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.     

Variable stabilities and recoveries of rat-liver RNA polymerases A and B according to growth status of the tissue.

1. The effect of growth status on the relative levels and recoveries of DNA-dependent RNA polymerase in rat liver nuclei was determined by two independent procedures: (a) measurement of RNA polymerase A and B activities in fraction IV [Roeder, R. G. and Rutter, W. J. (1970) Proc. Natl Acad. Sci. U.S.A. 65, 675--682] in the presence and absence of low concentrations of alpha-amanitin; (b) DEAE-Sephadex chromatography of fraction IV to resolve RNA polymerases A and B (and possibly other forms of the enzyme). 2. Growth was arrested in young rats (less than 100 g body weight) by hypophysectomy and stimulated by the administration of growth hormone or triiodothyronine. Under these conditions the rate of RNA synthesis in vivo or in isolated nuclei is known to be markedly depressed or stimulated relatively soon after hypophysectomy or hormone administration, respectively. RNA polymerases were obtained from animals under different growth conditions. There were no differences in the activities of nuclear RNA ploymerases per se, when these were separated from their endogenous template and assayed with heterologous denatured DNA. These reports contrast with earlier reports [Smuckler, E. A. and Tata, J. R. (1971) Nat. New Biol. 234, 37--39; Sajdel, E. M. and Jacob, S. T. (1971) Biochem. Biophys. Res. Commun. 45, 707--715]. 3. The discrepancy was resolved when a 'balance sheet' of enzyme recovery was established. Cessation of growth by hypophysectomy led to a marked reduction in the recovery of both forms A and B of the enzyme (less than 20% of the input RNA polymerase activity in fraction iv) following chromatography on DEAE-Sephadex. This effect was reversed within a short time after the administration of growth hormone (3--9 h) or triiodothyronine (18--24 h), leading to a doubling of the enzyme recoveries. These alterations which were more marked for RNA polymerase A, resulted in different elution profiles for RNA polymerases A and B upon chromatography. 4. It is concluded that the use of DEAE-Sephadex chromatography to compare the levels of RNA polymerases A and B isolated from tissues of different growth rate can give rise to over-estimates of apparent changes in their relative activities and that the measurement of enzyme activity in fraction IV is a better index of RNA polymerase levels. The relationship between growth rate of cells, the stability of RNA polymerases, and the importance of determining enzyme recoveries upon chromatography, are discussed.     

Inhibition of hepatic deoxyribonucleic acid-dependent ribonucleic acid polymerases by the exotoxin of Bacillus thuringiensis in comparison with the effects of α-amanitin and cordycepin

The action of Bacillus thuringiensis exotoxin, a structural analogue of ATP, on mouse liver DNA-dependent RNA polymerases was studied and its effects were compared with those of alpha-amanitin and cordycepin. (1) Administration of exotoxin in vivo caused a marked decrease in RNA polymerase activity of isolated nuclei at various concentrations of Mg(2+), Mn(2+) and (NH(4))(2)SO(4). A similar action was recorded after addition of exotoxin to isolated nuclei from control or exotoxin-treated mice. (2) Chromatographic separation of nuclear RNA polymerases from mice treated in vivo with exotoxin showed a drastic decrease of the peak of nucleoplasmic RNA polymerase, whereas the peak of nucleolar RNA polymerase remained unaltered. The same effect was observed after administration of alpha-amanitin in vivo, but cordycepin did not alter the relative amounts of the two main RNA polymerase peaks. (3) Administration of exotoxin in vivo did not alter the template activity of isolated DNA or chromatin tested with different fractions of RNA polymerase from control or exotoxin-treated mice. (4) Addition of exotoxin to isolated liver RNA polymerases inhibited both enzyme fractions. However, the alpha-amanitin-sensitive RNA polymerase was also 50-100-fold more sensitive to exotoxin inhibition than was the alpha-amanitin-insensitive RNA polymerase. Kinetic analysis indicated the exotoxin produces a competitive inhibition with ATP on the nucleolar enzyme, but a mixed type of inhibition with nucleoplasmic enzyme. The results obtained indicate that the B. thuringiensis exotoxin inhibits liver RNA synthesis by affecting nuclear RNA polymerases, showing a preferential inhibition of the nucleoplasmic alpha-amanitin-sensitive RNA polymerase.     

Nucleolar and nucleoplasmic RNA polymerase activity in different regions of rat brain during postnatal development.

RNA polymerase activities of whole nuclei, of isolated and purified nucleoli and of the nucleoplasmic fractions obtained from cerebral hemispheres, cerebellum and brain stem of rat at different days of postnatal development have been determined. In the whole nuclei the fraction of RNA polymerase which is sensitive to alpha-amanitin, is strongly affected by salt concentration; at low ionic strength most of the activity is resistant to the drug while at high ionic strength the enzymatic activity shows a greater sensitivity to the drug. In isolated nucleoli RNA synthesis is not inhibited at all by alpha-amanitin. The biosynthesis of RNA, at low ionic strength, is inhibited by low doses of actinomycin D, whereas at high ionic strength it is remarkably inhibited only by higher doses of the drug. The sensitivity of the reaction to alpha-amanitin and actinomycin D provide good evidence that UTP or GTP incorporation into RNA in purified nuclei and nucleoli, is dependent on RNA polymerases acting on DNA template and is not dependent on homopolymer formation. These results show that in the whole brain nuclei at low ionic strength there is a preferential synthesis of rRNA, whereas at high ionic strength the synthesis of heterogenous RNA predominates. In isolated nucleoli the synthesis of RNA is restricted to rRNA.     

Deoxyribonucleic acid-dependent ribonucleic acid polymerases from murine spleen cells. Increased amounts of the nucleolar species in leukaemic tissue

1. In the spleens of infected mice, the Friend leukaemia virus induces a sharp increase in the ability of subsequently isolated nuclei to incorporate exogenous UTP into an acid-insoluble product. Inhibitor studies indicate that the incremental RNA synthesis proceeds from a DNA template and that both nucleolar and nucleoplasmic activities are involved. 2. The partially purified DNA-dependent RNA polymerases from control and virus-infected tissue are indistinguishable with respect to chromatographic mobility, dependence on bivalent cations, ionic strength, pH and their susceptibility to alpha-amanitin. The RNA polymerases of the murine spleen resemble the enzymes of other mammalian tissue in these properties. 3. A comparison of the amount of polymerase solubilized from normal and infected tissue correlates with the activity observed in assays of the respective nuclei. These experiments indicated that the increase in nucleolar RNA synthesis after infection is mediated by increased extractable polymerase I activity whereas the change in nucleoplasmic RNA synthesis results from an alteration of chromatin or a chromatin-associated factor.     

Stimulation of the activities of solubilized pig lymphocyte RNA polymerases by phytohaemagglutinin

DNA dependent RNA polymerase has been solubilised from pig peripheral blood lymphocytes. Using α amanitin, an inhibitor of the nucleoplasmic polymerase B activity, we have found that 20 hrs following lymphocyte stimulation with phytohaemagglutinin (PHA) the activities of both polymerase A and polymerase B are increased. As previously observed with intact nuclei a greater stimulation of polymerase A activity is observed at this time. Since the activity of these enzymes was assayed using exogenous template this indicates that PHA stimulates RNA synthesis by regulating the amounts and/or the activities of the polymerases.     

Isolation and characterization of RNA polymerase B from the larval fat body of the tobacco hornworm, Manduca sexta.

DNA-dependent RNA polymerase B has been extensively purified from the larval fat body of the tobacco hornworm (Manduca sexta) by employing chromatography on ion-exchange columns of DEAE-Sephadex, DEAE-cellulose and phosphocellulose and centrifugation on glycerol gradients. The isolated enzyme after electrophoresis on acrylamide gels shows one main band and one minor band, both having enzyme activity sensitive to alpha-amanitin. The catalytic and physicochemical properties of the enzyme are similar to those of other eucaryotic B-type RNA polymerases. The enzyme has an apparent molecular weight of 530000, is inhibited 50% by alpha-amanitin at 0.04 microgram/ml and shows maximum activity on denatured DNA at 5 mM Mn2+ and 100 mM ammonium sulfate. An antibody was obtained that cross-reacts with the pure enzyme and forms a precipitin line. This antibody does not cross react with either Escherichia coli RNA polymerase or with wheat germ RNA polymerase but does react with one of the B polymerases isolated from wing tissue of the silkmoth, Antheraea pernyi.     

Poly(A) polymerase in quail oviduct. Changes during estrogen induction.

A nuclear poly(A) polymerase has been isolated from oviducts of immature quails. It could be purified 4300-fold. The enzyme depends specifically on ATP as substrate and requires Mg2+. The most effective primer for the enzyme is a polynucleotide, isolated from oviduct tissue. A poly(A) sequence to a maximum of 60 AMP residues is covalently linked per primer molecule. The poly(A)-rich product of the enzymatic reaction can be annealed to oligo(dT)-cellulose. The purest fraction does not contain any detectable poly(A)-degrading enzyme activity. Only very low activities of RNA polymerase are present. The poly(A polymerase activity in the assay with ATP is reduced by the ATP analogue, beta, lambda-ATP-methylene-diphosphonate. Both K-m and V are lowered. The ATP analogue is incorporated to a smaller extent into the poly(A) sequence, synthesized by the enzyme. Several other analogues of adenine, adenine nucleosides and adenine nucleotides are without effect on the enzymatic reaction. By these properties poly(A) polymerase can be distinguished from RNA polymerases form I and form II, isolated from the same tissue. Actinomycin D and alpha-amanitin failed to inhibit poly(A) polymerase activity. The activity of poly(A) polymerase has been determined during primary stimulation with the estrogen analogue diethylstilbestrol (daily injection for 5 days), after withdrawal of the hormone for 17 days and after secondary stimulation with the hormone analogue. The enzyme activity does not change during primary stimulation, withdrawal of the hormone or secondary stimulation. However the activity of a poly(A) degrading enzyme, localized in the nucleus, is reduced in oviducts from hormone-treated quails.     

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.     

Effect of an exotoxin from Bacillus thuringiensis on Deoxyribonucleic acid-dependent ribonucleic acid polymerase in nuclei from adult Sarcophaga bullata. Unusual behaviour of eukaryotic polymerases to inhibitors

The DNA-dependent RNA polymerase activities in nuclei isolated from adult Sarcophaga bullata are unusual in their responses to metal ions, ionic strength and inhibitors. There is an activity that is sensitive both to rifamycin and to alpha-amanitin. The activity is less sensitive to Bacillus thuringiensis exotoxin than is larval polymerase, and low concentration of exotoxin provoke a slight stimulation.     

Changes in "template-bound" and "free" RNA polymerase activities in isolated nuclei from Xenopus laevis embryos

Nuclei have been isolated from Xenopus laevis embryos and incubated under conditions allowing RNA synthesis to proceed for more than 3 h. The RNA molecules synthesized on the endogenous template are stable, heterogeneous in size and correspond to the activities of the three RNA polymerases.In these in vitro conditions we have determined the extent of activity of the three RNA polymerases during the embryonic development from blastula to swimming tadpole. Our results on isolated nuclei are in good agreement with the changes in RNA synthesis which take place during normal embryonic development.We have measured both the “template-bound” and the “free” activities of each of the three RNA polymerases during development. Amongst the total RNA polymerase activities engaged on the template, the proportion of polymerase I increases as development proceeds: at the blastula stage, there is practically no RNA polymerase I engaged on the template, whereas in swimming tadpoles, RNA polymerase I amounts to about 90% of the RNA polymerases bound to the DNA. Conversely, RNA polymerase I represents the major part of free RNA polymerases in blastula nuclei.Autoradiography of incubated nuclei shows that, at least in swimming tadpoles nuclei, both “free” and “template-bound” RNA polymerase I are localized in the nucleoli.The evolution of “template-bound” RNA polymerase II activity during development is quite different from that of RNA polymerase I: RNA polymerase II activity represents 75% of engaged polymerase activity in blastulae and only 47% at the swimming tadpoles stage.The results suggest that part of the “free” RNA polymerase I activity might progressively become “template-bound” during embryogenesis.     

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.     

DNA-Dependent RNA Polymerases from Artemia salina

Embryos and larvae of the brine shrimp, Artemia salina , provide a useful biological system for biochemical studies of animal development. Dormant encysted embryos can be cultured readily in the laboratory to provide large quantities of free-swimming nauplius larvae. The rate of synthesis of all classes of RNA in swimming larvae declines markedly between 24 and 72 h after immersion of dormant embryos in sea water. Nuclei were isolated from 24–72 h larvae and RNA polymerase activity was measured under conditions in which the nuclei remained intact. Total RNA polymerase activity of isolated nuclei decreased in parallel with RNA synthesis in vivo. RNA polymerases were solubilized from nuclei and fractionated by chromatography on DEAE-cellulose. The levels of both RNA polymerases I and II also decreased in parallel with RNA synthesis in vivo. The specific activity of highly purified RNA polymerase II was determined by comparison of enzyme activity with the mass of RNA polymerase II subunits displayed on SDS gels. The specific activities of RNA polymerase II preparations from 24 and 72 h larvae were identical. The number of polymerase II molecules was estimated from the mass of the subunits. The number of molecules per nucleus declined from 20,000 at 24 h to 3500 at 72 h.     

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.