalent cation stimulation, broad biphasic ammonium sulfate optima, and characteristic alpha-amanitin sensitivities which clearly distinguish them from the homologous enzymes, forms I and II. Polymerases IIIA and IIIB are both primarily localized in the nucleus (greater than 60%). The most notable characteristic of the class III enzymes is a unique sensitivity to inhibition by alpha-amanitin (50% inhibition at 15 mug/ml). HeLa cell enzyme I is not inhibited by the mushroom toxin even at very high concentrations (greater than 400 mug/ml), while HeLa cell polymerase II is inhibited by very low concentrations of amanitin (50% inhibition at 0.003 mug/ml). The three major classes of enzyme (I, II, III) exhibit characteristic sensitivities to alpha-amanitin whether assayed in nuclei, crude homogenates, or in a chromatographically purified state. Using a nuclear in vitro RNA synthesizing system to investigate the alpha-amanitin sensitivities of the synthesis of tRNA precursor (4.5S pre-tRNA) and 5S ribosomal RNA, it was found that the synthesis of these RNA species was inhibited 50% at 15 mug/ml of alpha-amanitin. The alpha-amanitin inhibition curves for the synthesis of pre-tRNA-5S ribosomal RNA in nuclei and the alpha-amanitin titration curves for the partially purified class III enzymes (IIIA and IIIB) are identical. These data, therefore, show that the in vivo functional role of the class III RNA polymerases (IIIA-IIIB) is the transcription of the genes coding for transfer RNA and 5S ribosomal RNA.
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PMID:HeLa cell deoxyribonucleic acid dependent RNA polymerases: function and properties of the class III enzymes. 125 52

The mouse compared with the rat, is more resistent to the acute toxic action of aflatoxin B1 and is refractory to its hepatocarcinogenic properties. Aflatoxin B1 inhibits DNA synthesis more strongly than RNA synthesis in the rat, and both nucleic acid syntheses more strongly in rat than in the mouse. Mouse hepatic microsomes, like those of the rat, are capable of metabolizing aflatoxin B1 in vitro in the presence of NADPH, to an active form which binds to DNA both in solution and in intact nuclei and also inhibits nuclear RNA synthesis. Non NADPH-dependent binding of aflatoxin B1 to nuclei is not effective in inhibiting RNA polymerase and is largely removed by washing with lipid solvents. Mouse nuclear RNA polymerases particularly Mn 2+ (NH4)2SO4 primed acitivity are more resistant to inhibition in vitro by activated aflatoxin B1 than are the corresponding enzyme activities in rat liver nuclei. This would appear to be due to the bound aflatoxin B1 being less efficient in the case of the mouse nucleus, in inhibiting RNA synthesis. Mouse liver slices exhibit a much lesser degree of inhibition of RNA synthesis by aflatoxin B1 than do rat liver slices. Accompanying this is a lower level of binding of aflatoxin B1 to subcellular particulate fractions in the mouse liver slice compared to the rat, this disparity being most marked in the case of the nuclear fraction. The suggestion is made that the resistance of RNA synthesis in the mouse liver, to aflatoxin B1, and perhaps also resistance to its toxicity, is dependent, not on a lower capacity to activate the toxin, but (a) on a less efficient inhibition of RNA synthesis by nuclear bound toxin, and (b) a detoxifying mechanism at least partially situated in the cytosol fraction.
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PMID:Some studies of the effects of aflatoxin B1 in vivo and in vitro on nucleic acid synthesis in rat and mouse. 126 46

The major cold shock protein from Bacillus subtilis (CspB) was overexpressed using the bacteriophage T7 RNA polymerase/promoter system and purified to apparent homogeneity from recombinant Escherichia coli cells. CspB was crystallized in two different forms using vapor diffusion methods. The first crystal form obtained with ammonium sulfate as precipitant belongs to the trigonal crystal system, space group P3(1)21 (P3(2)21) with unit cell dimensions a = b = 59.1 A and c = 46.4 A. The second crystal form is tetragonal, space group P4(1)2(1)2 (P4(3)2(1)2) with unit cell dimensions a = b = 56.9 A and c = 53.0 A. These crystals grow with polyethylene glycol 4000 as precipitant.
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PMID:Overproduction, crystallization, and preliminary X-ray diffraction studies of the major cold shock protein from Bacillus subtilis, CspB. 140 60

T3 and T7 phages package homologous DNA more efficiently than heterologous DNA and recombinant plasmids carrying DNA sequences necessary for DNA packaging (pac sequence). The pac sequence contains a promoter for phage RNA polymerase and transcription from the promoter is necessary for DNA packaging. T3 and T7 RNA polymerases are stringently specific for their own promoters. To examine the relationship between DNA packaging and transcription, we constructed a cleared in vitro system for packaging T3 or T7 DNA containing an ammonium sulfate fractionate of a high-speed supernatant of phage-infected cells. In the system, DNA packaging required GTP and was inhibited by the 3'-deoxy analog of GTP, ATP, or CTP. The DNA packaging activity paralleled the transcriptional activity, assayed by incorporation of [32P]UTP into acid-insoluble material. In the system, homologous DNA was packaged more efficiently than heterologous DNA, but heterologous DNA was packaged as efficiently as homologous DNA by the addition of heterologous phage RNA polymerase, demonstrating that the transcriptional specificity determines the DNA packaging specificity of T3 and T7.
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PMID:Transcription dependence of DNA packaging of bacteriophages T3 and T7. 141 5


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