DNA-dependent RNA synthesis in nuclear chromatin of fixed cells: relationship between dye-binding properties, nuclear protein content and RNA polymerase activity

Fixed mouse kidney epithelial cells have been examined for their capacity to synthesize RNA with their own RNA polymerases when supplied with ribonucleoside triphosphates. The endogenous polymerase activity of chromatin in fixed cells is clearly related to changes in the size and protein content of the nucleus. Cells with small nuclei which do not incorporate ³H-uridine in vivo show very little RNA polymerase activity at the ionic strength of the standard assay procedure. This activity can be enhanced by increasing the ionic strength of the assay medium. Changes in RNA polymerase activity also appear to be related to changes in the ability of chromatin to bind acridine orange (AO).     

RNA-dependent RNA polymerase activity in coronavirus- infected cells

An enzymatic activity which incorporates [3H]UMP into acid-precipitable material in the presence of endogenous template was found in the cytoplasm of porcine cells infected with the transmissible gastroenteritis virus of swine. This activity was not found in uninfected control cells, nor was it found in purified virus. The activity was associated with the mitochondrial fraction of infected cells, suggesting that the enzyme is membrane bound. The activity required the presence of all three ribonucleoside triphosphates in addition to [3H]UTP, and it was not inhibited by actinomycin D. The heated product was digested by RNase but not by DNase. Mg2+ was required for enzymatic activity, and its optimal concentration was approximately 5 mM. The size of the in vitro products was compared by electrophoresis with that of in vivo-synthesized virus-specified RNA to confirm the viral specificity of the polymerase activity. Virus-specified RNA from infected cells consisted of 10 species of single-stranded, polyadenylated RNA with molecular weights of 6.8 X 10(6), 6.2 X 10(6), 3.15 X 10(6), 1.40 X 10(6), 1.05 X 10(6), 0.94 X 10(6), 0.66 X 10(6), 0.39 X 10(6), 0.34 X 10(6), and 0.24 X 10(6). In vitro synthesized RNA consisted of a high-molecular-weight species, of apparently higher molecular weight than genomic RNA, and two single-stranded species that electrophoretically comigrated with the species of 1.40 X 10(6) and 0.66 X 10(6) molecular weight made in vivo.     

Alterations in myocardial RNA synthesis and RNA polymerase activity during normal growth

The effect of normal growth (hypertrophy) on myocardial nuclear activity was investigated using male Wistar rats at 21, 50, and 100 days of age. Cardiac mass increased sevenfold during this age range. The concentration of RNA (mg X g-1) was the highest at 21 days and decreased 48% by 50 days of age and 68% after 100 days of development. RNA synthesis, corrected for alterations in the specific activity of the cytoplasmic nucleotide pool, was the highest at 21 days of age. After 50 days of growth, uridine incorporation was decreased fivefold. With continual growth (100 days), RNA synthesis was still reduced compared with the 21-day animals. RNA polymerase activity in myocyte nuclei showed little change in activity from 21 to 100 days of age. However, in the nonmyocyte fraction, RNA polymerase decreased threefold after 50 days of development. Collectively, these data suggest that the large decrease in myocardial RNA synthesis cannot be accounted for by a change in nuclear RNA polymerase activity and that an alteration in chromatin template capacity may be involved during this form of cardiac growth.     

Effect of phytohaemagglutinin on the nuclear RNA polymerase activity of human lymphocytes

The DNA-dependent RNA polymerase activity of isolated nuclei from human peripheral blood has been shown to increase following stimulation with phytohaemagglutinin (PHA). Using the toxin α-amanitin it has been possible to demonstrate that within 4 h of the addition of PHA there is a two-fold increase in the amanitin-resistant polymerase activity (polymerase A) with little increase in the sensitive polymerase activity (polymerase B). 24 h following PHA stimulation the amanitin-resistant activity is stimulated 4–5 fold and the amanitin-sensitive activity less than two-fold. The susceptibility of this increased amanitin-resistant activity to low doses of actinomycin D both in vivo and in vitro indicates that the amanitin-resistant enzyme is mainly engaged in ribosomal RNA precursor synthesis. These changes in DNA-dependent RNA polymerase activity closely correspond to the observed changes in ribosomal and non-ribosomal RNA synthesis following lymphocyte stimulation.The increased polymerase A activity is diminished by a 1 h incubation of the cells with cycloheximide added 24 h after PHA whereas polymerase B activity remains unaffected. This indicates that the polymerase A activity observed after transformation is dependent on continuing protein synthesis.In our incubation conditions the polymerase activity observed in isolated nuclei appeared to be almost wholly attributable to elongation of nascent RNA molecules attached to the endogenous DNA template.     

The insensitivity of mushroom nuclear RNA polymerase activity to inhibiton by amatoxins

Nuclear fractions isolated from Amanita phalloides, Amanita muscaria and Agaricus bisporus were subjected to in vitro RNA synthesis assays in the presence of various concentrations of amatoxins. The mushroom nuclei were highly insensitive to inhibition by amatoxin when compared to assays of nuclear fractions isolated from the Oömycete fungus, Achlya ambisexualis and from rabbit brain.Abbreviations DTT dithiothreitol - PMSF phenylmethyl sulfonyl fluoride - MES 2[N-morpholino] ethane sulfonic acid Paper no. 1-78     

RNA polymerase activity in bovine spermatozoa

Washed mature spermatozoa from bulls incorporate ribonucleoside triphosphates into RNA using an endogenous template. Maximum incorporation was observed at 31 degrees C in the presence of MgCl2, all four ribonucleoside triphosphates, beta-mercaptoethanol, and glycine sodium hydroxide buffer at pH 9.0. The amount of synthesis was linearly dependent upon the concentration of spermatozoa and continued for at least 4 h. Digestion studies revealed the RNA to be present in a protected (intracellular?) location in the spermatozoa. The RNA synthesis was inhibited by ethidium bromide, rifampicin, acriflavine, actinomycin D, and caffeine, but not by alpha-amanitine or rifamycin SV. Fractionation of the spermatozoa by sonication and separation of the heads and tails by centrifugation through a discontinuous gradient revealed that more than half of the total RNA polymerase activity was associated with the tail fraction.     

Specificity and nature of the rapid steroid-stimulated increase in Sertoli cell nuclear RNA polymerase activity

This report explores the ability of various steroids to rapidly stimulate Sertoli cell RNA polymerase II activity and to compete with [3H]-androgens for nuclear and cytosol binding sites. Nuclear RNA polymerase II activity was significantly stimulated by a 1 nM concentration of the androgenic compounds testosterone, dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one). R1881 (methyltrienolone) and 5 alpha, 17 beta-diol and also by the potent progestins 6 alpha methylprogesterone and R5020 (17,21-dimethyl-19-nor-4-pregna-3,20-dione). Progesterone, 17 alpha-hydroxyprogesterone, estradiol, androsterone, and 5 alpha-androstan-3 beta, 17 beta-diol were ineffective at 1 nM. Cytosol binding and nuclear accumulation of [3H]-androgen was effectively reduced by 100 fold molar excess of those androgens and progestins which stimulated RNA polymerase II activity. These data suggest that androgens and progestins bind to at least some of the same proteins in the Sertoli cell and may elicit the rapid stimulation of RNA polymerase II activity via a common mechanism. Agarose gel electrophoresis of the nuclear RNA synthesized as a result of exposure to testosterone indicated that is was heterodisperse and in part polyadenylated. Electrophoresis of the poly A+-RNA demonstrated that testosterone administration increased the incorporation of [3H]-UTP into RNA that was larger than 28 S.     

Prostaglandin D2 lowers nuclear DNA polymerase activity in cultured mastocytoma cells

Prostaglandin D2 strongly inhibited growth of cultured mastocytoma P-815, 2-E-6 cells, which were established and cloned from mouse mast tumor cells. The inhibition was dose-dependent (IC50=2.09 x 10-5 M). Prostaglandin D2 also inhibited the DNA synthesizing activity of the cells dose-dependently. We next measured the activities of endogenous DNA polymerases extracted from untreated and prostaglandin D2-treated cells. Prostaglandin D2-treated cells were the same suggesting there was no gross change in the size of the enzyme. Prostaglandin D2 pretreatment of the cells reduced endogenous DNA polymerase beta activity to 68% of the control value; the sedimentation coefficients of the enzymes from treated and untreated cells were both 3.5 S. Interestingly, prostaglandin D2 had no direct inhibitory effect on the activity of either DNA polymerase alpha or beta. Our results indicate that the activities of DNA polymerase alpha and beta are lower in prostaglandin D2-treated mastocytoma cells. This finding accounts for the lower level of DNA synthesis in these cells.     

A new class of small nuclear RNA molecules synthesized by a type I RNA polymerase in HeLa cells

A new class of previously undetected small RNA molecules with a range of discrete sizes between 6S and 10S has been identified in HeLa cell nuclei. They differ in size and location from the previously described small nuclear RNA species (snRNA). These RNA molecules were initially found by selective RNA labeling in vitro in isolated nuclei. The in vitro products migrate in gel electrophoresis in the region from 6–10S with predominant components between 8S and 10S. They are labeled in the presence of very high concentrations of α-amanitin (150–400 μg/ml), suggesting they are synthesized by a type I polymerase. Unlike the major polymerase I product, ribosomal precursor RNA, however, these molecules are found in the nucleoplasm and their labeling is not affected by pretreatment of cells with low concentrations of actinomycin D (0.04 μg/ml). Their formation by a presumptive polymerase I type of enzyme is the basis of their tentative designation as small nuclear polymerase I (snPI) RNAs.The snPI RNA molecules appear to be associated with chromatin and the nuclear matrix. They can be selectively eluted from nuclei leaving most of hnRNA behind. This association is used as the basis of fractionation procedures which separate these molecules from hnRNA and permit the demonstration of the synthesis of at least the most predominant of these RNA molecules in vivo. w