Chromatin-bound RNA Research Articles

Dietary sugar promotes gene activation in intestinal cell chromatin of adult rats.

The effect of dietary sucrose on transcription activity of chromatin from intestinal cells of adult rats was investigated. Chromatin-bound RNA polymerase II activity was significantly enhanced after feeding sucrose to rats. RNA polymerase II transcribes the genes coding for pre-messenger RNA. The results indicate that feeding of sucrose increased the synthesis of pre-messenger RNA molecules coding for specific proteins which are involved in the adaptive processes of sucrose hydrolysis in intestinal cells.

Effects of partial hepatectomy on rna polymerase activities in mouse liver

1. 1. Chromatin-bound and poly[d(A-T)]dependent RNA polymerase 1 plus III and II activities of mouse liver were analysed 24 and 48 hr after partial hepatectomy. 2. 2. Chromatin-bound RNA polymerase I plus III activity showed an increase of 57%, at 24 hr and 51%, at 48 hr after partial hepatectomy. 3. 3. There was a decrease in chromatin-bound RNA polymerase II activity of 15% at 24 hr and 34% at 48 hr after partial hepatectomy. 4. 4. There was no significant changes in poly[d(A-T)] dependent RNA polymerase activities. 5. 5. Heparin caused an approximately 10-fold increase in chromatin-bound RNA polymerase II activity. The stimulation by heparin was significantly increased 48 h after partial hepatectomy. 6. 6. Anaesthesia and/or surgery had great influence on RNA polymerase activities. At 24 hr after operation, chromatin-bound RNA polymerase I plus III and II activities were depressed, and the liver cell chromatin was more susceptible to stimulation by heparin.

Non-random effect on RNA synthesis in liver chromatin by administration of dimethylnitrosamine to mice.

The effect of dimethylnitrosamine on functional activities of liver chromatin was studied in mice. After a single dose of dimethylnitrosamine injected i.v. (25 mg/kg body wt, 45 min before sacrifice) liver nuclei were isolated and incubated with micrococcal nuclease (EC 3.1.4.7) to an acid-solubility of 2.5% of total DNA. Chromatin was fractionated into a 1,200 g pellet P1, 102,000 g pellet P2 and supernatant fraction S2. Chromatin-bound RNA polymerase I plus III activity decreased 15% in the P1 and 25% in the P2 fraction. No changes in activity were observed in the S2 fraction. Chromatin-bound RNA polymerase II activity decreased 19% in the P1, 49% in the P2 and 32% in the S2 fraction. Heparin stimulated RNA polymerase II activity decreased 10% in the P1 and 44% in the P2 fraction. Formation of initiation in nuclear lysates with RNA polymerase from Escherichia coli increased after administration of dimethylnitrosamine suggesting an increase in the number of sites available for the start of new RNA chains. The results show that limited digestion of nuclei with endonuclease cleaves chromatin regions which are more affected by dimethylnitrosamine than the total chromatin suggesting a non-random effect of the hepatotoxin on chromatin. Modifications of the DNA template by dimethylnitrosamine is indicated by the change in number of initiation complexes.

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.

Lysine deficiency reduces transcription activity and concentration of chromatin proteins reversibly in rat liver.

The reversible effect of dietary lysine deficiency was studied in young adult rats. During 6 days on a lysine diet the rats maintained the same body weight. During 2 days of recovery body weight gain was that of the controls. Liver nuclei were isolated, incubated with micrococcus nuclease and chromatin fractionated in to a 2 000 X g pellet, 102 000 X g pellet and supernatant fraction. Chromatin-bound RNA polymerase 1 plus III activity decreased by 15% per mg of fractional and nuclear DNA and by 30% per total liver. The corresponding decrease of RNA polymerase II activity was 30% and 40%. Recovery from lysine deficiency was complete after 2 days of refeeding the amino acid. Chromatin proteins of the 102 000 X g pellet were characterized by polyacrylamide gel electrophoresis in sodium dodecylsulfate and by 2-dimensional gel electrophoresis. Quantitative but no qualitative differences between the proteins of the dietary groups were observed. Relative to DNA the non-histone proteins decreased in the lysine deficient group by 43% and histones by 10%. It is concluded that RNA synthesis is restored to its original level within 2 days of refeeding lysine after 6 days of lysine deficiency.

Cortisone acetate does not change free and chromatin-bound RNA polymerase II activities early after injection

FEBS LettersVolume 149, Issue 1 p. 41-43 Full-length articleFree Access Cortisone acetate does not change free and chromatin-bound RNA polymerase II activities early after injection Yasuji Okai, Yasuji Okai Department of Immunology, OEH University Medical School, Yawatanishi-ku, Kitakyushu, 807, JapanSearch for more papers by this author Yasuji Okai, Yasuji Okai Department of Immunology, OEH University Medical School, Yawatanishi-ku, Kitakyushu, 807, JapanSearch for more papers by this author First published: November 22, 1982 https://doi.org/10.1016/0014-5793(82)81066-1Citations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume149, Issue1November 22, 1982Pages 41-43 ReferencesRelatedInformation

DNA-binding property of free and chromatin-bound RNA polymerase II: Analysis by DNA—Sephadex G-25 chromatography

At present, the biological determinants that localize RNA polymerase molecules in the cell nucleus are not fully elucidated. Since it seemed possible that the enzyme’s DNA-binding property might be one of the determinants, we examined DNA-binding activity of the extracted RNA polymerase II by affinity chromatography of native DNA that was covalently coupled to Sephadex G-25 [ 1,2]. We found that: (1)

Calcium effects on free and chromatin-bound RNA polymerase II reactions

Calcium effects on free and chromatin-bound RNA polymerase II reactions

Wounding-induced enhancement of the activity of chromatin-bound DNA-dependent RNA polymerases in sweet potato root.

Chromatin fractions were isolated from intact and wounded sweet potato root tissues. The synthesis of RNA by the chromatin fractions was dependent on four ribonucleoside triphosphates and a divalent cation such as Mg2+ and Mn2+, Mn2+ being most effective. Whereas phosphate did not interfere with the polymerase reaction, it was totally blocked by pyrophosphate. The reaction was inhibited by DNase and actinomycin D as well as RNase and trypsin. The RNA polymerases of sweet potato root needed SH-groups for catalysis. Activity of chromatin-bound RNA polymerases (EC 2.7.7.6) promptly increased in the 6 hr after wounding and then decreased gradually up to 24 hr. Under the present experimental conditions it was mostly due to the activity of RNA polymerase I. RNA polymerase II contributed only about 5 to 15% to the total activity. The increase in the activity after wounding was completely inhibited by cycloheximide. Plant hormones such as 2,4-dichlorophenoxyacetic acid, gibberellic acid and dibutyryl cyclic adeno...

Characteristic distribution of two forms of chromatin-bound RNA polymerase II in rat liver nuclei.

We have shown that, in rat liver nuclei, the chromatin-bound RNA polymerase II is released as two different forms on digestion with micrococcal nuclease or DNase I (peak 1 and peak 2). To elucidate the origin of the two forms of the enzyme, we examined their distribution in fractionated chromatins obtained by mild micrococcal nuclease digestion of the nuclei. About half of the total peak 2 activity was recovered in a nuclease-sensitive chromatin fraction which contained DNA enriched in the sequences appearing in the polysomal polyadenylated mRNA. On the other hand, four-fifths of the total peak 1 activity was recovered in a nuclease-resistant chromatin fraction which contained DNA comprising only two thirds of the transcribed sequences. Furthermore, during the nuclease digestion, peak 2 activity was rapidly released from the chromatin, whereas peak 1 activity was gradually released. These results indicate that the two forms of RNA polymerase II are distributed differently in the cell nuclei.

A simple solubilization method for loosely and tightly chromatin-bound RNA polymerase II from rat liver nuclei.

Rat liver chromatin-bound RNA polymerase II could be differentially solubilized into two distinct populations, loosely and tightly bound enzymes, by a simple method. By this method the recovery of the solubilized enzyme from the chromatin fraction could be increased considerably as compared with the procedure of Yu (1). The two chromatin-bound enzymes had different properties: (a) Loosely bound enzyme was easily extractable from chromatin with relatively mild ionic condition (0.5 M NaCl); the tightly bound enzyme had to be solubilized by more drastic conditions such as sonication or nuclease treatment. (b) Loosely bound enzyme could not efficiently transcribe the chromatin template, but the tightly bound enzyme was active toward the same template. The latter enzyme is involved in the tight complex with the RNA synthesis activating factors. (c) Cycloheximide treatment in vivo suggests that the two enzymes have different turn-over rates. Therefore, with this simple solubilization method the functionally different two chromatin-bound RNA polymerase II activities can be estimated.

DNA-bound RNA polymerases during poliovirus infection: Reduction in the number of form II enzyme molecules

The anionic detergent Sarkosyl, known to dissociate most of the proteins from chromatin, was employed to determine the relative number of chromatin-bound RNA polymerases at different times of poliovirus infection. Both this approach and one using a coupled strategy employing Sarkosyl and 32P-labeled cordycepin indicate that the number of polymerase II molecules engaged in chain elongation decreases as infection proceeds. At 2 hr after polio infection, the irreversible inhibition of RNA polymerase II parallels the appearance of a viral-induced polypeptide in the nuclei of infected cells ( C. Fernández-Tomás (1982) Virology 116, 629–634). The possibility that the inhibition may occur at the level of chain initiation is discussed.

Reduced Transcription Activity of Rat Liver Chromatin after Protein Restriction and Selective Digestion of Nuclei with Micrococcus Nuciease

Restriction of protein intake in the diet has been shown to produce a change in transcription activity as determined in vitro. Conditions for a reduced transcription activity were investigated with selective digestion of nuclei with micrococcus nuclease (EC 3.1.4.7). Liver nuclei from young male rats fed a diet containing either 20 or 3% casein for 6 days were digested with 1.3 µg of micrococcus nuclease protein/mg of nuclear DNA (specific enzyme activity 15,000 or 150 units/mg of protein). Part of the chromatin-bound RNA polymerase activity was transferred from the 2,000 × g to the 102,000 × g pellet. Independent of the type of endonuclease used, the specific activity of chromatin-bound and soluble RNA polymerase I plus III was similar in the two groups of rats. After protein restriction RNA polymerase II activity was significantly diminished in the 2,000 × g pellet, and was unchanged in the 102,000 × g pellet. Heparinstimulated and soluble RNA polymerase II activities were significantly reduced. Number and length of RNA chains synthesized by chromatin-bound RNA polymerase I plus III remained unchanged by dietary treatment. After a low protein diet, RNA polymerase II in the absence and presence of heparin synthesized an unchanged number of RNA chains with reduced length. A selective digestion of chromatin with micrococcus nuclease is needed to show the reduction in RNA polymerase II activity after protein restriction.protein restriction RNA polymerase

Increased Transcription Activity of Rat Liver Chromatin after Protein Restriction and Limited Digestion of Nuclei with Micrococcus Nuclease

Protein restriction has been shown to produce either an enhancement or a reduction of transcription activity in vitro. Conditions for an enhanced transcription activity were investigated. Young male rats were fed a complete diet containing either 20% or 3% casein for 6 days. Body weight changed + 7.4 g/day and -0.5 g/day, respectively. Liver wet weights were 6.8 and 3.7 g and the DNA amounts/g were 1.31 and 1.67 mg. Liver nuclei were incubated without or with 1 unit micrococcus nuclease (EC 3.1.4.7) per milligram of nuclear DNA, and chromatin was fractionated into a 2,000 × g, a 102,000 × g pellet and a supernatant fraction. In the livers of rats fed a low protein diet, chromatin-bound RNA polymerase I plus III and II activity/mg of fractional and nuclear DNA and soluble RNA polymerase activity were increased, while heparin-stimulated RNA polymerase II activity remained unchanged. An increased number of chains was synthesized by RNA polymerase I plus III without change in chain length and incorporation rate per chain. The length and incorporation rate per chain increased, while the number of chains synthesized by RNA polymerase II did not. After stimulation by heparin, an increased number of short chains was synthesized at a lower rate of incorporation per chain. In the complex chromating structures the capacity for RNA synthesis was determined by specific enzyme activity, RNA chain number and length.protein restriction transcription activity rat liver nuclei

Auxin-induced changes in chromosomal protein phosphorylation in wounded potato tuber parenchyma

White potato tuber tissue reacts upon wounding with a rapid increase in activity of both chromatin-bound DNA-dependent RNA polymerase I and II as well as protein phosphokinase. This enhancement is more pronounced if 0.1 mM of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is added shortly after wounding. The effect of the hormone on protein kinases becomes evident only after a lag phase of about 10h and lasts throughout the wound-healing period. Different protein kinases with different substrate specificity (i.e. histone, phosvitin, casein phosphokinases) are distinctly more active in auxin-treated tissues. The phosphate is apparently introduced into proteins via seryl and threonyl bonds. Acyl or histidyl phosphates are not involved.The properties of protein phosphokinases are virtually identical in wounded and auxin-treated tissues. However, the pattern of chromosomal proteins and the pattern of their phosphorylation in hormone-treated tissues is different from those in wounded ones. A drastic stimulation of phosphorylation of both high and low-molecular weight chromosomal proteins is characteristic for auxin-treated cells.

Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates.

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I.

A new method for determining the number of RNA polymerases active in chromatin transcription

A new method is described for determining the number of RNA chains which are being actively propagated in an in vitro transcription system. This method employs [ 32P]-α-cordycepin triphosphate which terminates RNA chain propagation upon incorporation of the nucleotide analogue into an RNA transcript. This method is quicker and subject to fewer problems than end group analysis using [ 3H]nucleoside triphosphates. The auxin-induced increase in soybean RNA polymerase I activity was examined using this method. At 12, 24, and 48 hours after auxin treatment, the increase in chromatin-bound RNA polymerase I activity is predominantly due to a greater rate of RNA chain elongation rather than to an increase in the number of elongating RNA chains.

Release of transcriptionally active chromatin from liver nuclei by micrococcus nuclease

Chromatin-bound and free RNA polymerase activity was measured in nuclei before and after treatment with micrococcus nuclease. Isolated rat liver nuclei were incubated with the enzyme at concentrations of 0.2, 1.0, or 20 units/mg nuclear DNA. Chromatin was fractionated by differential centrifugation into a low-speed pellet, a high-speed pellet, and a supernatant fraction. In the low-speed pellet increasing concentrations of nuclease resulted in decreasing absorbance at 260 nm. A diminution in total chromatin-bound RNA polymerase I and II activities was also observed. The high-speed pellet showed a 2-fold increase in absorbance, total RNA polymerase I activity rose 4-fold, and RNA polymerase II activity 40-fold. An enrichment of chromatin-bound enzymes was also found in the high-speed supernatant fraction. Free poly[d(A-T)] dependent RNA polymerases were distributed in all 3 chromatin subfractions but the majority was found in the high-speed supernatant fraction. The results showed that transcriptionally active chromatin was released from total chromatin by treatment with micrococcus nuclease. The transcriptionally active regions were verified by their bound RNA polymerase activity.

Gibberellic Acid Activates Chromatin-bound DNA-dependent RNA Polymerase in Wounded Potato Tuber Tissue

Chromatin-bound DNA-dependent RNA polymerases react upon wounding of white potato tuber tissues with an increase in activity, which is additionally enhanced to 300% in the presence of 0.1 micromolar gibberellic acid (GA(3)). 2,4-Dichlorophenoxyacetic acid is only weakly effective and indoleacetic acid not at all. Wounding and treatment with GA(3) affect template availability of chromatin only slightly. The hormone has no effect on chromatin-bound RNA polymerases, if added in vitro.The enzymes from intact, wounded, and hormone-treated tissues possess similar characteristics: their activity is dependent on the presence of all four ribonucleotides and a divalent cation such as Mg(2+) or Mn(2+). However, the sensitivity of the enzymes from different preparations toward alpha-amanitin differs. Total RNA polymerase activity of chromatin was inhibited by alpha-amanitin to about 44% in intact, to about 22% in wounded, and only 15% in GA(3)-treated tissues. The relative activities of polymerases I and II were estimated by varying the (NH(4))(2)SO(4) and alpha-amanitin concentrations in the assay system. It is evident that GA(3) preferentially stimulates polymerase I and hence ribosomal RNA synthesis. RNA polymerase II is but slightly affected by GA(3). Nearest neighbor frequency analysis revealed that the RNA synthesized by the enzymes from the intact tuber is different from that of wounded or GA(3)-treated tissues.

Cyclic AMP binding protein from Jerusalem artichoke rhizome tissues

A cyclic AMP binding protein has been purified to electrophoretic homogeneity from Jerusalem artichoke rhizome tissues. Its MW is ca. 240 000 and the apparent constant of cyclic AMP binding to the protein is 2.3 × 10 −7 M. When tested using Millipore filter assay, cyclic AMP binding activity was enhanced by protamine and histone, but not by casein and phosvitin. Of several purine derivatives tested, only 5′-AMP and adenosine inhibited significantly the binding of cyclic AMP by the protein. The protein also binds adenosine and this binding is not affected by cyclic AMP or by other purine derivatives. The apparent binding constant for adenosine is 1.0 × 10 −6 M. The binding protein did not show protein kinase activity. In addition, it did not affect the chromatin-bound DNA dependent RNA polymerase of homologous origin, either in the presence or absence of cyclic AMP. The binding protein is devoid of the following activities: cyclic AMP phosphodiesterase, 5′-nucleotidase, adenosine deaminase and ATPase.