DNA-dependent RNA-polymerases from Artemia embryos. characterization of polymerases I and II from nauplius larvae.

Partial purification and characterization of DNA-dependent RNA-polymerases from nauplius larvae of the brine shrimp, Artemia salina, are described. Fractionation of solubilized RNA-polymerases on columns of DEAE-cellulose yielded partially purified preparations of RNA polymerases I and II. The properties of these enzymes were found to be similar to properties of corresponding enzymes from other animal sources. A significant change in the relative amounts of polymerases I and II occurs between 36 and 72 hr of development. Polymerase activity obtained from 36-hr nauplii consisted of approximately equal amounts of polymerases I and II, whereas polymerase II accounted for more than 80% of the activity recovered from 72-hr nauplii. Total polymerase activity was lower at 72 than at 36 hr. The significance of these changes in relation to the decrease in RNA synthesis in vivo that occurs after 36 hr is discussed.     

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 polymerase activities during embryogenesis in the bug, Oncopeltus fasciatus

Using α-amanitin to inhibit polymerase II activity in intact nuclei from Oncopeltus embryos, it is demonstrated that there is no difference in relative amounts of α-amanitin-resistant (Form I) and α-amanitin-sensitive (Form II) polymerases at two stages of embryonic development (70 and 140 hr), although the total polymerase activity is considerably higher at the earlier stage. However the RNA made under these circumstances (presumably due to Form I activity) appears to be, as expected, largely ribosomal.When the RNA polymerase activities are solubilized and separated, there is a substantially higher level of Form I activity in 70-hr embryos over that in 140-hr embryos. It is suggested that this high level of polymerase activity is correlated directly with the high level of ribosomal RNA synthesis at this stage.     

Conservation of RNA polymerase during maturation of the Rana pipiens oocyte.

DNA-dependent RNA polymerase was extracted from oocytes of the frog, Rana pipiens. The bulk of the enzyme activity was present in the germinal vesicle and the amounts of each major form of such activity did not significantly change during oocyte maturation. Therefore, either nuclear polymerase activity is conserved after breakdown of the oocyte nucleus during maturation or, alternatively, de novo synthesis of the enzymes must occur during oocyte maturation concomitant with degradation. We have measured rates of protein synthesis in oocytes and determined a maximum rate of synthesis for RNA polymerases. Our kinetic studies show that no more than 20, 10, and 5% of RNA polymerases type I, IIa, and IIb, respectively, could be synthesized during steroid-induced oocyte maturation. These results thus show that the bulk of RNA polymerase accumulates in the germinal vesicle during oogenesis, is dispersed into the cytoplasm during maturation, and, since only limited synthesis seems to be occurring, the polymerase is available during embryogenesis.     

The effect of antimycin A on cytochromes b561, b566, and their relationship to ubiquinone and the iron-sulfer centers S-1 (+N-2) and S-3.

Three nuclear RNA polymerases and one poly(A) polymerase were isolated from the yeast, Saccharomyces cerevisiae. The ability of cordycepin triphosphate to inhibit each was determined. RNA polymerase II was significantly more sensitive to this compound than the other polymerases. RNA polymerase I was relatively insensitive, being inhibited less than 20% by 40 μm cordycepin triphosphate. The calculated apparent K_i values of RNA polymerases II and III and poly(A) polymerase were, respectively, 0.3, 3.0, and 4.6 μm. Inhibition was competitive with regard to ATP. These data do not support the idea that, in yeast, poly(A) addition to preformed RNA in vivo is the primary site of cordycepin action.     

RNA polymerase activities of Calliphora vicina larvae in response to ecdysteroids

The effect of ecdysterone on RNA polymerase activities, partially purified from fat body nuclei of Calliphora vicina third instar larvae, has been investigated. Three different forms of DNA-dependent RNA polymerase activities have been isolated and resolved on the basis of chromatographic behaviour and α-amanitin sensitivity. RNA polymerase I and II are similar to the analogous polymerases from other eukaryotic organisms in their enzymatic properties, such as salt and metal-ion optima and in their DNA requirements. The activities of polymerase I and II could be correlated with the endogenous ecdysteroid concentrations of third instars. The enzyme activities increase two-fold in white puparia compared to five-day-old larvae. After injection of ecdysterone in five-day-old larvae the enzyme activities are stimulated within 3 hr to the same amount as in white puparia. The enzyme stimulation coincides with the increase in RNA synthesis. Our results indicate the existence of a hormone dependent mechanism for regulating the intracellular activities of RNA polymerases I and II.     

Specific interference of metalaxyl with endogenous RNA polymerase activity in isolated nuclei fromPhytophthora megasperma f. sp.medicaginis

The systemic fungicide metalaxyl preferentially inhibits [³H]uridine incorporation into RNA by mycelium ofPhytophthora megasperma f. sp.medicaginis. Even at high concentrations of metalaxyl inhibition is not complete but circa 80%. Neither uptake of [³H]uridine nor its conversion into UTP is inhibited, indicating that interference with RNA synthesis takes place. Synthesis of RNA that lacks poly(A) sequences is more affected than that of poly(A)⁺ RNA. Metalaxyl has no effect on the activity of RNA polymerases present in mycelial extracts fromPhytophthora nor on that of polymerases I and II that have been partially purified with a procedure involving precipitation with polyethyleneimine, selective elution of RNA polymerases from the polyethyleneimine precipitate, ammonium sulfate fractionation, and DEAE-Sephadex chromatography. RNA polymerase II in mycelial extracts is half-maximally inhibited by α-amanitin at concentrations below 0.01 ¼g/ml. Both metalaxyl and α-amanitin inhibit endogenous RNA polymerase activity of isolated nuclei ofPhytophthora. According to their sensitivity to metalaxyl and α-amanitin, three types of endogenous activity can be distinguished: (a) an α-amanitin-sensitive type, the activity of which is stimulated by ammonium sulfate; (b) an α-amanitin-insensitive but metalaxyl-sensitive type; and (c) a type insensitive to both metalaxyl andα-amanitin. The first type of activity is characteristic of RNA polymerase II; the identity of the latter two remains to be elucidated. Metalaxyl andα-amanitin do not have any effect on free nuclear polymerases when assayed at a concentration of 50 mM ammonium sulfate with poly[d(A-T)] as exogeneously added template in the presence of actinomycin D to inhibit endogenous RNA polymerase activity. At 250 mM ammonium sulfate the free polymerase activity becomes α-amanitin sensitive but remains metalaxyl insensitive. Metalaxyl apparently inhibits RNA synthesis by specific interference with template-bound andα-amanitin-insensitive RNA polymerase activity. Endogenous polymerase activity of nuclei isolated from a metalaxyl-resistant mutant ofP. megasperma f. sp.medicaginis is not inhibited by metalaxyl, indicating that interference with RNA synthesis is the primary action of metalaxyl and that modification of the target site may lead to resistance.     

Wounding-induced Enhancement of the Activity of Chromatin-bound DNA-Dependent RNA Polymerases in Sweet Potato Root

Chromatin fractions were isolated from intact and wounded sweet potato root tissues. The synthesis of RNA by the chromatin fractions was dependent on four ribonucleoside triphosphates and a divalent cation such as Mg²⁺ and Mn²⁺, Mn²⁺ being most effective. Whereas phosphate did not interfere with the polymerase reaction, it was totally blocked by pyrophosphate. The reaction was inhibited by DNase and actinomycin D as well as RNase and trypsin. The RNA polymerases of sweet potato root needed SH-groups for catalysis. Activity of chromatin-bound RNA polymerases (EC 2.7.7.6) promptly increased in the 6 hr after wounding and then decreased gradually up to 24 hr. Under the present experimental conditions it was mostly due to the activity of RNA polymerase I. RNA polymerase II contributed only about 5 to 15% to the total activity. The increase in the activity after wounding was completely inhibited by cycloheximide. Plant hormones such as 2,4-dichlorophenoxyacetic acid, gibberellic acid and dibutyryl cyclic adenosine 3′,5′-monophosphate stimulated the increase in RNA polymerases three to four times after wounding. Ethylene partially suppressed the wound-induced increase of RNA polymerases.     

Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase Activity in Cells Infected with Influenza Virus

Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase activity was assayed on nuclear preparations of chick embryo fibroblast cells at various times after infection with an influenza A virus (fowl plague virus) and was compared with the activity of uninfected cells. Polymerase activity was increased by about 60% by 2 hr after infection, and this increase coincided with an increase in RNA synthesis in infected cells, as determined by pulse-labeling with uridine. No difference could be detected between the polymerases of infected and uninfected cells as to their requirements for DNA primer, divalent cations, and nucleoside triphosphates, and they were equally sensitive to addition of actinomycin D to the reaction mixture. It is possible that host cell DNA-dependent RNA polymerase is involved in the replication of influenza virus RNA.     

Changes in DNA-dependent RNA polymerase in sporulating yeast

Diploid yeast cells can be made to undergo sporulation and meiosis in a relatively synchronous fashion. Earlier studies on this process showed that the rate of RNA synthesis reaches a maximum at 6 hr and has declined drastically by 10 hr [5]. Starting at about 6 hr a new peak of RNA polymerase activity appears between polymerases Ib and II upon DEAE Sephadex chromatography. This peak appears to reach a maximum at 8.5 hr and to have decreased by 10 hr. The new peak is intermediate between polymerases Ib and II in its sensitivity to α-amanitin. It does not appear in a non-sporulating (αα) diploid grown under sporulating conditions.     

Role of Adenovirus Structural Proteins in the Cessation of Host-Cell Biosynthetic Functions

Two of the adenovirus capsid proteins, the fiber and the hexon, complexed with either KB cell or type 5 adenovirus deoxyribonucleic acid (DNA). Maximal binding occurred at 0.01 m NaCl; increasing the ionic strength of the reaction mixture to 0.2 m NaCl resulted in a decrease in the association of either antigen to DNA. Variations of pH between 6.3 and 8.4 did not affect the binding of fiber antigen to DNA. Below pH 7.5, however, there was a small decrease in the ability of the hexon to bind nucleic acid. The association between the adenovirus structural proteins and DNA was reversible and was independent of whether the DNA was native or denatured. The fiber or hexon protein inhibited the DNA-dependent ribonucleic acid (RNA) polymerase and the DNA polymerase from KB cells. On a weight basis, the fiber protein inhibited enzymatic activity to a greater extent than the hexon. Increasing the template DNA concentration decreased this inhibition. The inhibition of the DNA-dependent RNA polymerase activity by either antigen could be reversed by increasing the ionic strength of the reaction mixture. After infection of KB cells with type 5 adenovirus, the levels of DNA and RNA polymerases remained unchanged for 15 to 20 hr. Thereafter, the specific activity of both enzymes decreased. By 30 hr postinfection, the polymerase activities were only about 30% of the enzyme activities in uninfected cells.     

EARLY EVENTS IN LYMPHOCYTE TRANSFORMATION BY PHYTOHEMAGGLUTININ : I. DNA-Dependent RNA Polymerase Activities in Isolated Lymphocyte Nuclei

The DNA-dependent RNA polymerase activities of isolated nuclei from lymphocytes were examined after stimulation with phytohemagglutinin (PHA). The nuclear fraction was prepared with Mg⁺⁺ or Mn⁺⁺ to distinguish between polymerase I (nucleolar) and polymerase II (nucleoplasmic). Distinction between polymerases II and III was obtained by the addition of α-amanitin to the reaction mixture. The results indicated that within 15 min after exposure to PHA the activity of polymerase I increased. Polymerase II activity increased after 1 hr. The enhancement was linear for 6 hr and then leveled off for the subsequent 48 hr. Small increase in polymerase III activity was observed at 48 hr. Inhibition of protein synthesis at the time of exposure to PHA did not prevent the increase in activities during the initial 6 hr. These results imply that the initial increase in enzymatic activities is dependent upon preexisting polymerase molecules and/or factors.     

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.     

Arabinosyl nucleosides. XIII. Influence of arabinofuranosylthymine on DNA-, RNA- and protein-synthesizing systems in vitro

The natural metabolite of the sponge Cryptotethya crypta, arabinofuranosylthymine (araThd), is intracellularly phosphorylated to araTTP. The present study demonstrates that araTTP inhibits both isolated DNA polymerases α and the DNA polymerase β from L5178y cells competitively with respect to the analogous substrate dTTP. The affinity of araTTP is higher to the DNA polymerase α than to the DNA polymerase β.The activity of mammalian DNA-dependent RNA polymerases I, II and III as well as the incorporation rate of a protein cellfree system is not affected by high doses of araTTP.