RNA Polymerase Activity Of Nuclei Research Articles

Action of the products of the deglycosylation of the nuclear glycoprotein of rabbit brain neurons on the polymerase activity of the neuronal nuclei

The influence of a glycoprotein and its fragments on the RNA polymerase activity of nuclei has been studied. Both the glycoprotein itself and its fragments suppress the RNA polymerase activity of the systemin vitro, and the carbohydrate moiety suppresses the activity more strongly than the protein moiety.

DNA and RNA polymerase activities of nuclei and hypotonic extracts of nuclei isolated from tomato golden mosaic virus infected tobacco leaves.

Nuclei and hypotonically leached extracts of nuclei prepared from tomato golden mosaic virus (TGMV)-infected Nicotiana benthamiana leaves have been used in in vitro DNA and RNA polymerisation reactions. The synthesis of virus-specific DNA was resistant to aphidicolin, sensitive to N-ethylmaleimide and dideoxy TTP, and stimulated by KC1 and ATP. Variably virion (+) and complementary (-) strand DNA of both the A and B genomic components were synthesised. Virus-specific RNA was synthesised in reactions which were initiated prior to nuclei isolation and leaching. From inhibitor studies and salt requirements RNA synthesis appeared to be catalysed by a DNA-dependent RNA polymerase type II enzyme. Both components of the TGMV genome were transcribed in a bidirectional fashion with a prevalence in some experiments of transcripts derived from DNA component A.

Effect of triiodothyronine (T 3) on ribonucleic acid polymerase activity in developing tadpole hindlimb

1. 1. Optimum conditions were studied tor the determination of RNA polymerase activity in nuclei isolated from Rana catesbeiana tadpole hindlimbs. 2. 2. Tadpole nuclei were tested at 15°C in the presence of spermicline (1.5 mM) bovine serum albumin (1.0%) and a high concentration of nucleoside triphosphates (1.0 mM). 3. 3. Tadpole nuclei exhibited a 60–70% higher total RNA polymerase activity with maximum activity of RNA polymerase I at 4 hr and RNA polymerase II at 8 hr after triiodothyronine injection. 4. 4. The results support a nuclear mechanism for the differentiation of tadpole hindlimbs induced by triiodothyronine.

Hormonal stimulation of 3H-orotic acid incorporation into RNA by serum-free cultured hepatocytes

Insulin and dexamethasone greatly stimulate the incorporation of 3H-orotic acid into RNA. Such a stimulation is associated to an increase in the uptake of the labelled precursor into the acid soluble fraction as well as in the specific radioactivity of the nucleoside plus nucleotide pool suggesting that hormone supplementation does not affect RNA synthesis by cultured cells. The lack of effect of insulin and dexamethasone on the level of total RNA polymerase activity in nuclei isolated from cultured hepatocytes is in line with this assumption. The hormone stimulated uptake of orotic acid is dependent on protein synthesis since it is completly abolished by cycloheximide.

Anabolic steroid metabolism in skeletal muscle

Three hundred and sixty male albino rats weighing 180 to 200 g were used to determine the effect of anabolic steroid hormones on adaptive changes in the synthesis of ribosomal RNA both in sedentary animals and in animals involved in a training programme. One injection of Retabolil (0.1 mg/100 g body weight) increased the α-amanitin insensitive RNA polymerase activity of nuclei from skeletal muscles. Fourteen h after this hormone injection the enzyme activity was 45% higher than in control animals and it remained at this level for 4 days. Under these conditions a selective binding of 19-nortestosterone with cytoplasmic proteins of skeletal muscle was found. Physical training increased the RNA polymerase activity by 50% ( P < 0.05). It was found that the testosterone binding capacity of a cytoplasmic extract from trained animals was 70% greater than that of the control animals ( P < 0.05). Four injections of Retabolil during training resulted in an additional increase of RNA polymerase activity of 40% ( P < 0.05) but reduced the testosterone binding capacity of the cytoplasmic proteins that occurred with training by 21%. These results demonstrate the effect of anabolic hormones in the regulations of RNA synthesis in skeletal muscle nuclei in the process of their adaptation to systematic physical training.

The effect of retabolil and training on activity of RNA polymerase in skeletal muscles

One hundred and twenty male albino rats weighing 180--200 g were used to determine the effect of anabolic steroid hormones on adaptive changes in the synthesis of ribosomal RNA both in sedentary animals and in animals involved in a training program. One injection of Retabolil (0.1 mg/100 g body weight) increased the alpha-amanitin insensitive RNA polymerase activity of nuclei from skeletal muscles. Fourteen h after this hormone injection the enzyme activity was 45% higher than in control animals and it remained at this level for 4 days. Under these conditions a selective binding of 19-nortestosterone with cytoplasmic proteins of skeletal muscle was found. Physical training increased the RNA polymerase activity by 50% (P less than 0.05). It was found that the testosterone binding capacity of a cytoplasmic extract from trained animals was 70% greater than that of the control animals (P less than 0.05). Four injections of Retabolil during training resulted in an additional increase of RNA polymerase activity of 40% (P less than 0.05) but reduced the testosterone binding capacity of the cytoplasmic proteins that occurred with training by 21%. The findings demonstrate the effect of anabolic hormones in the regulation of RNA synthesis in skeletal muscle nuclei in the process of their adaptation to systematic physical training.

Transcription and circular dichroism of chromatin in lymphoblastoid cell lines during proliferation and quiescence

RNA polymerase activity of nuclei and positive circular dichroism ellipticity in the 250–300 nm region of chromatin were studied in three lymphoblastoid cell lines: HRI, an Epstein-Barr Virus (EBV) positive virus-producing line; Raji, an EBV positive non-producer line; and SU-AMB-1, and EBV negative line. Raji consistently exhibited the highest and SU-AMB-1 the lowest polymerase activity and ellipticity. All lines showed higher RNA polymerase activity and positive ellipticity when exponentially growing than when stationary. In Raji it was shown that changes in RNA polymerase activity and chromatin ellipiticity were parallel. The changes in RNA polymerase activity of nuclei and circular dichroism ellipticity correlated with those of the proliferative state but not with the number of viral genomes or the production of virus or viral antigens.

Estrogen withdrawal in chick oviduct: Characterization of RNA synthesized in isolated nuclei using a mercurated precursor

1. As a prerequisite for analyzing the effect of estrogen on transcription in chick oviduct, we describe suitable methods for the synthesis (under conditions restricting reinitiation), and isolation of RNA transcripts from oviduct nuclei in vitro, utilizing mercurated UTP (Hg-UTP) as an RNA precursor and chromatography on sulphydryl-Sepharose (SH-Sepharose) to recover mercurated RNA (Hg-RNA). The techniques described include treatment of Hg-RNA with p- hydroxymercuribenzoate , to improve the efficiency of binding to SH-Sepharose, and elution of Hg-RNA from SH-Sepharose after treatment with 60% formamide at 90°C, to eliminate contamination by aggregated nucleic acid. 2. RNA synthesized by endogenous form B RNA polymerase (using either UTP or Hg-UTP as precursor) was recovered in nuclear lysates in the form of 30–85-S heterogeneous RNA · protein complexes, and after removal of protein, was 10–12 S in size. 3. The nature of RNA transcripts synthesized in vitro was examined by hybridization. More than 90% of the RNA was complementary to “unique” DNA sequences, and 50–60% of the hybridized RNA could be competed with homologous, steady-state nuclear RNA, indicating a significant degree of homology between in vitro transcripts and in vivo RNA. The level of homology was similar whether RNA synthesis was performed in low salt, or in high salt in the presence of heparin. Possible reasons for only partial competition in these experiments are discussed. 4. Withdrawal of estrogen from chicks leads to a 50% reduction in endogenous RNA polymerase activities in nuclei within 48 h. Similar levels of competition with Hg-RNA transcripts for “unique” DNA were obtained using oviduct nuclear RNAs isolated before or after estrogen withdrawal, and even with liver nuclear RNA. Thus, in oviduct, those sequences present in primary transcripts, and analyzed under our experimental conditions, are present in different hormonal states and also in other chick tissues.

Transcription and RNA polymerase stimulatory activity in nuclei isolated from soybean

Nuclei were isolated from light and dark grown soybean first leaves for use in studies on non-histone chromatin proteins. The ratio of DNA : RNA : protein for nuclei isolated from light grown and dark grown tissue was 1 : 0.30 : 2.81 and 1 : 0.33 : 4.02 respectively. The endogenous RNA polymerase activity of both types of nuclei was enhanced by exogenous DNA, and the addition of α-amanitin at a final concentration of 5 μg/ml resulted in 52% and 48% inhibition of RNA polymerase activity in nuclei from light and dark grown tissue respectively. The nuclei from dark grown tissue were nearly twice as active in RNA synthesis as the nuclei from light grown tissue on a DNA basis. Chromatin prepared from isolated nuclei had a 260/280 ratio if 1.63 for both light and dark grown tissue. The non-histone protein fraction of chromatin from both types of tissue was subjected to DNA-cellulose chromatography, which showed that material bound to the column and eluted at 0.6 M NaCl is capable of stimulating E. coli RNA polymerase 2–3-fold. The preparation stimulates transcription to a greater degree using native rather than denatured calf thymus DNA, and is more active at higher template concentrations. The non-histone protein fraction from soybean nuclei also stimulates purified soybean RNA polymerase II 2.5-fold when primed by soybean DNA, but to a lesser degree when primed by calf thymus DNA.

Evaluation of RNA polymerase activities in nuclei isolated from slow and fast skeletal muscles

Summary The optimal incubation conditions were determined for the assay of the α-amanitin-resistant, DNA-dependent RNA polymerase A and the α-amanitin-sensitive, DNA-dependent RNA polymerase B in nuclei isolated from rat skeletal muscles. Significantly higher levels of activity of RNA polymerase B were found in the nuclei isolated from the slow-twitch soleus compared with nuclei from the fast-twitch extensor digitorum longus.

Effect of estradiol on rat uterus DNA-dependent RNA polymerases. Studies with whole nuclei.

Immature female Sprague-Dawley rats (19-21 days old 50 gm) were used to systematically study DNA-dependent RNA polymerase activity in nuclei from the uterus after administration of 1 mcg estradiol dissolved in .1 ml of 9% NaC1 solution. Controls received only the saline solution. Experimental conditions included an excess of nucleoside 5-triphosphate and short-time assays (6 minutes) at low temperature to obtain the maximal velocity of the reaction and to minimize RNase activity. Nucleotide incorporation was measured in the absence (low ionic strength medium) or the presence (high ionic strength medium) of .25 M ammonium sulfate under various divalent cation concentrations either in the presence or absence of alpha-amanitin. The alpha-amanitin-resistant RNA polymerase activity of nuclei was identical when measured under low ionic strength conditions with either Mg or Mn and under high ionic strength in the presence of Mn (all at 4 mM concentrations). At high ionic strength alpha-amanitin-sensitive activity is about 10 times greater than activity measured at low ionic strength Mn. In all the experiments initiation of new RNA chains was negligible. Estradiol administration leads to: a 50% increase in alpha-amanitin-resistant activity within 1-2 hours under all experimental conditions; a 100% increase by 2 hours in alpha-amanitin-sensitive activity under low ionic strength conditions; but no change in alpha-amanitin-sensitive activity at 6 hours under high ionic strength conditions. Since enzyme activity measured under high ionic strength conditions mainly reflects the number of enzyme molecules engaged in transcription these results suggest that estradiol leads to an early increase in number of alpha-amanitin-resistant molecules engaged in the process of transcription while the number of alpha-amanitin-sensitive molecules remains constant. The increase in alpha-amanitin-sensitive activity obtained under low ionic strength conditions can be interpreted as either an increase in template activity or an activation of the molecules already engaged in the process of transcription or both.(AUTHORS MODIFIED)

Thermosensitivity of Nuclear RNA Polymerase during the in vitro Senescence of Mouse Fibroblasts

Embryonic mouse fibroblasts divide approximately twelve times in vitro prior to cessation of mitotic activity. During this period of cellular senescence the thermosensitivity of the RNA polymerase activity of isolated nuclei has been examined as a means of detecting the possible accumulation of defective enzyme molecules, as has been found by other workers for several cytoplasmic enzymes during the ageing of human fibroblasts in vitro. The total RNA polymerase activity of nuclei isolated from old (10th generation) cells is more thermoresistant than that of young (2nd generation) cells. However, the net RNA polymerase activity of nuclei from non-dividing (confluent) cells is more thermoresistant than that of exponentially growing cells of the same age. When allowance is made for the state of growth of the cultures, little difference is seens in the thermosensitivity of the activities of nuclei from old and young cells. Neither is there any difference between the thermosensitivity of the net activity of an established line of murine fibroblasts (L-cells) and cells in primary culture. Preheating nuclei increases the inhibition of their total RNA polymerase activity by alpha-amanitin, indicating that RNA polymerase II is the most heat resistance species present. There appears to be no difference between the thermosensitivity of the alpha-amanitin sensitive and resistance species of the enzyme in the nuclei of old and young cells. It is concluded that old cells resemble non-dividing young cells in containing a higher proportion of RNA polymerase II in their nuclei, resulting in greater thermoresistance of the total RNA polymerase activity over that of exponentially growing cells. However, there appears to be no increase in thermosensitivity of the enzymes with age.

Auxin-induced enhancement of RNA polymerase activity in soybean as a function of development

Studies on chromatin and solubilized RNA polymerase from control and 2,4-D treated whole hypocotyls indicate that the activity of RNA polymerase I is enhanced by the auxin treatment while the activity of polymerase II is essentially the same in control and treated tissue. However, different portions of the hypcotyl respond differently to the auxin treatment. The enhancement of solubilized polymerase I activity is greater in the lower half of the hypocotyl than it is in the upper half. In the hook region an inhibition of RNA solubilized polymerase activity is observed. Similarly, 2,4-D treatment results in an inhibition of chromatin bound RNA polymerase activity in nuclei isolated from first leaves. Chromatography on DEAE-Sephadex also reveals a selective enhancement of polymerase I in the upper and lower halves of the hypocotyl.

Similarities between 5α-dihydrotestosterone-receptor complexes from human and rat prostatic tissue: Effects on RNA polymerase activity

Protein macromolecules specifically binding [ 3H]5α-dihydrotestosterone ([ 3H]DHT) have been identified in cytosol and in nuclei prepared from human benign hypertrophie prostate. These macromolecules have similar properties to receptor proteins from other androgen-dependent tissues, as regards sedimentation coefficients on sucrose gradients and steroid specificity. Cytosol preparations from androgen-dependent tissues were able to transfer [ 3H]DHT in a recoverable protein-bound form to nuclei of other androgen-dependent tissues but not to nuclei of androgen-independent tissues. No transfer of radioactive steroid from cytosol of these latter tissues to any nuclei could be achieved. Labelled cytosol preparations from androgen-dependent tissues could stimulate the RNA polymerase activity of nuclei from androgen-dependent tissues but not that of nuclei from androgen-independent tissues. Cytosol preparations from these latter tissues could not affect RNA polymerase activity. Under suitable ionic conditions, human cytosol preparations containing DHT could stimulate both α-amanitin-sensitive and -insensitive RNA polymerase activities of human prostatic nuclei. However, rat ventral prostatic DHT-cytosol protein complexes were equally as efficient in performing this function, suggesting the possible involvement of specific DHT-receptor complexes in this process. It is therefore suggested that receptor molecules from androgen-dependent tissues may not be species specific but may share properties which would facilitate research into the understanding and ae.tiology of pathological conditions.

The effects of oestradiol-17beta on the ribonucleic acid polymerases of immature rabbit uterus.

Measurements of the endogenous RNA polymerase activities of nuclei isolated from immature rabbit uteri have shown that prior treatment of the animals with oestradiol-17beta has a profound effect on the apparent activities of both RNA polymerases A and B. Within 1 h of hormone treatment, the activity of RNA polymerase A is increased and continues to rise until about 4h when it reaches a plateau and remains steady until at least 8h. The activity of RNA polymerase B increases sharply after oestradiol treatment reaching an early maximum at 30-45 min. Thereafter this activity declines until by 1-2h it approaches control values but a second increase in activity then occurs with a maximum at 3-4h. Treatment of the rabbits with alpha-amanitin before the administration of oestradiol inhibits the hormone-induced stimulation of RNA polymerase A activity in isolated nuclei but when the administration of alpha-amanitin is delayed until after the early rise of RNA polymerase B activity, the oestradiol-induced stimulation of RNA polymerase A is retained. Similar results have been obtained in experiments with cycloheximide suggesting that the stimulation of RNA polymerase A activity by oestradiol is dependent on the hormone-induced stimulation of RNA polymerase B and the subsequent synthesis of protein using the RNA product of the early increase in RNA polymerase B activity. Measurement of the activities of RNA polymerases A and B after isolation of the enzymes from immature rabbit uterine nuclei before and after oestradiol treatment failed to show any differences. Therefore it would appear that the changes in the observed activities of RNA polymerases A and B in isolated nuclei are consequences of changes in the structure and function of chromatin rather than the results of modifications in the RNA polymerases themselves.

&lt;italic&gt;Brief Communication:&lt;/italic&gt; Estrogen-Dependent In Vitro Stimulation of RNA Synthesis in Hormone-Dependent Mammary Tumors of the Rat&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;

Incubation of estradiol in vitro at 25 degrees C with homogenates of carcinogen-induced mammary tumors of ovariectomized rats stimulated the magnesium-dependent RNA polymerase activity of nuclei of the hormone-dependent (HD) (regressing) tumors, but had no effect on this activity in nuclei of hormone-independent (HI) (growing) tumors. Furthermore, recombination of the nuclei and cytosol fractions of HD and HI tumors indicated that the in vitro effect of estradiol on subsequent tumor nuclear RNA synthesis required the estrogen receptor-containing cytosol but was specific to nuclei of the HD tumor. This constituted the first direct in vitro effect of estrogen on a specific biochemical process in an HD mammary tumor.

Role of DNA-dependent RNA polymerases II and III in transcription of the adenovirus genome late in productive infection.

DNA-dependent RNA polymerases I, II, and III were isolated and partially purified from KB (human) cells 18 hr after infection with adenovirus 2. As reported previously for the enzymes from other animal cells, RNA polymerase II was completely sensitive to low concentrations of alpha-amanitin (50% inhibition at 0.02 mug/ml), RNA polymerase III was completely sensitive to high concentrations of alpha-amanitin (50% inhibition at 20 mug/ml) and RNA polymerase I was totally resistant to concentrations of alpha-amanitin less than or equal to 200 mug/ml. RNA synthesis by the endogenous RNA polymerase activities in nuclei isolated from infected cells was completely sensitive to alpha-amanitin, thus suggesting that RNA polymerase I is not involved in viral DNA transcription even though it is present in these cells. The alpha-amanitin inhibition curve was biphasic and showed inflection points at about 0.02 and 20 mug/ml, suggesting the participation of both RNA polymerases II and III in the synthesis of RNA in these nuclei. Furthermore, at least a large fraction of the synthesis of the nuclear precursors to viral mRNA, monitored by hybridization to viral DNA, showed the same sensitivity to alpha-amanitin as did RNA polymerase II; and the synthesis of both viral 5.5S RNA and (presumably cellular) 5S RNA in the isolated nuclei exhibited the same sensitivity to alpha-amanitin as did purified RNA polymerase III. Thus, these data provide strong supporting evidence for previous studies which suggested the involvement of an RNA polymerase II in transcription of the adenovirus genome and demonstrate the role of an RNA polymerase III activity in the synthesis of viral 5.5S RNA and cellular 5S RNA.

Changes in RNA-Synthesizing Activity and Template Activity in Nuclei From Cotyledons of Developing Pea Seeds

Using Pisum sativum grown under controlled conditions, the pattern of cotyledon development has been defined in terms of fresh and dry weight, number of cells, and content of chlorophyll, starch, DNA, RNA and protein. The onset of storage-protein synthesis has been determined using antibodies to purified vicilin and legumin. During growth by cell expansion, DNA per nucleus of the parenchymatous cotyledon cells increases to an average of 50 C. Measurements of the endogenous RNA polymerase activity of nuclei and the template activity of chromatin isolated at various stages during seed development, suggest that the DNA beyond 2 C level does not contribute in a major way to RNA synthesis, although its template activity per unit of DNA is not diminished.

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

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

Properties of RNA Polymerases from B16 Melanoma

Some of the properties of RNA polymerases from melanotic and amelanotic B16 melanomas have been studied. Chromatin-bound RNA polymerase is activated by Mg ++ more the Mn ++ . Addition of ammonium sulfate was found to activate the enzyme. Preincubation with phospholipase A or phospholipase C as well as extraction with ether or iso-octane decreased the RNA polymerase activity in the presence of Mg ++ but not that in the presence of Mn ++ . The polymerase activity in the presence of high salt concentration (ammonium sulfate) was not affected by the phospholipases or by solvent extraction. Preincubation in the presence of trypsin was found to activate enzymatic activity in the presence of Mn ++ to a greater extent than in the presence of Mg ++ . RNA polymerase activity of melanoma mitochondria was decreased by treatment with phospholopase A or phosholopase C as well as by extraction with ether or iso-octane. Phospholipase D as well as wheat-germ lipase did not have any effect on the RNA polymerase activities of nuclei or mitochondria. α-amanitin was found to inhibit the nuclear RNA polymerase activity in the presence of Mn ++ at high salt concentration but not in the presence of Mg ++ . The RNA polymerase activity of melanoma mitochondria was not inhibited by alpha;-amanitin. The RNA polymerase activity in melanoma nuclei and mitochondria was inhibited by actinomycin D but rifamycin and rifampicin did not have any inhibitory action. Of the various tissues studied, the properties of the RNA polymerase from melanoma closely resembled those of the RNA polymerases from liver.