Increased levels of rat hepatic nuclear free and engaged RNA polymerase activities during liver regeneration.

Rat liver nuclei, seventeen hours after partial hepatectomy, showed a two to three-fold increase in total RNA synthesis $\text{in vitro}$ over the sham operated controls. When tested with exogenous synthetic template, this was found to be mainly a reflection of increased levels of both the nuclear free and engaged RNA polymerase activities $\text{per se}$. It was also observed that there was a greater stimulation of the species of RNA polymerase that are α-amanitin resistant than sensitive (3.2 μg/ml). This observation was further confirmed by DEAE-Sephadex column chromatography of the solubilized nuclear free and engaged RNA polymerases and found RNA polymerase I and IIIa were the major species greatly stimulated during this period of liver regeneration. These data suggest not only that there exists a sensitive equilibrium between the nuclear free and engaged RNA polymerases; they also suggest the possibility that RNA polymerase itself may play a positive role in the regulation of gene expression.     

An improved method for the quantitative isolation of rat liver nuclear RNA polymerases.

Rat liver nuclear RNA polymerases exist in two functional states, one of which is active towards the endogenous chromatin template (engaged enzyme), while the other is inactive (free enzyme) (Yu, F.L. (1974) Nature 251, 344-346). This paper reports the direct separation of these two populations of RNA polymerases from isolated rat liver nuclei by a simple extraction procedure. It is estimated that as much as 50% of the total nuclear RNA polymerase activity in normal rat liver may exist in the form of the free enzyme. Evidence is also presented to indicate that the free enzyme activity is easily lost when the nuclear isolation procedure involves the use of an isotonic buffer medium, or when the isolated nuclei are subjected to sonication as is required for the solubilization of the nuclear RNA polymerases by the conventional method. Based on these new findings, it is proposed that nuclei be isolated directly in hypertonic sucrose and that the free enzyme be extracted before the nuclei are subjected to sonication to solubilize the engaged enzyme. This method circumvents the loss of the free RNA polymerase population and, as a result, the total yield of the nuclear RNA polymerases is greatly increased. The possible functional role of the free RNA polymerase in gene expression is discussed.     

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.     

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.     

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.     

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.     

RNA synthesis in nuclei isolated from early embryos of Xenopus laevis.

1. Rates of RNA synthesis in isolated Xenopus embryo nuclei decrease from blastula through gastrula and neurula stages to hatching tadpoles. 2. In blastula and gastrula nuclei, net synthesis of RNA continues for over 30 min, both in the presence of KCl at 0.4 M and in its absence. In nuclei from later stages, net synthesis continues for only about 10 min in the absence of KCl. 3. At low ionic strength, RNA synthesis in all nuclei is greater with optimum Mg-2+ (6 mM) than with optimum Mn-2+ (1 mM). At high ionic strength the reverse is true. 4. An unusual feature, which gradually disappears as development proceeds, is that curves relating RNA synthesis to KCl concentration show a peak at 0.1 M KCl. In blastula nuclei, RNA synthesis is more rapid at 0.1 M KCl than at 0.4 M. 5. This peak at low ionic strength is not observed in the presence of the initiation inhibitor rifamycin AF/013. It is concluded that the peak arises from initiation of RNA synthesis by an excess of RNA polymerases bound non-specifically to the isolated nuclei. The residual synthesis, representing elongation of chains that were initiated in vivo, still declines as development progresses. 6. In blastula nuclei, over half of the RNA synthesis is effected by polymerase II (inhibited by alpha-amanitin), the proportion remaining roughly constant with increasing ionic strength. In neurula nuclei, the proportion rises from about one-half to three-quarters. The initiation-dependent peak in blastula and gastrula nuclei is contributed by both alpha-amanitin-sensitive and alpha-amanitin-resistant enzymes.     

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.     

A factor in sea urchin eggs inhibits transcription in isolated nuclei by sea urchin RNA polymerase III

Isolated nuclei from sea urchin embryos synthesize RNA at a rate comparable to other animal cell nuclei. All three RNA polymerases are active as judged by alpha-amanitin sensitivity and hybridization to specific cloned DNAs. Extracts were prepared from sea urchin eggs and embryos by extraction with 0.35 M KCl. None of the crude extracts had a large effect on total RNA synthesis. However, extracts from sea urchin eggs inhibited RNA polymerase III activity in nuclei from blastula and gastrula embryos. There was no effect on the synthesis of ribosomal RNA by RNA polymerase I or on the synthesis of two RNA polymerase II products, histone mRNA and the sea urchin analogue of U1 RNA. The inhibitor is present in two different species of sea urchin and has been 50-fold purified by diethylaminoethylcellulose and hydroxylapatite chromatography. The inhibitor is not present in extracts prepared from sea urchin blastula embryos.     

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.     

RNA synthesis by nuclei and nucleoli from ischemic liver cells

Nuclei and nucleoli were isolated from rat livers subjected to an interruption of the blood supply for periods of different duration, as well as after restoration of the blood supply. They were assayed for RNA synthesis under conditions of diverse ionic strengths, and in the presence of an exogenous template, such as poly d (A–T), and actinomycin to inactivate the endogenous template; α-amanitin was made used of to distinguish polymerase I and polymerase II dependent RNA synthesis. Nuclei and nucleoli from ischemic livers showed a severe impairment of RNA synthesis, which is likely to be due to decreased initiation frequency of the engaged polymerases, while free polymerases were essentially unchanged. Both form I and II polymerase were equally involved. After restoration of the blood supply RNA synthesis recovered with an overshooting well above normal levels of activity, lasting for at least 24 hours. Increased RNA synthesis was not followed by thymidine incorporation into DNA.