Endogenous RNA Polymerase Activity Research Articles

Influenza virus RNA polymerase may be activated inside the virion.

Influenza A and B virions are packaged with their polymerases to catalyse RNA-dependent RNA polymerase activity. Since there is no evidence to rule in or out the permissiveness of influenza virions to triphosphate ribonucleotides, we functionally evaluated this. We found the means to stimulate influenza A and B RNA polymerase activity inside the virion, called natural endogenous RNA polymerase (NERP) activity. Stimulation of NERP activity increased up to 3 log10 viral RNA content, allowing the detection of influenza virus in otherwise undetectable clinical samples. NERP activation also improved our capacity to sequence misidentified regions of the influenza genome from clinical samples. By treating the samples with the ribavirin triphosphate we inhibited NERP activity, which confirms our hypothesis and highlights that this assay could be used to screen antiviral drugs. Altogether, our data show that NERP activity could be explored to increase molecular diagnostic sensitivity and/or to develop antiviral screening assays.

Effect of radiation on the functioning of neuron chromatin of mice exposed in the zone of the Chernobyl nuclear power plant

Circadian fluctuations in the activity of endogenous RNA polymerases were examined in the nuclei ofLv spinal ganglion neurocytes, spinal α-motoneurons, and in neurons of the sensorimotor and visual cortex of mice exposed to radiation in the zone of the Chernobyl power plant. The circadian organization of the functioning of the genetic apparatus of neurons in various compartments of the nervous system was altered in irradiated mice. A shift of the acrophases of nucleoplasmic and nucleolar labeling in the course of the 24-hour cycle was observed after a 40-day exposure under conditions of a high radioactive background. The degree of synchronization of the level of transcription of ribosomal and structural genes of the studied cell populations diminished. The mesors and amplitudes of circadian rhythms of chromatin matrix activity decreased. The status of the histone component of chromatin also changed in exposed animals.

Effect of different conditions of hyperbaric oxygenation on morphology and transcription of cortical neurons of rats with experimental stroke

The protective action of hyperbaric oxygenation (I-IBO) in stroke has been described by many research workers [3, 7, 8], but its clinical use in the treatment of acute cerebral ischemia still rests on an inadequate basis. The aim of this investigation was to study the trend of the morphological changes and transcription activity of pyramidal neurons in the frontal cortex of rats with acute ischemia, depending on the conditions of HBO. METHODS Experiments were carried out on 120 noninbred albino rats weighing 200 g. Two models of ischemia were used: moderately severe -- after fight-sided ligation (group I), and severe, with bilateral ligation of the common carotid arteries (group II) [1, 2, 6]. The operation was performed under pentobarbital anesthesia. The state of the animals in group I immediately after the operation was satisfactory, and their behavior and general appearance were not visibly different from those of intact animals, yet a few hours later these rats were found to have mild mono- and hemipareses. The animals of group II were in a severe state after the operation. With the passage of time the state of the animals worsened and their mortality increased, so that after 24 h it was 100%. The effect of a single application of HBO on the morphologic and functional state of the frontal cortical neurons of the rats was investigated on these models, when three different conditions of HBO were used: exposure for 1 h to 2.0 atm (absolute atmospheres) with oxygen, and in group II, the same but with air, and exposure for 30 rain to 1.2 atm with oxygen, which is the situation closest to that observed clinically [3]. The sessions were given 2 h after the operation. The use ofHBO, whatever the conditions, was accompanied after 10-15 min by appreciable improvement of the animals' state. In the animals of group II the cyanosis disappeared, behavior and external appearance returned to normal, and their general state remained good for several hours after HBO. The survival rate of the rats after the operation in this group, following the use of HBO toward the end of the 1st day, amounted to 50% after exposure to 1.2 atm and 30% after exposure to 2.0 atm with oxygen. The animals' state 24 h after HBO at 1.2 arm was rather better than after exposure to 2.0 arm. S~uctural changes and transcription activity of the large and medium-sized pyramidal neurons in the frontal cortex of the ligated rats was assessed 15 rain after a session of HBO and 24 h later. The material was taken from 3-5 animals (except in group II after 24 h, when it was only from 2-3 survivors). Intact rats, rats undergoing a mock operation, and rats undergoing ligation but not treated with HBO, and killed 2.5 h after the operation, served as the control. The number of large and medium-sized preserved pyramidal neurons was counted in several fields of vision and the results averaged. To determine transcription (template) activity of the chromatin of the neurons studied, the histoautoradiographic method of detecting activity of endogenous RNA-polymerases in fixed neurons in sections was used [ 10]. The RNA

Antifungal modes of action of metalaxyl, cyprofuram, benalaxyl and oxadixyl in phenylamide-sensitive and phenylamide-resistant strains of Phytophthora megasperma f. sp. medicaginis and Phytophthora infestans

The activity of the phenylamide fungicides metalaxyl, cyprofuram, benalaxyl and oxadixyl against phenylamide-sensitive and phenylamide-resistant strains of Phytophthora megasperma f. sp. medicaginis and Phytophthora infestans has been compared in several tests. Metalaxyl showed the highest activity amongst the four fungicides against mycelial growth of sensitive strains on agar media. Benalaxyl and cyprofuram showed the highest activity against resistant strains. RNA synthesis of sensitive strains, measured as [ 3H]uridine incorporation, was inhibited by about 80% ( P. megasperma) and by about 40% ( P. infestans) by all phenylamides at concentrations of 1 μg/ml. At higher concentrations inhibition remained at this level, indicating that only part of the RNA synthesis was inhibited. RNA synthesis of resistant strains was completely insensitive to metalaxyl and oxadixyl at concentrations as high as 200 μg/ml. Cyprofuram, however, at concentrations above 1 μg/ml ( P. megasperma) and 10 μg/ml ( P. infestans) was inhibitory and at 200 μg/ml this compound almost completely inhibited the phenylamide-sensitive part of the RNA synthesis of both strains. In contrast to the other phenylamides, benalaxyl affected [ 3H]uridine uptake into mycelium at concentrations higher than 10 μg/ml, and at 100 μg/ml inhibition of uptake was almost total with both sensitive and resistant strains of both Phytophthora species. Inhibition of [ 3H]uridine uptake obviously is a feature of a second mechanism of action of benalaxyl. Endogenous RNA polymerase activity of isolated nuclei from sensitive strains of P. megasperma was inhibited at phenylamide concentrations as low as 0·1 μg/ml whereas that from resistant strains was not affected at concentrations as high as 100 μg/ml. Similarly, endogenous nuclear RNA polymerase activity of sensitive P. infestans strains appeared to be more sensitive to the phenylamides than that of resistant P. infestans strains. This suggests that resistance that developed in field strains of P. infestans has a basis similar to that of mutagen-induced resistance in laboratory strains of P. megasperma. Tests of the fungicidal activity of the phenylamides determined in a lucerne seedling assay ( P. megasperma) and a detached potato leaf assay ( P. infestans) clearly differentiated between sensitive and resistant strains. Resistance levels to cyprofuram and benalaxyl were as high as those to metalaxyl and oxadixyl, indicating that the additional effect of a second mechanism of action of benalaxyl and the property of cyprofuram to inhibit RNA synthesis of resistant strains at higher concentrations are of limited practical importance for counteracting phenylamide resistance.

Studies on the mechanism of action of cymoxanil in Phytophthora infestans

Colony growth and germ tube emergence of sporangia and encysted zoospores of Phytophthora infestans were highly sensitive to cymoxanil (ED 50 0.5–1.5 μg/ml), whereas differentiation of sporangia and zoospore release were insensitive at concentrations up to 100 μg/ml. Treated sporangia did not show distorted germ tubes. Oxygen consumption for glucose oxidation by germinating sporangia and zoospore motility were not inhibited at concentrations up to 100 μg/ml. Cymoxanil hardly affected the uptake of radiolabeled precursors of DNA, RNA, and protein at concentrations up to 100 μg/ml. Incorporation of [ 14C]phenylalanine into protein was completely insensitive. RNA synthesis as measured by [ 3H]uridine incorporation was differentially inhibited in the various developmental stages of the fungus. Inhibition did not occur at differentiation of sporangia, whereas at cyst and sporangial germination and mycelial growth this process was inhibited 20–45% at a concentration of 100 μg cymoxanil/ml. Endogenous RNA polymerase activity of isolated nuclei was not inhibited by cymoxanil. DNA synthesis as measured by [ methyl- 3H]thymidine incorporation was inhibited 20–80% at the various stages of development at cymoxanil concentrations higher than 10 μg/ml. Metalaxyl, a specific inhibitor of ribosomal RNA synthesis, inhibited [ 3H]uridine incorporation 40–60% at all developmental stages. The data suggest that although DNA synthesis is affected more than RNA synthesis, inhibition of both biosynthetic processes is a secondary effect. The primary mode of action of cymoxanil thus remains unknown.

RNA Polymerase Activity During Breakage of Seed Dormancy by Low Temperature Treatment of Fruits ofAcer platanoides(Norway Maple)

Slater, R. J. and Bryant, J. A. 1987. RNA polymerase activity during breakage of seed dormancy by low temperature treatment of fruits of Acer platanoides (Norway maple).—J. exp. Bot. 38:1026-1032. Endogenous RNA polymerase activity has been characterized in nuclei isolated from embryo axes of Acer platanoides. Optimal activity was recorded at 40 mol m~3 MgCl2 and 50 mol m~3 (NH4)2S04 and total activity could be inhibited by up to 30% by a-amanitin. Stratification of fruits leads to a stimulation of RNA polymerase activity. A minimum of 3 d cold treatment is required with at least 3-fold stimulation recorded after 10 d at 4 °C. The increased enzyme activity is resistant to a-amanitin

Inactivation of a Single Eucaryotic Gene Irradiated in Vitro in Transcriptionally Active Chromatin Form.

The transcriptional activity of endogenous RNA polymerases has been used to study the inactivation of a single eucaryotic gene irradiated in chromatin form in aqueous solution. The gene codes for the ribosomal RNA precursor in Tetrahymena where it exists in multiple copies as free extrachromosomal molecules. The gene chromatin is isolated with intact RNA polymerases on the coding strand. Irradiation with 10-MeV electrons reduces the in vitro RNA synthesis on the isolated chromatin in a dose-dependent manner which allows the amount of DNA damage to be determined. Irradiation of the gene chromatin in dilute solution under various gas atmospheres demonstrates that OH radicals account for most of the inactivation. Under these conditions, $e_{{\rm aq}}^{-}$ is unimportant, while the results suggest that H may contribute to inactivation of gene transcription.

Periodical changes in chromatin properties in non-proliferating cells of germinating Pisum sativum L.

Within 32 h of germination three stage changes of chromatin properties (DNA availabity to acridine orange, the activity of endogenous RNA polymerases I and II and the rate of RNA synthesis) in cells of pea hypocotyl and cotyledons have been observed. An increase of chromatin activity with maxima at 6—9 h for the first stage, 16–20 h for the second stage and 28 h for the third stage is followed by its decrease at 12, 24 and 32 h of germination. The first and the second stages occur prior whereas the third one—after radicle protrusion. These changes occured simultaneously to those found previously in pea proliferating tissues (V. Troyan et al., Plant Sci. Lett., 33 (1984) 213). It is concluded that the periodicity of changes in chromatin properties in the whole seed cells might be a characteristic feature of the seed germination process of different plant species.

A differential basis of antifungal activity of acylalanine fungicides and structurally related chloroacetanilide herbicides in Phytophthora megasperma f. sp. medicaginis

The acylalanine fungicides CGA 29212 and metalaxyl inhibit colony growth of Phytophthora megasperma f. sp. medicaginis at much lower concentrations than structurally related chloroacetanilide herbicides. Metolachlor, among the latter, shows the highest antifungal activity, followed by propachlor. Dimethachlor and alachlor are only weakly inhibitory. A metalaxyl- and CGA 29212-resistant strain of P. megasperma f. sp. medicaginis shows cross-resistance to metolachlor and propachlor, but levels of resistance are much lower than observed with CGA 29212 and metalaxyl. Cross-resistance does not occur to dimethachlor and alachlor. All compounds except metalaxyl inhibit uptake of [ 3H]uridine by mycelium, propachlor being most effective. Effects are similar with both a metalaxyl-sensitive and a -resistant strain. CGA 29212, metalaxyl, and metolachlor inhibit incorporation of [ 3H]uridine into RNA by mycelium of the sensitive strain at concentrations not inhibitory to uptake. Metalaxyl slightly affects incorporation by mycelium of the resistant strain; the other compounds have a more pronounced effect but only at concentrations inhibitory to uptake. Metalaxyl, CGA 29212, and propachlor do not induce leakage of radioactivity from mycelium of both strains when added at high concentrations to cultures previously incubated with [ 3H]uridine; under these conditions incorporation by mycelium of the metalaxyl-resistant strain is significantly more affected by CGA 29212 and propachlor than by metalaxyl. Endogenous RNA polymerase activity of isolated nuclei from a metalaxyl-sensitive strain is inhibited by CGA 29212, metalaxyl, and metolachlor but not by propachlor, dimethachlor, and alachlor. Neither compound has any effect on endogenous RNA polymerase activity of isolated nuclei from a metalaxyl-resistant strain. CGA 29212 and metolachlor evidently have a metalaxyl-type of action. The presence of cross-resistance of the metalaxyl-resistant strain to propachlor also indicates a metalaxyl-type of action for this compound, although this could not be confirmed by an inhibitory effect of propachlor on endogenous RNA polymerase activity. In addition to a metalaxyl-type of action, CGA 29212, metolachlor, and propachlor have a second one that is also present in dimethachlor and alachlor, which lack the metalaxyl-type of action. The second mechanism of action, involving inhibition of [ 3H]uridine uptake, is most prominent with propachlor and might be related to the primary mechanism of action in plants of the chloroacetanilide herbicides.

Response of isolated nuclei to phospholipid vesicles: Analysis of the nuclear proteins after treatment with phosphatidylserine and phosphatidylcholine and comparison with heparin

Phospholipid liposomes affect the histone pattern of isolated rat liver nuclei. Multilamellar vesicles (MLV) obtained with phosphatidylserine (PS) release a large amount of the lysine rich histones, while those obtained with phosphatidylcholine (PC) do not induce significant changes with respect to controls. This different response has been compared to the effects obtained with Heparin, which slightly modifies the relative ratio of the histone fractions. These data might account for the mode by which phospholipids induce transitions of the chromatin structure and changes of the endogenous RNA polymerase activity.

THE MAJOR HISTONE‐LIKE PROTEIN FROM THE NONPHOTOSYNTHETIC DINOFLAGELLATE CRYPTHECODINIUM COHNII (PYRROPHYTA) IS PRESENT IN STATIONARY PHASE CULTURES1

ABSTRACTAn improved method is described for the isolation of nuclei from the nonphotosynthetic dinoflagellate Crypthecodinium cohnii (Seligo) Chatton in Grassé. The new method results in an increase in yield from about 13% to 45% or higher, with no loss in purity or activity of endogenous RNA polymerase. Most of this increase in yield is from the ability of 25% Ficoll to cause many cells to shed their walls, and from the stabilizing effect of hexylene glycol during cell disruption. The histone‐like protein, previously undetectable in chromatin from stationary phase cells, can now be demonstrated in chromatin from stationary as well as log phase cells. Preliminary experiments indicate that this protein inhibits transcription.

The periodicity of the change in the rate of RNA synthesis, endogenous RNA polymerase activity and DNA availability to acridine orange during the first cell cycle in the root meristematic cells of germinating Pisum sativum L.

During the 32 h of germination three stages of the increase in the transcriptional activity of chromatin of pea root meristematic cells occurred. The first two stages of chromatin activation took place during the pre-replicative period of the cell cycle and the third occurred at the S-period. The feature of the first stage, from 3 to 12 h of germination, was a 20–30% increase in the template availability of DNA. At the second stage, from 12 to 24 h, the RNA polymerase II activity being significantly elevated. The increase of the RNA polymerase I activity occurred at the S-period after 24 h of germination. The change patterns of DNA availability in the cells with different amounts of DNA (2C and 4C) are practically the same.

RNA polymerase activity and template activity of chromatin after butyrate induced hyperacetylation of histones in Physarum.

We have studied the effect of sodium-n-butyrate on endogenous RNA polymerase in Physarum polycephalum. 1 mM butyrate strongly reduces RNA polymerase activity measured in isolated nuclei or chromatin; both RNA polymerase A as well as the alpha-amanitin sensitive RNA polymerase B are equally affected. Despite a concomitant hyperacetylation of histone H4 the template activity of chromatin, as analyzed by in vitro transcription of the chromatin with exogenous RNA polymerase from E. coli or RNA polymerase II from wheat germ, remains unaltered as compared to untreated control chromatin, indicating that there is no positive correlation between histone acetylation and template activity of chromatin for transcription in this organism. The results further indicate, that butyrate acts primarily as a quick but reversible inhibitor of protein synthesis in Physarum; the fast decrease of endogenous RNA polymerase activity after butyrate treatment is due to inhibition of enzyme synthesis rather than inactivation of other factors necessary for transcription.

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.

Modulation of RNA Polymerase Activities in the Intestine of Adult Rats by Dietary Sucrose

The effect of sucrose feeding on endogenous intestinal RNA polymerase activities and on chromatin structure was studied in rats. Adult rats were given a 70% sucrose solution for 15 hours following a 48-hour starvation period. Comparison was made with rats starved for 63 hours and with ad libitum nourished animals. Chromatin-bound RNA polymerase I activity was significantly reduced by starvation. Sucrose feeding provoked a significant rise in the activity, but the level found in the nourished rats was not reached. The free poly[d(A-T)]-dependent RNA polymerase I activity of the sucrose-fed rats exceeded that of the starved and the nourished animals. Chromatin-bound RNA polymerase II activity was enhanced most markedly by sucrose feeding. The balance between the chromatin-bound and free enzymes was shifted towards the chromatin-bound state when compared to the starved and nourished rats. Starvation caused a reduction in the size of oligonucleosomes but sucrose feeding restored almost entirely the original pattern obtained in the nourished animals. These results reflect modifications in the structure of chromatin after sucrose feeding. The present report demonstrates that the adaptive processes triggered in the intestine by dietary sucrose are associated with changes in gene expression.

Characterization and transcription of bovine thyroid chromatin

1. 1. Thyroidal chromatin, isolated from bovine thyroid nuclei, has been characterized and the endogenous RNA polymerase activity was studied. 2. 2. The template activity of chromatin was investigated for exogenous homologous RNA polymerase I and II. 3. 3. The influence of chromosomal proteins on the transcription process was described.

Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin.

After treatment of SV40 minichromosomes with DNA topoisomerase I, the superhelicity in the bulk of the DNA extracted from minichromosomes is known to remain unchanged. However, we found that the DNA extracted from a small fraction of SV40 minichromosomes (2-5%), was almost completely relaxed, and covalently closed as shown by agarose gel electrophoresis. Thus, the DNA in these 2-5% of SV40 minichromosomes was probably torsionally strained (TS). The proportion of such TS minichromosomes is close to the estimated proportion of transcriptionally active minichromosomes. The distribution of the TS minichromosomes in sucrose gradient coincided with the distribution of transcriptionally active complexes. Both sedimented faster than the majority of minichromosomes. Furthermore, after treatment with topoisomerase I the relaxed minichromosomes could be quantitatively separated from the bulk of material by recentrifugation in a sucrose gradient. A major part of the endogenous RNA polymerase activity was recovered in the relaxed fraction. These data suggest that TS-minichromosomes correspond to transcriptionally active chromatin. After relaxation with topoisomerase I the TS minichromosomes lacked histones.

Transcriptional activation and structural alteration of wheat chromatin during germination and seedling‐growth

The chromatin was isolated from embryos or young seedlings of wheat, Triticum aestivum L. cv. Mukakomugi, and the template activity, the endogenous RNA polymerase activity, the susceptibility to DNase 11 and polylysine binding capacity were assayed. Both transcriptional activities of chromatin were very low for the first 12 h of germination, increased from 12 to 24 h, and then increased markedly during the next 48 h of post‐germination. During the 72 h of germination, the template activity and the endogenous RNA polymerase activity increased 10‐fold and 40‐fold, respectively, while they decreased from 72 h to 96 h of seedling growth. Seventy‐two h seedling chromatin was more susceptible to endonuclease and was bound with more polylysine than the germ chromatin. These results suggest that the structure of the chromatin available for transcription changes during seed germination and seedling‐growth.

Divalent cation-activated RNA synthesis in toluene-treated Escherichia coli.

Novel RNA polymerase activities (termed type II reaction) can be found in toluene-treated Escherichia coli with Ca2+, Fe2+, or endogenously bound cations, probably Mg2+. These activities are distinguishable from the well characterized DNA-dependent RNA polymerase (type I reaction) by: (i) their divalent cation requirements, i.e., the classical enzyme is activated by exogenously added Mn2+, Mg2+, or CO2+ ions; (ii) their relative resistance to inhibition by actinomycin D, rifampicin, and streptolydigin; (iii) their selective synthesis of low molecular weight RNA; (iv) their sensitivity to inhibition by arabinonucleoside 5'-triphosphates or deoxyribonucleoside 5'-triphosphates; and (v) the strict requirement for ATP in Ca2+ and bound cation-activated reactions. The Ca2+-activated and endogenous RNA polymerase activities are inhibited by orthophosphate. The properties of the type II RNA polymerase(s) are compared with those of polynucleotide phosphorylase, and dnaG gene product, and the RNA polymerase described by Ohasa and Tsugita.

Clomiphene and tamoxifen action in the rat uterus.

The antioestrogens clomiphene and tamoxifen exhibit both agonistic and antagonistic properties in the rat uterus. The effect of these antioestrogens on RNA polymerase activities in the rat uterus was investigated. Both compounds stimulated an early increase in the activity of endogenous RNA polymerase B similar to that observed after oestradiol treatment. A secondary stimulation of activity of RNA polymerase B was observed after treatment with oestradiol and both antioestrogens. In addition, the activity of endogenous RNA polymerase A was increased initially at 1 h by all three compounds but this activity was maintained at 24 h only by oestradiol.