Endogenous RNA Polymerase Activity Research Articles

The RNA polymerase activity of the preimplantation mouse embryo

The activation of the mouse embryo genome has been studied during early cleavage, in vivo. Individual embryos, prepared as whole mounts, were assayed for endogenous RNA polymerase activity. RNA synthesis was detected by autoradiography as the incorporation of [3H]UMP into an acid-insoluble product. No RNA polymerase activity could be detected in the pronuclei of one-cell embryos. Radioactive incorporation was first evident in the nuclei of two-cell embryos. This appeared to be confined to the nucleoplasm and could be abolished by alpha-amanitin but not by low concentrations of actinomycin D. Polymerase activity which was not affected by alpha-amanitin was first detected in the four-cell embryo, predominantly at the peripheries of the nucleoli. Nucleolar labelling increased markedly between subsequent cleavages, reaching a peak in early morulae. In one- and two-cell embryos, label incorporation could be found in the nucleus of the persisting polar body.

Separation of transcriptionally active from less active rat ventral prostate chromatin

Rat ventral prostate chromatin, prepared by a method in which marked intentional shearing was avoided, and analysed by sucrose density gradient centrifugation. yielded two major components. 1. 1. L chromatin, the quantitatively minor, less dense and/or structurally less compact fraction remained near the top of the gradient. H chromatin, containing 85–90% of the chromatin DNA, migrated into the more dense regions of the gradient, and in some experiments, was resolved into two fractions, H 1 and H 2. 2. 2. The H fraction exhibited a typical chromatin absorption profile; the absorption maximum of L chromatin was shifted towards 270 nm. 3. 3. The Tm app of these fractions was: whole chromatin, 83 °C; H 1, 85 °C; H 2, 80.5 °C, and L, 62.5 °C compared with 69 °C for calf thymus DNA. 4. 4. The ratio of L chromatin protein to DNA was 5 to 1, compared with 1.7 for unfractionated chromatin. The ratio of RNA to DNA for H and L fractions was 0.055 and 0.075. 5. 5. L chromatin was associated with TCA-precipitable radioactivity after ( a) in vivo administration of 14C-uridine or orotic acid; ( b) after in vitro incubation with E. coli RNA polymerase and subsequent separation on the gradient, and ( c) exhibited 10–20-fold greater template activity for E. coli polymerase than H chromatin when it was first separated on a gradient and then incubated for RNA synthesis. 6. 6. Residual endogenous RNA polymerase activity (cpm/unit A 260, nucleoplasmic enzyme II, 0.2 M ammonium sulfate, Mn 2+) associated with L chromatin was 5 times that of H. 7. 7. Both H and L chromatin were associated with similar amounts of radioactivity derived from injected 3H-testosterone, but L chromatin had a specific radioactivity (per μg DNA) 10 times that of H chromatin. 8. 8. The fraction of ‘minimally-sheared’ chromatin excluded from Bio-Gel A50m represented 10–20% of the total A ̀ 260 , exhibited a melting profile and Tm app similar to L fractions isolated on sucrose gradients, included 80% of the remaining in vivo-labelled RNA and was a better template for E. coli RNA polymerase than unfractionated chromatin. L chromatin from a sucrose gradient was also excluded from Bio-Gel A50m. By these four criteria, the two fractions are similar, and may be virtually identical. 9. 9. ‘Minimally-sheared’ chromatin did not degrade 14C-labelled rat liver RNA or DNA, and nucleoside triphosphatase activity, although present, did not interfere with the assay for RNA polymerase. 10. 10. Total proteins and histones from ‘minimally-sheared’ chromatin prepared after swelling overnight, and from ‘rapidly prepared’ chromatin subjected to more shearing were compared by SDS polyacrylamide gel electrophoresis (pH 7.2, total proteins) and electrophoresis at pH 3.5 (histones). No differences in the binding of Coomassie blue by histones or by total proteins from these preparations were evident. 11. 11. SDS-polyacrylamide gel electrophoresis of total protein from unfractionated chromatin and from the fraction excluded from Bio-Gel A50m exhibited many similarities in the high molecular weight region of the gels, and some quantitative differences in the region from about 1.5–4.5 × 10 4 D. Properties 1–11 are consistent with the seperation of a transcriptionally more active L from of chromatin (euchromatin) from less active H hetrochromatin.

Nuclear receptors for thyroid hormone: evidence for nonrandom distribution within chromatin.

Chromatin receptor proteins appear to mediate some actions of thyroid hormone. In this study, sheared mammalian chromatin containing [125I]triiodothyronine (T3) bound by these receptors was separated using sucrose gradient velocity sedimentation. T3-receptor complexes were distributed throughout the chromatin fractions, but were enriched in the slowly sedimenting fractions. The latter contain most of the template capacity for RNA synthesis and most of the endogenous RNA polymerase activity but a minor portion of the total DNA. Formaldehyde treatment of chromatin containing receptor-bound [125 I ]T3 resulted in fixation of radioactivity, as evidenced by its migration with chromatin after equilibrium density gradient sedimentation in both cesium chloride and Conray. This fixation implies that the T3 receptor protein is closely associated with chromatin. These results suggest that proteins involved in the regulation of gene function may be nonrandomly distributed within chromatin subfractions, and are consistent with a direct role for thyroid hormone in regulating genetic expression.

Activation of Nuclear RNA Polymerase by Sarkosyl

Treatment of mouse nuclei with the anionic detergent, sarkosyl, causes a several‐fold enhancement of endogenous RNA polymerase activity. The stimulatory effect of sarkosyl is due to two factors: a two‐fold increase in the initial activity of RNA polymerase II, and a prolongation of the total RNA polymerase reaction. Inhibition of RNase by sarkosyl could not account for either of these effects. Whereas sarkosyl and ammonium sulfate both stimulate RNA polymerase II, the effects are non‐additive, The RNA synthesized in the presence of sarkosyl sedimented primarily at 16‐23 S. whereas in the absence of detergent the RNA sedimented at 4–5 S. Sarkosyl caused the release of nearly all of the protein from cellular DNA, but apparently did not release initiated RNA polymerase. The stimulatory effect of sarkosyl on the initial rate of RNA synthesis catalyzed by RNA polymerase II can be explained by either of two extreme alternatives, or some combination of them. Either 50% of the enzyme is present in a totally repressed state, or all of the enzyme is partially inhibited by protein or other sarkosyl‐sensitive material. Copyright © 1975, Wiley Blackwell. All rights reserved

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.

Isolation, fractionation and template activity of the continuously-condensed chromatin of Euglena gracilis

A technique for isolating whole chromatin from nuclei of the lower eukaryote Euglena gracilis is presented. This chromatin, which appears under the electron microscope as uniformly condensed fibers, can, nevertheless, be subfractionated into distinct heterochromatic and euchromatic fractions. The euchromatin, comprising about 14% of the total DNA of the nucleus, contains over 80 % of the total endogenous RNA polymerase activity measured. The K m for this enzyme is higher than that found for prokaryotes, but falls in the range found for other eukaryotes. Stability constants, calculated from cation-chromatin binding data, suggest that internal carboxyl groups of chromosomal proteins, at least, are involved in the condensation of Euglena chromatin. The relationship between Euglena chromatin and that of higher eukaryotes is discussed.

Modification of the template capacity of liver chromatin for form-B ribonucleic acid polymerase by food intake in rats under controlled feeding schedules.

Nuclei from liver of rats accustomed to eating during the first 8h of a daily 12h dark period demonstrate an increased capacity to synthesize RNA 6H after the beginning of the feeding period. 2. This increase is accompanied by a higher yield of extractable form-B DNA-dependent RNA polymerase activity. 3. The endogenous RNA polymerase activity associated with nuclear chromatin is also stimulated by food intake. Both purified and chromatin-associated form-B enzyme activities exhibit different ionic strength requirements after food intake. 4. The sensitivity of exogenous (added) form-B-enzyme to changes in ionic strength changes after feeding when chromatin is used as template. 5. Chromatin extracted from the liver of fed rats is a better template for form-B-enzyme than chromatin extracted from starved rats.

Template capacity of mouse TLT hepatoma ghromatin fractions

1. 1. Isolated mouse TLT hepatoma chromatin has no measurable protease activity, whereas some of the chromatin fragments separated on exclusion agarose columns possess endogenous RNA polymerase activity. 2. 2. The template capacity of the template active chromatin fractions is 5–50-fold greater than usual when measured using a portion of E. coli RNA polymerase, apparently modified by purification and aging. 3. 3. Any contaminating nuclease activity in partially-purified RNA polymerase had minimal modifying effects on template capacity; the addition of pancreatic DNase but not RNase essentially eliminates the template capacity of chromatin fractions. 4. 4. Under conditions avoiding chromatin aggregation, similar template capacity patterns of chromatin fractions are obtained when the RNA incorporation reaction is executed either prior to, or subsequent to, the fractionation of the sheared chromatin.

Effect of plasma membrane contacts between cells on uridine incorporation and RNA polymerase activity

The effect of lateral plasma membrane contacts between neighbouring cells on uridine incorporation and endogenous RNA polymerase activity has been studied in kidney epithelial cells growing on a solid substrate in culture. A gradual increase of intercellular membrane contacts was parallelled by a gradual decrease in the rate of uridine incorporation. In contrast, the endogenous RNA polymerase activity remained unaltered when cells established membrane contacts between each other. The results are interpreted as reflecting a decreased uptake of uridine through the plasma membrane rather than a decline in RNA synthesis in cells with intercellular membrane contacts.

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.

Transcription of high-molecular-weight RNA from hen-oviduct chromatin by bacterial and endogenous form-B RNA polymerases.

With the aid of the ribonuclease inhibitor, heparin, techniques were developed to isolate high‐molecular‐weight RNA products synthesised by Micrococcus and homologous form B RNA polymerases using hen oviduct chromatin as template. These methods are suitable for analysis of RNA transcripts synthesised in vitro by a variety of enzymes and templates. A highly sensitive assay for ribonuclease, based on the ability of the latter to degrade ovalbumin mRNA, confirmed that chromatin and enzyme samples contained significant amounts of ribonuclease activity. Heparin completely inhibits initiation of both bacterial and homologous enzymes, but has no effect on elongation. The average rates of elongation of RNA polymerases on chromatin DNA could therefore be determined. These values were 3–6 nucleotides per second for bacterial enzyme and 1–2 nucleotides per second for homologous enzyme. By allowing initiation of bacterial enzyme prior to the addition of heparin, RNA chains approaching 2000 nucleotides in length (molecular weight 0.7 X 106) were synthesised after 1 min; at later times, these chains exceeded 5000 nucleotides in length. Hen oviduct chromatin contains endogenous RNA polymerase activity which represents predominantly the elongation of form B enzymes. RNA chains ranging from 100 to 2000 nucleotides long are still attached to these enzymes in isolated chromatin. By allowing elongation of these chains in vitro, products ranging from 2000 to 10000 nucleotides in length (molecular weight 0.7 to 3.0 X 106) were synthesised. Estimates of the number of endogenous RNA polymerases bound to chromatin DNA suggest that approximately 10% of the total form B RNA polymerase molecules per cell are retained in the isolated chromatin fraction.

Modification of the template capacity of chick-oviduct chromatin for form-B RNA polymerase by estradiol.

1 Within 2 h of administration of estradiol in vivo to immature chickens, ENA synthesis by both form A and B RNA polymerases is stimulated in isolated oviduct nuclei. 2 24 h after hormone treatment this effect is enhanced, and is accompanied by four- and two-fold increases in the level of extractable form A and B enzymes in the nucleus. 3 The endogenous RNA polymerase activity associated with nuclear chromatin is also stimulated by estradiol. 90% of this activity, as shown by the use of the drugs α-amanitin and rifampicin AF/0–13, represents from B enzymes elongating RNA chains. 4 Analysis of RNA chains synthesised by the endogenous RNA polymerases of chromatin from estradiol-treated animals (E-chromatin) or control animals (C-chromatin) indicates that there is 2 to 3-times the number of elongating RNA chains in E-chromatin than in C-chromatin. 5 E-chromatin is twice as efficient as template for purified hen magnum form B RNA polymerase compared with C-chromatin. Two independent methods demonstrate that this effect is not due to differential ribonuclease content in the chromatins. 6 The results suggest that at early times during the differentiation of oviduct cells by estradiol the chromatin template is modified, thus providing additional binding sites for RNA polymerase molecules.

Transcription of brain chromatin by ribonucleic acid polymerases from brain nuclei and from Escherichia coli.

1. Transcription of ox brain chromatin by brain nuclear RNA polymerase II and Escherichia coli RNA polymerase was studied. 2. The soluble chromatin prepared from brain nuclei contained DNA, RNA, histone and non-histone proteins. Such chromatin preparations did not display any endogenous RNA polymerase activity, when assayed in the presence of concentrations of KCl as high as 0.4m. 3. The chromatin-templated activity of brain nuclear polymerase II was stimulated by KCl, with an optimum around 0.25m. 4. The template activity of brain chromatin for brain nuclear polymerase II and E. coli enzyme was about 20-25% of that of pure DNA. This greatly repressed templatecapacity of chromatin was probably due to the acid-soluble chromosomal proteins. 5. Brain nuclear polymerase II was 3-4 times more active with dehistonized chromatin than with pure DNA as template, whereas bacterial enzyme was almost equally active with either of these two templates, reflecting the specificity of the transcriptional control mechanisms in mammalian cells.

Properties of cell nuclei isolated from various regions of rat brain: divergent characteristics of cerebellar cell nuclei.

Abstract— Cell nuclei were isolated in yields ranging from 38 to 61 per cent from six anatomically defined brain regions of the albino rat. To provide basic information for further studies of altered genomic activity in brain cell nuclei, various properties of these isolated nuclei were measured, including counts of their number, estimates of the distribution of sizes, amounts of RNA, DNA and protein, and endogenous RNA polymerase activity. DNA content per nucleus approximated the accepted value of 6 pg per diploid set of chromosomes. Distributions of nuclear size showed a sensitivity to the concentration of divalent cation, with a shift toward larger nuclear diameters as the Mg concentration was reduced. Cell nuclei from hippocampus, hypothalamus‐preoptic region, cerebral cortex, amygdala and midbrain plus brainstem were generally similar in yield, distribution of size, and RNA, DNA and protein content. Cell nuclei from cerebellum differed from those of other brain regions, in all of these parameters. The cerebellum contained a high content of DNA and had an enormous number (8 × 108 per g wet wt.) of cell nuclei of predominantly very small size and characterized by lower ratios of RNA, histones and non‐histone protein to DNA and lower endogenous activity of RNA polymerase than nuclei from other brain structures. These properties correlated well with properties of cerebellar tissue, namely, high content of small granule neurons and low ratio of RNA to DNA, and suggest that the small cerebellar nuclei may have relatively inactive genomes. The relationship of ‘large’ and ‘small’ cell nuclei to cell types in the brain is discussed.

Transcription of mammalian chromatin by mammalian DNA-dependent RNA polymerases.

The transcription of rat‐liver chromatin has been studied using partially purified from AI and highly purified form B DNA‐dependent RNA polymerases isolated from rat liver. Chromatin contains endogenous RNA polymerase activity. This activity is evident only when the RNA polymerase assays are carried out at high ionic strength and its appearance as the ionic‐strenght increases is thought to be due to the removal of chromatin‐associated proteins which block further transcription by the bound enzyme. This activity is insensitive to the rifampicin derivative AF/0‐13 which is shown to inhibit initiation of RNA synthesis by mammalian RNA polymerases. From AI polymerase is virtually inactive in the transcription of chromatin whereas the from B RNA polymerase (α‐amanitin sensitive) actively transcibes chromatin. This activity has two salt concentration optima: (a) 0.01 M (NH4)2SO4 or 0.03 M KCI; (b) 0.16 M (NH4)2SO4 or 0.25 MKCI. Both these activities are inhibited by rifampicin AF/0‐13. A comparison of the activity of the rat‐liver from B polymerase and the Micrococcus lysodeikticus RNA polymerase demonstrates that chromatin is a more efficent template for the rat‐liver enzyme. Evidence is presented that the rat‐liver form B and the Micrococcus lysodeikticus RNA polymerases bind to and transcribe from different sites on the chromatin DNA.

Nucleolar and extranucleolar chromatin of rat Novikoff hepatoma cells: Separation and partial characterization of RNA polymerase activities

The endogenous DNA-dependent RNA polymerase activity of rat ascites nucleoli was characterized in situ and compared with the activity assayed in whole nuclei and extranucleolar chromatin. Optimal activity of the nucleolar polymerase required Mg 2+ and low salt concentrations (0.08–0.10 M NaCl or 0.04 M (NH 4) 2SO 4). High salt (0.3–0.5 M NaCl or 0.3–0.4 M (NH 4) 2SO 4) inhibited the nucleolar polymerase activity but was stimulatory in whole nuclei and extranucleolar chromatin. The response of the nucleolar RNA polymerase to salt has some similarities to the bacterial polymerase system and suggests an enzyme pool not associated with the DNA template in nucleoli.

On the site of cortisol inhibition of thymus ribonucleic acid synthesis

Cortisol, in doses shown to inhibit RNA synthesis by thymus aggregate enzyme, did not alter maximum template activity of chromatin isolated from thymus glands of steroid-treated rats when assayed in the presence of E. coli RNA polymerase. Cortisol was shown, however, to decrease the amount of endogenous thymic RNA polymerase activity extractable from purified nuclear preparations. The magnitude of this effect was similar to the decrease in [ 3H]-UMP incorporation seen with intact nuclei or aggregate enzyme preparations when assayed without added bacterial RNA polymerase. When aggregate enzyme was obtained from cortisol-treated rats and assayed at low ionic strength (no NH 4Cl added to assay system) in the presence of added E. coli RNA polymerase, the usual cortisol-induced inhibition of RNA synthesis disappeared; when this same preparation was assayed in the presence of 0.3 M NH 4Cl, however, the inhibitory effect of cortisol was paradoxically converted to a stimulatory effect. Removal (by extraction) of endogenous RNA polymerase from control aggregate enzyme-like preparations was also shown to cause a stimulation in [ 3H]-UMP incorporation when the resulting preparation was assayed in the presence of 0.3 M NH 4C1 and added E. coli RNA polymerase. The paradoxical stimulation in [ 3H]-UMP incorporation seen after cortisol treatment was therefore explained on the basis that the presence of less endogenous RNA polymerase might allow greater access of the more efficient E. coli RNA polymerase to the template. These results are compatible with the suggestion that cortisol may inhibit thymic RNA synthesis by decreasing the amount, availability or activity of thymic RNA polymerase rather than by an effect on template activity.

RNA synthesis on native DNA complexes isolated from Streptomyces griseus and Escherichia coli.

1. A native DNA fraction was isolated from the young vegetative mycelium of Streptomyces griseus strain No. 52-1 and was compared with a similar fraction of Escherichia coli B. The endogenous RNA polymerase activities of these DNA fractions were examined. 2. Determining the base composition of the 14C labelled RNA synthesized in vitro, it was found that the conversion of 14C UTP into CMP residue of RNA is negligible in the DNA fraction of S. griseus, while it is significant in the DNA fraction of E. coli. By the proof of density gradient centrifugation, the RNA synthesized in vitro was mainly of messenger size in both species. 3. Template activity of the native DNA fractions was investigated in the presence of isolated E. coli RNA polymerase. When measuring the endogenous RNA polymerase and template activities of the DNA complexes at lower and higher ionic strength it was established that DNA complexes of E. coli under both ionic conditions behaved essentially as free DNA strands did, while endogenous and exogenous RNA polymerase activities in DNA complexes of S. griseus are inhibited in medium of lower ionic strength. Our results indicate the presence of some additional compound in the S. griseus aggregate which is absent in E. coli. This compound could function at lower ionic strength while it would be inactivated at higher ionic strength.

L-Asparaginase: Inhibition of endogenous RNA polymerase activity regenerating liver

The administration of l-asparaginase at the time of 70% hepatectomy of rats delays the onset of DNA synthesis and mitosis in the residual hepatocytes. The effect of this treatment on the increased RNA synthetic capacity of isolated regenerating hepatocyte nuclei was studied. Treatment with E. coli or agouti serum asparaginase delayed for 10–12 hr the increased incorporation of nucleotides into RNA under conditions which are reported to reflect endogenous RNA polymerase activity. No effect upon the increased capacity of nuclear template to react with exogenous RNA polymerase was demonstrated. These data demonstrate intranuclear increase of RNA polymerase activity is delayed by asparaginase treatment for a period of time similar to the delay in DNA synthesis and mitosis. The temporal relationship between these events suggests a possible functional association between them but the inhibition of polymerase activity represents only one of several effects upon the liver and may not be of unique importance.