Nuclear RNA Polymerase Activity Research Articles

Paradoxical effect of the long chain omega‐3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on nuclear transcription in vitro

Long chain unsaturated fatty acids are intimately involved in both the cause and prevention of various human diseases, e.g. obesity and diabetes, cardiovascular diseases, inflamation and allergy, and cancers including breast cancer. However, the long chain omega‐3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are believed to have protective effects against the above listed diseases. The mechanism(s) is not clear. But it is reasonable to believe that these beneficial effects are most likely through a common pathway deeply rooted at the gene level. This is to report the effect of EPA and DHA on nuclear transcription in vitro. We found that when EPA or DHA was added to the isolated rat liver nuclei, the nuclear RNA synthesis was stimulated over 2‐fold. This effect was determined to be a result of an increased rate of elongation of the nuclear RNA synthesis, and the rate at the initiation was not affected. Surprisingly, we found also that both EPA and DHA were able to strongly inhibit the enzyme activity of the isolated “free” nuclear RNA polymerase per se. We believe this is the first report on the paradoxical effect of EPA and DHA on the stimulation of the “engaged” and inhibition of the “free” nuclear RNA polymerase activity [Yu, FL. (1974) Two functional states of the RNA polymerases in the rat hepatic nuclear and nucleolar fractions. Nature 251, 344–346]. We hope that this finding will inspire further investigations into the mechanism of this paradoxical effect.

Prevention of chemical carcinogen DNA binding and inhibition of nuclear RNA polymerase activity by organosulfur compounds as the possible mechanisms for their anticancer initiation and proliferation effects

This report examines the transcriptional roles and DNA binding properties of the three major organosulfur compounds (OSCs) from garlic, diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS). We found DADS and DATS, but not DAS, could be activated by the versatile epoxide-forming oxidant dimethyldioxirane (DMDO) and could strongly inhibit nuclear RNA synthesis in vitro. We also found that when incubated together with [ 3 H ]-labeled 17β-estradiol (E 2) for activation by DMDO, DADS and DATS, but not DAS, were able to prevent the binding of [ 3 H ]E 2 to DNA. This preventive effect of DADS and DATS was confirmed when liver microsomes were used, and further verified by 32 P post-labeling analysis. Additionally, we discovered that the DMDO treated DADS and DATS, but not DAS, were able to directly inhibit the enzyme RNA polymerase per se. These novel findings provide new insights into the potential mechanisms of the preventive effects of OSCs on tumor initiation and promotion.

Age-related changes of protein- and RNA-synthetic processes in experimental hyper- and hypothyroidism.

The rate of liver and plasma protein synthesis and the activity of liver RNA polymerases 1 and 2 were investigated in rats of various age under experimental hyper- and hypothyroidism. The rate of plasma protein synthesis decreased with age more dramatically than that of liver proteins. Hyper- and hypothyroidism exerted opposite effects on protein synthesis in rats: stimulation and inhibition, respectively. The manifestation of these effects was age related. The thyroid status of animals also influenced the balance of protein synthesis. Thyroxin administration caused preferential incorporation of a label into blood plasma proteins. Changes of thyroid status of old animals insignificantly affected the absolute values of the label incorporation into proteins and the ratio of the label incorporation into local and secreted liver proteins. Age-related decrease of total hepatic nuclear RNA-polymerase activity was due to reduction of the template-bound functionally active forms of RNA-polymerases 1 and 2. Administration of thyroxin caused initial redistribution of the enzyme activity between template-bound and free fractions accompanied by the increase of template bound RNA-polymerases. Prolonged hormonal stimulus also caused an increase of free RNA-polymerases, which reflects the increased synthesis of these enzymes. Mecrazolyl administration reduced the activity of RNA-polymerase 1 and 2. All age groups were characterized by preferential reduction of the bound form. RNA-polymerase 2 activity decreased to a greater extent than that of RNA-polymerase 1. The data suggest age-determined reactions of the body to altered thyroid status.

Effect of Methyl Ester of Jasmonic Acid and Benzylaminopurine on Growth and Protein Profile of Excised Cotyledons of Cucurbita Pepo (Zucchini)

Treatment of excised marrow (Cucurbita pepo L., zucchini) cotyledons with methyl ester of jasmonic acid (MeJA) had no effect on their growth in darkness. On the other hand, MeJA induced the synthesis of three polypeptides (69, 60 and 43 kDa) and stimulated the accumulation of other polypeptides (97.4 and 53 kDa). These changes in the polypeptide profile were accompanied by a suppression of total protein and RNA synthesis as well as the activity of nuclear RNA polymerases. In contrast to MeJA, N6-benzylaminopurine (BAP) significantly enhanced cotyledon growth and stimulated protein and RNA synthesis. Furthermore, BAP, when applied together with MeJA, was able to counteract some effects of MeJA including the appearance of specific MeJA-induced polypeptide bands.

Effect of low-dose gamma radiation on nuclear RNA-polymerase activity and protein synthesis in liver cells of different-age chicks

The effect of low-dose (0.05 Gy) gamma irradiation of chicks in the preincubation period on subsequent liver cell RNA-polymerase activity and protein synthesis is studied at different ages. It is shown that irradiated chicks demonstrate an increased level of RNA-polymerase and increased incorporation of labeled amino acids into cytoplasmic proteins. Apparently, low-dose ionizing radiation induces activation of the liver cell genome, this being one of the main mechanisms of the general stimulatory effect of low doses.

RNA polymerases and transcriptional activity of the fat body during the final larval instar of the tobacco hornworm Manduca sexta

AbstractThe activities of RNA polymerases I and II in the fat body of the tobacco hornworm larva increased during feeding and then declined during wandering in the fifth instar (Sridhara and Gilbert, Dev Biol 45:7–21 [1975]). RNA polymerase II activity was much higher than that of RNA polymerase I. To determine how the differences in the activities of the two enzymes and their developmental changes correlate with changes in RNA and protein, the contents of these macromolecules were determined during the same period. RNA and protein increased by about 8 to 10‐fold during the feeding period. Activities of RNA polymerases I and II in nuclei, as well as those present in “free” (not transcribing) and “engaged” (transcribing) pools, were measured. Incorporation of labeled uridine into RNA by fat body cells in vitro was correlated to nuclear enzyme activities. The amounts of RNA polymerases I, II, and III were determined by the application of an ELISA with subunit‐specific monoclonal antibodies. Results showed that even though the in vitro activity of RNA polymerase II and its amounts are higher than those of RNA polymerase I and III, rRNA and tRNA syntheses predominated over mRNA synthesis in vivo. The activities and amounts of RNA polymerase II in the “free” pool were higher than those for RNA polymerase I. The “free” pools of the two enzymes increased as the larva progressed through the fifth instar. There appears to be little correlation between RNA polymerase activities and RNA synthesis during wandering, as RNA synthesis by the tissue and nuclear RNA polymerase activities declined faster than the amounts of the enzymes. The amount of RNA remained fairly steady during the same period. 20‐hydroxyecdysone did not affect either the RNA polymerase activities or RNA synthesis of the fat body cultured in vitro. © 1995 Wiley‐Liss, Inc.

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.

Effect of all- trans-retinoic acid on nuclear RNA polymerase activity in chemically-induced rat mammary tumors

In the present study the effect of all-trans-retinoic acid (RA) on nuclear RNA polymerase activity in N-methyl-N-nitrosourea (MNU)-induced mammary tumors was investigated. Three experimental protocols were used. (1) The tumor mince was incubated with 1 μM RA for 30 min at 30°C; the RNA polymerase activity was measured in the purified nuclei and compared with control nuclei. (2) In order to evaluate the influence of retinoic binding protein on enzyme activity, mammary tumor nuclei were incubated with RA bound cytosolic retinoic acid binding protein complex (RA-CRABP) at 25°C for 30 min. This step allows the complex to translocate into the nuclei. The enzyme activity in these nuclei was compared with the nuclei pre-incubated with buffer or cytosol. (3) Finally, the influence of the addition of RA-CRABP complex directly into the RNA polymerase reaction mixture was determined and compared with appropriate controls. Results indicated that the RNA polymerase activity in the nuclei of RA treated tissue as well as in the nuclei subsequent to the translocation step was significantly reduced. However, the addition of RA-CRABP into the reaction mixture did not alter the enzyme activity. These results suggest that alteration of RNA polymerase activity may be an essential step in the retinoid action in mammary tissues.

Induced diploid gynogenesis (suppression of meiosis I) in the loach: [formula omitted]. Institute of Developmental Biology, USSR Academy of Sciences, Moscow, USSSR

An abrupt activation of RNA transcription occurs at the early to mid blastula stage in loach (Misgurnus fossilis) embryos. This phenomenon was examined by assay of the RNA polymerase activity and the template efficiency of the nuclei isolated from different developmental stages.The nuclei isolated at the early blastula stage, 1 h before the activation of nuclear RNA synthesis, already show RNA polymerase activity. The RNA polymerase activity of the nuclei, expressed on the basis of DNA content, increased 3–5-fold from the early blastula to the mid blastula stage, and decreased at later stages.On the other hand, the template efficiency of the osmotically-shocked nuclei remained essentially constant from the early to mid blastula stage, before increasing at later stages.The results suggest that the activation of RNA synthesis which occurs in loach embryos between early blastula and mid blastula stages, is accounted for by an increase in the amount of nuclear RNA polymerase activity, rather than by an increase in the overall availability of nuclear DNA templates being transcribed.

Mechanism of Apparent Transcription Inhibition by Methyl Mercury in Cerebellar Neurons

We have investigated the mechanism of inhibition of RNA synthesis by methyl mercury (MeHg) in isolated neonatal rat cerebellar cells. Each of the three component steps involved in the incorporation of exogenous [3H]uridine into cellular RNA was examined separately in whole-cell and/or subcellular preparations. Nuclear RNA polymerase activity was measured in preparations containing both free nuclei and whole cells. Incorporation of [3H]UTP into nuclear RNA was found to be unimpaired at concentrations of MeHg that inhibited whole-cell incorporation of [3H]uridine by greater than 75%. Cellular uptake of [3H]uridine was assayed in cerebellar cells treated with KCN to deplete ATP levels and block subsequent phosphorylation reactions of transported uridine. Uptake activity under these conditions was unaffected by MeHg. Measurement of intracellular phosphorylation of [3H]uridine indicated that inhibition of this activity closely paralleled that of RNA synthesis. Quantitation of individual uridine nucleotides by polyethyleneimine-cellulose TLC revealed reduced levels of UTP and UDP whereas levels of UMP were elevated, suggesting that impairment of phosphorylation was not the result of cellular ATP depletion but, more likely, a direct effect on phosphouridine kinase enzymes. This mechanism of MeHg-induced inhibition of RNA synthesis was confirmed by assays of uridine phosphorylation using cell-free extracts in which exogenous ATP was supplied.

Initiation of transcription in nuclei is inhibited by captan

Captan ( N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) was examined for its effects on bovine liver nuclear RNA synthesis. Transcription was measured by [ 3H] UTP incorporation into either acid insoluble product or isolated RNA. Captan (1 mM) was found to inhibit RNA synthesis 50% in intact nuclei and 70% in a hypotonic lysate of nuclei. Individual RNA polymerase activities were distinguished in both intact nuclei and hypotonic lysate by α-amanitin sensitivity. Captan inhibited RNA polymerase I and II activities to an equal extent in both intact and lysate systems. The sulfhydryl compound dithiothreitol (DTT) protected the RNA polymerase activities from inhibition by captan. Initiation of transcription in intact nuclei was measured by [γ- 32P] ATP incorporation into purified RNA and was found to be inhibited 75% by 1 mM captan. This report describes the inhibition of nuclear RNA polymerase activities by captan and suggests a possible mechanism for its toxic effect on eukaryotic polymerases.

Nuclear RNA polymerase activity in tumor-host livers

This study has evaluated changes in RNA synthesis in livers under the distant influence of a malignant tumor. A transplantable-induced sarcoma (MCG 101), transplanted on inbred adult mice (C57BL/6J), was used. Activities of DNA-dependent RNA polymerase (EC 2.7.7.6) were measured in relation to RNA content and translational activity. Liver nuclei from freely fed sarcoma-bearing mice had increased RNA synthesis. As a consequence of this, RNA content per DNA was increased in liver tissue. This was independent of depressed food intake and malnutrition. Elevated RNA synthesis, proportional to the tumor burden was due to an increased proportion of chromatin-engaged RNA polymerase I and II activities. RNA polymerase III activity (template-engaged form) was unchanged when evaluated in isolated nuclei, but appeared to be increased in partially purified extracts of nuclei. RNA content in tumor-host liver was a composite of increased levels of rRNA and tRNA, whereas the levels of poly(A)+ mRNA could not be measured as increased. Overall translational activities in vitro of mRNA from liver tissue of tumor-bearing, pair-weighed, and freely fed tumor-free controls were qualitatively and quantitatively different. mRNA from tumor-bearing mice directed an increased synthesis, particularly of larger proteins (above 55,000 daltons) compared with control animals. The results support the conclusion that previous evidence of elevated net protein synthesis in tumor-host liver is accompanied by increased transcription of genes coding for RNA and also for some or several hepatic proteins.

The Effect of Hydroxycamptothecin in the Activity of RNA and DNA Polymerases Prepared from Murine Hepatoma Cells

Hydroxycamptothecin (HCPT) is an antitumor alkaloid isolated from Camptotheca acuminata indigenous to China. It could reduce the activity of nuclear RNA polymerase II and I(III) of hepatoma cells. HCPT at 25-100 microM caused a remarkable inhibition on DNA polymerase alpha whilst only a slight inhibition on beta. The inhibitory action on alpha was restored by increasing amounts of enzyme or DNA template, but unchanged by varying amounts of substrate. It is suggested that HCPT may exert a stronger inhibition on DNA replication process.

The inhibition of HeLa cell RNA synthesis following infection with vaccinia virus.

Vaccinia virus WR induces an immediate and rapid inhibition of HeLa S3 cell RNA synthesis as determined by pulse-labelling with [3H]uridine. The inhibition was independent of the purity of the infecting virus preparation and the multiplicity of infection over the range of 4 to 200 pk.f.u./cell. Inhibition was not evident in cells pre-treated with cycloheximide or following infection with u.v.- or heat-inactivated virus, suggesting that viral protein synthesis was required. There was no apparent selective inhibition of any particular species of RNA. Following infection, the uptake of [3H]uridine into cellular pools and the subsequent biosynthesis of UTP proceeded at the same rate as in mock-infected control cells. The rate of degradation of pre-labelled RNA was not enhanced in infected cells compared to controls. Analysis of the nuclear DNA-dependent RNA polymerase (EC 2.7.7.6) activities revealed a progressive and eventually total loss of RNA polymerase B activity, no obvious effect on RNA polymerase A and the presence of a viral RNA polymerase, the possible significance of which is discussed.

Analysis of activated androgen receptors in rat brain and anterior pituitary and ventral prostate glands: nuclear binding and RNA polymerase activity.

Cytosolic androgen receptors from neocortex, hypothalamus and anterior pituitary and ventral prostate glands were analysed by miniature isoelectric focusing and sucrose density-gradient centrifugation before and after precipitation of [3H]dihydrotestosterone (DHT)-bound complexes with ammonium sulphate. In the hypothalamus and neocortex (NH4)2SO4 precipitation appeared to cause heterodisperse peaks, and in the case of the ventral prostate there was a clear shift to a more basic isoelectric point. After sucrose density-gradient centrifugation all cytosols sedimented as large aggregates which appeared to dissociate into subunits in 0.4 M-KCl gradients. The functional significance of these altered forms was tested by nuclear uptake studies of cytosolic [3H]DHT-bound complexes, which could only be detected in brain and pituitary nuclei after prior precipitation with (NH4)2SO4, which also significantly increased extraction of ventral prostate [3H]DHT-bound complexes from the nucleus. The nuclei apparently responded to the (NH4)2SO4-precipitated and redissolved complexes by increased RNA polymerase activity. These results are consistent with the possibility that the neural androgen receptor is altered before interaction with the genome, and this alteration may be necessary for the action of the hormone to be expressed.

Alterations in myocardial RNA synthesis and RNA polymerase activity during normal growth.

The effect of normal growth (hypertrophy) on myocardial nuclear activity was investigated using male Wistar rats at 21, 50, and 100 days of age. Cardiac mass increased sevenfold during this age range. The concentration of RNA (mg X g-1) was the highest at 21 days and decreased 48% by 50 days of age and 68% after 100 days of development. RNA synthesis, corrected for alterations in the specific activity of the cytoplasmic nucleotide pool, was the highest at 21 days of age. After 50 days of growth, uridine incorporation was decreased fivefold. With continual growth (100 days), RNA synthesis was still reduced compared with the 21-day animals. RNA polymerase activity in myocyte nuclei showed little change in activity from 21 to 100 days of age. However, in the nonmyocyte fraction, RNA polymerase decreased threefold after 50 days of development. Collectively, these data suggest that the large decrease in myocardial RNA synthesis cannot be accounted for by a change in nuclear RNA polymerase activity and that an alteration in chromatin template capacity may be involved during this form of cardiac growth.

RNA synthesis in primary cultures of adult rat hepatocytes.

The ability of hepatocyte monolayers to synthesize RNA was investigated by measuring [3H]orotic acid incorporation into RNA and the total nuclear RNA polymerase activity as a function of the time in culture. The results demonstrate that primary cultures of hepatocytes maintained in a chemically defined serum- and hormone-free medium are able to synthesize RNA actively. This ability increases within the first 2 d of culture, despite the concomitant decrease in [3H]orotic acid uptake, and decreases only after 3 d. Factors such as serum, insulin, and dexamethasone, known to improve maintenance of functional hepatocytes, markedly stimulate the uptake of labeled precursor without apparently affecting the rate of RNA synthesis by cultured cells. It is suggested that the culture of adult rat hepatocytes provides a useful experimental model for the studies of hormonal regulation of transcription in liver.

Cytoplasmic steroid effects on nuclear RNA polymerase activity in canine mammary carcinomas

DNA-dependent RNA polymerase activity has been studied in isolated nuclei from canine mammary tumours. Initial experiments showed high levels of RNase activity in this tissue; accordingly, routine assays were terminated before loss of acid-precipitable radioactivity was evident. RNA polymerase A and B activity in isolated nuclei were shown to be increased by addition of receptor-containing cytosol previously incubated with oestradiol-17β, dihydrotestosterone or R5020. Where no receptor was present, as measured by saturation binding assays and sucrose density gradient analysis, there was no corresponding increase in polymerase activity. The steroid antagonists tamoxifen and cyproterone did not elicit any response even when their corresponding steroids produced a 1- to 2-fold stimulation of polymerase activity. Steroid-induced effects proved to be dose-dependent, with half maximal responses for oestradiol-17β 8 × 10 −8 M, R5020 2 × 10 −6 M and dihydrotestosterone 9 × 10 −6 M.

Differential effect of 2,3,7,8-tetrachlorodibenzo- p-dioxin on nuclear RNA polymerase activity in the rat liver and thymus

An increase in RNA polymerase B activity in hepatic cell nuclei was observed after a single intravenous injection of 1 μg/rat of TCDD. The enzyme activity was about 70% above the control value at 1 hr after injection and then declined rapidly. A secondary increase was evident at 24 hr. Prior to the secondary increase in RNA polymerase B activity, there was an increase in RNA polymerase A activity which was about 125% above the control value. The initial increase in RNA polymerase B activity was sensitive to both α-amanitin and actinomycin D injected 30 min before TCDD administration. In the thymus, an increase in RNA polymerase B activity was observed 4 hr after injection (25% above control value) but thereafter it declined and at 24 hr it was about 30% below the control value. RNA polymerase A activity was inhibited as early as 1 hr after injection but had returned to the control value at 4hr and then paralleled RNA polymerase B activity. Thus, TCDD stimulated RNA synthesis in the rat liver but the drug inhibited RNA synthesis in the rat thymus. Since these effects were detected as early as 1 hr after TCDD administration it is proposed that TCDD action requires transcriptional response and that this response may represent a primary site of TCDD action in the cell. The cellular entities or mechanisms which TCDD uses to alter transcription in the cell nucleus remain to be determined.

Specificity and nature of the rapid steroid-stimulated increase in sertoli cell nuclear RNA polymerase activity

This report explores the ability or various steroids to rapidly stimulate Sertoli cell RNA polymerase II activity and to compete with [ 3H]-androgens for nuclear and cytosol binding sites. Nuclear RNA polymerase II activity was significantly stimulated by a 1 nM concentration of the androgenic compounds testosterone, dihydrotestosterone (17β-hydroxy-5α-androstan-3-one), R1881 (methyltrienolone) and 5α-androstan-3α,17β-diol and also by the potent progestins 6-αmethylprogesterone and R5020 (17,21-dimethyl-19-nor-4-pregna-3,20-dione). Progesterone, 17α-hydroxyprogesterone, estradiol, androsterone, and 5α-androstan-3β,17β-diol were ineffective at 1 nM. Cytosol binding and nuclear accumulation of [ 3H]-androgen was effectively reduced by 100 fold molar excess of those androgens and progestins which stimulated RNA polymerase II activity. These data suggest that androgens and progestins bind to at least some of the same proteins in the Sertoli cell and may elicit the rapid stimulation of RNA polymerase II activity via a common mechanism. Agarose gel electrophoresis of the nuclear RNA synthesized as a result of exposure to testosterone indicated that it was heterodisperse and in part polyadenylated. Electrophoresis of the poly A +-RNA demonstrated that testosterone administration increased the incorporation of [ 3H]-UTP into RNA that was larger than 28 S.