Cyclic GMP and lymphocyte proliferation: effects on DNA-dependent RNA polymerase I and II activities.

Cyclic GMP (guanosine 3′:5′-cyclic monophosphate) accumulation and Ca²⁺ influx have been correlated with early nuclear events associated with increases in RNA polymerase I activity and decreases in RNA polymerase II activity in phytohemagglutinin-stimulated lymphocytes. In the present study we demonstrate that cyclic GMP in the presence of Ca²⁺ stimulates RNA polymerase I activity in lymphocyte nuclei isolated from both non-stimulated and phytohemagglutinin-stimulated lymphocytes. In addition, cyclic GMP in the presence of Ca²⁺ decreases RNA polymerase II activity in both non-stimulated and phytohemagglutinin-stimulated lymphocyte nuclei. These observations suggest that cyclic GMP and Ca²⁺ may represent components of a plasma membrane-to-nucleus “mitogen signal sequence”.     

Comparative studies on polyguanylate polymerase and polyadenylate polymerase activities in the DNA-dependent RNA polymerase I fraction from cauliflower.

The properties of poly(G) polymerase and poly(A) polymerase activities in the DNA-dependent RNA polymerase [nucleosidetriphosphate: RNA nucleotidyltransferase EC 2.7.7.6] I fraction from cauliflower (Brassica oleracea var. botrytis) were comparatively investigated. The pH optimum, the effect of ionic strength, the effect of substrate concentration on the rate of synthesis, the effect of divalent metal ion concentration, and the time course of synthesis at different temperatures were all different for the three polymerase activities. The enzyme fraction preferentially utilized denatured DNA. Synthetic poly(C) and poly(U) were more effectively utillized for the synthesis of polyguanylate and polyadenylate, respectively. Further, it was found that poly(G) and poly(A) formed in vitro by the enzyme fraction had chain length of 25-28 and 84-89 nucleotides, respectively, and that poly (adenylate-gluanylate) chain was hardly formed when ATP and GTP were added together as substrates in the same reaction medium.     

DNA-dependent RNA polymerase I from human placental nuclei

This paper describes a large-scale method for solubilisation and purification of DNA-dependent RNA-Polymerase I from mature human placenta. The solubilisation method involves homogenization of the whole human placenta, isolation of cell nuclei, sonication of separated nuclei at high ionic strength and ammonium sulfate precipitation. The purification method consists of chromatography of RNA-Polymerase I activity on DEAE-Sephadex A-25 and Phosphocellulose P-11, and glycerol-density gradient centrifugation. In result, RNA-Polymerase I of human placenta nuclei has been shown to be completely resistant to alpha-amanitin. Besides dependence of RNA-Polymerase I on different Mg2+ and Mn2+ concentrations, glycerol concentration and ionic strength was studied. Using our results, an optimal RNA-Polymerase I assay mixture was developed. The subunit composition of RNA-Polymerase I was investigated by dodecylsulfate-gel electrophoresis. The RNA-Polymerase I molecule of human placenta consists of 13-14 polypeptides.     

Purification of DNA-dependent RNA polymerase II from Saccharomyces cerevisiae

A rapid procedure for the purification of RNA polymerase II from Saccharomyces cerevisiae is described. Total RNA polymerase activity was solubilized from whole cells by sonication in 0.32 M (NH4)2SO4 and RNA polymerase II purified by polyethylenimine fractionation, ammonium sulfate precipitation, and chromatography on DEAE-cellulose, DEAE-Sephadex, and phosphocellulose. The procedure may be completed in 2.5 days and the resultant enzyme is judged to be greater than 90% pure.     

DNA-dependent RNA polymerase II from nuclei of suspension-cultured tobacco cells

From isolated nuclei of suspension cultured cells of Nicotiana tabacum. DNA-dependent RNA polymerase II (E.C. 2.7.76) has been purified to homogeneity as evidenced by polyacrylamidegel electrophoresis under non-denaturing conditions. The purified enzyme had a specific activity of more than 15 nmol min^-1·mg^-1 with denatured calf thymus DNA as template. Sodium-dodecyl-sulfate gel electrophoresis and protein highperformance liquid chromatography revealed a subunit composition of four proteins with molecular weights of 165 000, 135 000, 35 000 and 25 000 and with a stoichiometry of 1:1:2:2. The RNA polymerase did not exhibit any detectable proteinkinase activity. The 25 000 subunit binds ADP in a molar ratio of 1:1; it could not be decided whether this subunit has an ATPase activity or is merely an acceptor of ADP.Abbreviations HPLC high-performance liquid chromatography - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate This contribution is dedicated to Professor Fritz Cramer on the occasion of his 60th birthday     

Isolation and characterization of DNA-dependent RNA polymerase I of Tetrahymena pyriformis

A procedure for the separation and purification of DNA-dependent RNA polymerases [EC 2.7.7.6] from macronuclei of Tetrahymena pyriformis is described. We have used it to isolate and characterize the class I enzyme. RNA polymerase I was identified by its resistance against alpha-amanitin and its location in nucleoli. The purified enzyme consists of at least 12 major subunits with approximate molecular weights of 180,000, 118,000, 37,500, 36,000, 29,000, 27,500, 20,000, 18,500, 15,600, 14,500, 13,500, and 12,600. Chromatography on DEAE-Sephadex separated two forms of RNA polymerase I which differed in the presence of an additional polypeptide of 25 kDa. Independently of this polypeptide, the enzyme was found to segregate on DNA cellulose into a binding and a non-binding fraction. This type of heterogeneity was found to be unrelated to differences in molar ratios or molecular weights of the enzyme subunits. The catalytic properties of all enzyme subfractions were very similar and complied with the general characteristics of RNA polymerase I [cf. Roeder, R.G. (1976) in RNA Polymerase (Losick, R. & Chamberlin, M., eds.) pp. 285-329, Cold Spring Harbor Publ. Co., New York].     

DNA-dependent poly (G) synthesizing activities in DNA-dependent RNA polymerase fractions from cauliflower inflorescence

DNA-dependent RNA polymerase fractions isolated from cauliflower inflorescence were found to be accompanied with poly (A) and poly (G) synthesizing activities in the presence of denatured calf-thymus DNA. The dialysis against Tris-HCl buffer in the absence of Mg⁺⁺, dithiothreitol (DTT) and EDTA resulted in a great increase of poly (A) polymerase activity while RNA polymerase and poly (G) synthesizing activities decreased.     

Localization of DNA-dependent RNA polymerase activities in fixed human fibroblasts by autoradiography

The sites of activity of DNA-dependent RNA polymerases may be demonstrated in the cell nucleus by incubating ethanol/acetone fixed cells with ATP, GTP, CTP and ³H-UTP. The amount of ³H-UMP bound to the chromatin in an RNase-digestible, acid-insoluble form is determined by autoradiography. The activity of one polymerase lies in the nucleolus and is inhibited by 0.05–0.5 μg/ml actinomycin D, another is present in the nucleoplasm and is blocked by α-amanitin. A large increase in α-amanitin-sensitive polymerase activity may be stimulated by performing the enzyme assay in the presence of a high concentration of ammonium sulphate.     

DNA-dependent RNA polymerase of thermoacidophilic archaebacteria

Among 979 non-glycerol growers of the yeast Schizosaccharomyces pombe, 40 strains were found to be deficient in the mitochondrial ATPase activity. Three of them exhibited an alteration in either the alpha or beta subunits of the F1ATPase. The alpha subunit was not immunodetected in the A23/13 mutant. The beta subunit was not immuno-detected in the B59/1 mutant. The existence of these two mutants shows that the alpha and beta subunits can be present independently of each other in the inner mitochondrial membrane. The beta subunit of the mutant F25/28 had a slower electrophoretic mobility than that of the wild-type beta subunit. This phenotype indicates abnormal processing or specific modification of the beta subunit. All mutants showed reduced activities of the NADH-cytochrome c reductase and of the cytochrome oxidase and a decreased synthesis of cytochrome aa3 and cytochrome b. This pleiotropic phenotype appears to result from specific modifications in the mitochondrial protein synthesis. The mitochondrial synthesis of four polypeptides (three cytochrome oxidase and one cytochrome b subunits) was markedly decreased or absent while three new polypeptides (Mr = 54000, 20000 and 15000) were detected in all the mutants analysed. This observation suggests that a functional F1ATPase is necessary for the correct synthesis and/or assembly of the mitochondrially made components of the cytochrome oxidase and cytochrome b complexes.