Comparison of the effects of polyamines on the activities of RNA polymerases from murine normal tissues and transplantable tumors

Nuclear DNA-dependent RNA polymerases I, II and III were purified from kidney, liver and spleen from Swiss mice (Mus musculis) and from seven transplantable murine tumors. In the presence of the optimal concentration of (NH4)2SO4 for each polymerase, 1-8 mM spermidine or spermine stimulated most polymerases several fold, and generally, enzyme I was stimulated more than either enzyme II or III. Spermine was more efficacious than spermidine as a stimulant of polymerase activity except for polymerase III from three tumors. Tumor polymerases I (or II) and the corresponding normal tissue enzymes responded similarly to the polyamines. Stimulation of a RNA polymerase by a polyamine could not be correlated with the growth rate of the tissues of polymerase origin or with the tissue's RNA polymerase or RNA synthetic activities.     

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”.     

Viral RNA synthesis and levels of DNA-dependent RNA polymerases during replication of adenovirus 2.

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin alpha-amanitin was used to determine the relative and absolute levels of RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication of from replication or from functional modifications of the RNA polymerases or from a combination of these effects.     

Viral RNA Synthesis and Levels of DNA-Dependent RNA Polymerases During Replication of Adenovirus 2

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin α-amanitin was used to determine the relative and absolute levels of RNA synthesis by RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was monitored by hybridization to viral DNA, and of viral 5.5S RNA, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication or from functional modifications of the RNA polymerases or from a combination of these effects.     

Zinc deficiency and the Euglena gracilis chromatin: formation of an alpha-amanitin-resistant RNA polymerase II

Both the single DNA-dependent RNA polymerase found in zinc-deficient (-Zn) Euglena gracilis and the RNA polymerase III from zinc-sufficient (+Zn) cells have been isolated by methods previously used to purify polymerases I and II [Falchuk, K. H., Mazus, B., Ulpino, L., & Vallee, B. L. (1976) Biochemistry 15, 4468; Falchuk, K. H., Mazus, B., Ulpino, L., & Vallee, B. L. (1977) Biochem. Biophys. Res. Commun. 74, 1206]. Like class II polymerases, the enzyme from -Zn organisms elutes from DNA-cellulose and phosphocellulose with 0.6 M NaCl and 0.35 M NH4Cl, respectively. It is inhibited by 8-hydroxyquinoline, 8-hydroxyquinoline-5-sulfonic acid, alpha,alpha'-bipyridyl, dipicolinic acid, and 1,10-phenanthroline (OP); 4,7-phenanthroline, the nonchelating analogue, does not inhibit. The pKI(OP) of this enzyme is identical with that of polymerase II but distinct from those of polymerases I and III. Elemental analysis confirms that zinc is the functional metal while copper, manganese, iron, and magnesium are absent. However, the -Zn enzyme is at least 4 orders of magnitude more resistant to alpha-amanitin (alpha-A) than the class II polymerase. Further, its response to alpha-A is unlike that of either polymerase I or polymerase III. Thus, -Zn cells contain a single, alpha-amanitin-resistant (alpha-Ar) RNA polymerase, whose behavior otherwise resembles that of the alpha-amanitin-sensitive polymerase II.     

Mouse Brain DNA-Dependent RNA Polymerases After Chronic Morphine Treatment

Abstract: Chronic morphine pellet implantation was found to decrease the specific activity of two forms of mouse brain RNA polymerase I and to alter the requirements of Mg²⁺ and Mn²⁺ for the activities of RNA polymerases II and III. DNA-dependent RNA polymerases were partially purified from small dense nuclei isolated from brains of naive and morphine tolerant-dependent mice, and three RNA polymerases were separated on a DEAE-Sephadex A-25 column. The three fractions, referred to as peak I, peak II, and peak III, were studied, characterized, and identified as being RNA polymerases I, II, and III, respectively. Chronic-morphine pellet implantation resulted in a lower specific activity of RNA polymerase I, but the specific activities of RNA polymerases II and III were not affected. This effect was prevented by preimplantation of a naloxone pellet and thus was narcotic-specific. Chronic morphine treatment lowered the concentration of Mg²⁺ required for optimal activity of RNA polymerase II and elevated the Mn²⁺-Mg²⁺ activity ratios of RNA polymerases II and III. A second DEAE-Sephadex A-25 column chromatography of the peak I RNA polymerase was carried out, revealing five component activity peaks. Two of these contained lower specific activities as a result of chronic morphine pelletimplantation. These specific changes in RNA polymerase function in morphine tolerance-dependence may be associated with the elevated chromatin template activities, altered chromatin phosphorylation, and elevated rates of cell-free translation that have been reported by others.     

Steroidogenic effect of atrial natriuretic factor in isolated mouse Leydig cells is mediated by cyclic GMP.

The effects of different atrial natriuretic peptides on cyclic GMP formation and steroidogenesis have been studied in Percoll-purified mouse Leydig cells. Rat atrial peptides rANP (rat atrial natriuretic peptide), rAP-I (rat atriopeptin I) and rAP-II (rat atriopeptin II), in the presence of a phosphodiesterase inhibitor, stimulated cyclic GMP formation in a concentration-dependent manner. In the presence of saturating concentrations of the peptides, a 400-600 fold stimulation of cyclic GMP accumulation was observed. Among the peptides, rAP-II appeared to be the most potent. ED50 values (concentration causing half-maximal effect) for rAP-II, rANP and rAP-I were 1 X 10(-9) M, 2 X 10(-9) M and 2 X 10(-8) M respectively. A parallel stimulation of cyclic GMP formation and testosterone production by the cells was observed after incubation of the cells with various concentrations of rAP-II. In the presence of a saturating concentration of rAP-II (2 X 10(-8) M), maximum stimulation of intracellular cyclic GMP content was obtained within 5 min of incubation. Testosterone production by mouse Leydig cells could be stimulated by 8-bromo cyclic GMP in a concentration-related manner. At a 10 mM concentration of the cyclic nucleotide, steroidogenesis was stimulated to a similar extent as that obtained with a saturating concentration of human chorionic gonadotrophin (5 ng/ml). On the basis of these results we conclude that cyclic GMP acts as a second messenger in atrial-peptide-stimulated steroidogenesis in mouse Leydig cells. The steroidogenic effect of atrial peptides appears to be species-specific, since none of these peptides stimulated testosterone production by purified Leydig cells of rats, though in these cells a 40-60-fold stimulation of cyclic GMP formation in response to each of the three peptides was observed. However, 8-bromo cyclic GMP could stimulate testosterone production in rat Leydig cells. Therefore we conclude that the lack of steroidogenic response in rat Leydig cells to atrial-natriuretic-factor-stimulation results from an insufficient formation of cyclic GMP in these cells. This species difference would appear to result from a lower guanylate cyclase activity in rat Leydig cells.     

Isolation and partial characterization of the multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in the fungus Podospora anserina

Three DNA-dependent RNA polymerases have been isolated and partially purified from the mycelium of the fungus Podospora anserina. Separated by DEAE-Sephadex chromatography, they have been designated RNA polymerases I, II, and III according to their order of elution. Their catalytic properties and alpha-amanitin sensitivity are in agreement with those of the homologous enzymes found in other eukaryotic organisms. The three enzymes exhibit rather sharp monophasic ammonium sulfate dependence with optima which are, respectively, 0.035 M, 0.050 M, and 0.075 M. Enzyme I has the largest Mn2+/Mg2+ activity ratio, shows a marked preference for native DNA, and is insensitive to alpha-amanitin. Enzyme III uses poly(dA-dT) in preference to native DNA as template and is only partially sensitive to alpha-amanitin. Enzyme II is sensitive to alpha-amanitin, but high concentrations of the toxin are required for inhibition compared to other eukaryotic class II enzymes. Three similar RNA polymerases with comparable levels of activity were found in the temperature-dependent VR strain when cellular incompatibility, leading to a rapid cessation of RNA synthesis, was induced.     

Effect of 3-methylcholanthrene administration on hepatic ribonucleic acid polymerase activities

The effect of the in vivo administration of 3-methylcholanthrene upon rat hepatic RNA polymerase activities was investigated. Aggregrate RNA polymerase activity assayed in liver nuclei was stimulated by 33% over control. Characterization of the individual RNA polymerase activities by virtue of their differential sensitivity to α-amanitin revealed that RNA polymerase I activity was maximally increased by 70% at approx. 16 h post-administration of the polycyclic hydrocarbon; RNA polymerase II activity was stimulated by 33%. The kinetics of RNA polymerases I and II stimulation differed in that the nucleolar enzyme's activity increased earlier and peaked later. RNA polymerase III activity was not significantly different from control. Phenobarbital, another inducer of the mixed function oxidases, had essentially no effect on the activity of hepatic RNA polymerases. Solubilization of the RNA polymerases followed by separation on diethylaminoethyl (DEAE)-Sephadex allowed for a comparison of the treated and control enzymatic activities using a common exogenous template. While no qualitative difference was evident, RNA polymerases I and II isolated from 3-methylcholanthrene-treated rats again were more active than control, indicating an effect of the polycyclic hydrocarbon at the level of the enzyme.     

Phosphorylation of yeast DNA-dependent RNA polymerases in vivo and in vitro. Isolation of enzymes and identification of phosphorylated subunits.

Yeast DNA-dependent RNA polymerases I, II, and III are phosphorylated in vivo. Yeast cells were grown continuously in 32Pi and the RNA polymerases were isolated by a new procedure which allows the simultaneous purification of these enzymes from small quantities (35 to 60 g) of cells. Each of the RNA polymerases was phosphorylated. The following phosphorylated polymerase polypeptides were identified: polymerase I subunits of 185,000, 44,000, 36,000, 24,000, and 20,000 daltons; a polymerase II subunit of 24,000 daltons; and polymerase III subunits of 24,000 and 20,000 daltons. The incorporated 32P was acid-stable but base-labile. Phosphoserine and phosphothreonine were identified after partial acid hydrolysis of purified [32P]polymerase I. A yeast protein kinase that co-purifies with polymerase I during part of the isolation procedure was partially purified and characterized. This protein kinase phosphorylates the subunits of the purified polymerases that are phosphorylated in vivo and, in addition, a polymerase I subunit of 48,000 daltons and a polymerase II subunit of 33,500 daltons. Phosphorylation of the purified enzymes with this protein kinase had no substantial effect on polymerase activity in simple assays using native yeast DNA as a template. Preincubation of purified polymerase I with acid or alkaline phosphatase also had no detectable effect on polymerase activity.     

Immunological relationships between Artemia RNA polymerases and between RNA polymerases II from different eukaryotic organisms

Summary Rabbit antibodies against Artemia RNA polymerase II have been raised and utilized to study the immunological relationships between the subunits from RNA polymerases I, II and III from this organism and RNA polymerase II from other eukaryotes. We describe here for the first time the subunit structure of Artemia RNA polymerases I and III. These enzymes have 9 and 13 subunits respectively. The anti-RNA polymerase II antibodies recognize two subunits of 19.4 and 18 kDa common to the three enzymes, and another subunit of 25.6 kDa common to RNA polymerases II and III. The antibodies against Artemia RNA polymerase II also react with the subunits of high molecular weight and with subunits of around 25 and 33 kDa of RNA polymerase II from other eukaryotes (Drosophila melanogaster, Chironomus thummi, triticum (wheat) and Rattus (rat)). This interspecies relatedness is a common feature of eukaryotic RNA polymerases.Abbreviations RNAp RNA polymerase - DPT diazophenylthioether - SDS sodium dodecylsulfate     

Comparison of binding and cyclic GMP accumulation by atrial natriuretic peptides in endothelial cells

Rat 125I-labeled atrial natriuretic factor (ANF (8-33)) was used to identify ANF receptors on cultured bovine aortic endothelial cells. Specific binding of 125I-ANF at 37 degrees C to confluent endothelial cells was saturable and of high affinity. Scatchard analysis of the equilibrium binding data indicated that endothelial cells contain a single class of binding sites with a Kd of 0.1 +/- 0.01 nM. This particular clone of endothelial cells had 16000 +/- 1300 receptors per cell. The order of potency for competing with 125I-ANF binding was human atrial natriuretic peptide (hANP) = atrial natriuretic factor (ANF (8-33)) greater than atriopeptin II greater than atriopeptin III greater than atriopeptin. The weakest competitor, atriopeptin I, had a K1 of 0.45 nM, which was only 6-fold higher than the K1 for hANP and ANF (8-33). ANF (8-33) and hANP in the presence of 0.5 mM isobutylmethyl-xanthine produced a 15-20-fold increase in cyclic GMP content at 10 pM and a maximal 500-fold elevation of cyclic GMP at 10 nM. The concentrations required to elicit a half-maximal increase in cyclic GMP for hANP, ANF (8-33), atriopeptin I, atriopeptin II and atriopeptin III were 0.30, 0.35, greater than 500, 4.0 and 5.0 nM, respectively. Although atriopeptin I acted as a partial agonist, it was unable to antagonize the effect of ANF (8-33) on cyclic GMP formation. These findings suggest that endothelial cells have multiple and functionally distinct ANF-binding sites.     

Multiple forms of DNA-dependent RNA polymerases in Xenopus laevis. Rapid purification and structural and immunological properties

DNA-dependent RNA polymerases I, II, and III (EC 2.7.7.6) were isolated from Xenopus laevis ovaries. The soluble enzymes were precipitated with polyethyleneimine and subjected to chromatography on heparin-Sepharose, DEAE-Sephadex, and phosphocellulose. RNA polymerase I was subjected to an additional chromatographic step on CM-Sephadex. The procedure required 40 h and produced purified RNA polymerase forms IA, IIA, and III in yields of 5 to 40%. The specific activities of RNA polymerases IIA and III (on native DNA) were comparable to those reported from other eukaryotic sources, whereas that of form IA was severalfold greater than the specific activities reported for other purified class I RNA polymerases. The complex subunit compositions of chromatographically purified RNA polymerases IA, IIA, and III were distinct when analyzed by polyacrylamide gradient gel electrophoresis under denaturing conditions, although all three classes contained polypeptides with Mr = 29,000, 23,000, and 19,000. Antibodies prepared against RNA polymerase III showed common antigenic determinants within the class I, II, and III enzymes. The sites responsible for the cross-reaction are located, at least in part, on the common 29,000-dalton polypeptide.     

Inhibition of isolated yeast and mycelial phase RNA polymerases of Histoplasma capsulatum by rifamycin derivatives

Various derivatives of rifamycin were shown to inhibit the RNA polymerases of the yeast and mycelial phases of Histoplasma capsulatum. The relative potency of each of the derivatives against the isolated polymerases was the same as the potency of each against the viable organism. RNA polymerase PC III from the yeast phase was more susceptible to the rifamycin derivatives than yeast phase polymerases PC I and PC II and the biggest differences in susceptibility were seen with the derivative AF/ABDP (2,6-dimethyl-4-benzyl-4-demethyl-rifamycin). The susceptibility pattern of the mycelial polymerase activity was identical to the yeast polymerase PC III.     

DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene

Three nuclear DNA polymerases have been described in yeast: DNA polymerases I, II, and III. DNA polymerase I is encoded by the POL1 gene and is essential for DNA replication. Since the S. cerevisiae CDC2 gene has recently been shown to have DNA sequence similarity to the active site regions of other known DNA polymerases, but to nevertheless be different from DNA polymerase I, we examined cdc2 mutants for the presence of DNA polymerases II and III. DNA polymerase II was not affected by the cdc2 mutation. DNA polymerase III activity was significantly reduced in the cdc2-1 cell extracts. We conclude that the CDC2 gene encodes yeast DNA polymerase III and that DNA polymerase III, therefore, represents a second essential DNA polymerase in yeast.     

Isolation of ribonucleic acid polymerases I, II, and III from Saccharomyces cerevisiae.

A procedure for the simultaneous purification of RNA polymerases I, II, and III from Saccharomyces cerevisiae is described. High yields of each enzyme activity are obtained, allowing the preparation of approximately 10 mg of polymerase I, 25 mg of polymerase II, and 12 mg of polymerase III from 1.2 kg of cells (wet weight). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates RNA polymerase I contains polypeptides with molecular weights 185 000, 137 000, 41 000, 35 000, 28 000, 24 000, 20 000, 16 000, 14 500, and 12 300; RNA polymerase II contains subunits with molecular weights 170 000, 145 000, 41 000, 33 500, 28 000, 24 000, 18 000, 14 500, and 12 500; and RNA polymerase III contains polypeptides with molecular weights 160 000, 128 000, 82 000, 53 000, 41 000, 37 000, 34 000, 28 000, 24 000, 20 000, 14 500, and 10 700.