Contemporaneous isolation of deoxyribonucleic acid-dependent ribonucleic acid polymerase and poly(A) polymerase from rat liver mitochondria.
Ontology highlight
ABSTRACT:
SUBMITTER: Gallerani R
PROVIDER: S-EPMC1163853 | biostudies-other | 1976 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Rats were fed for 6 days on a diet containing either 3 or 20% high-quality protein. Nuclei were isolated from liver and DNA-dependent RNA polymerases (EC 2.7.7.6) extracted with 1 M-(NH4)2SO4. The proteins were then precipitated with 3.5 M-(NH4)2SO4 and after dialysis applied to a DEAE-Sephadex column. The column was developed with a gradient of (NH4)2SO4. Polymerase I separated well from alpha-amanitin-sensitive polymerase II. The enzyme activities were compared between the two dietary groups. Rats that had received 3% protein showed a lower polymerase I activity per g wet wt. of liver, per mg of DNA and per mg of protein. Polymerase II was lower in activity per g wet wt. of liver and per mg of DNA, but was higher per mg of protein. Polyacrylamide-gel electrophoretograms showed a higher proportion of contaminating proteins in polymerase II fractions isolated from 20%-protein-fed rats. The data explain the lower activity obtained per mg of protein in these rats. It is concluded that a decrease in dietary protein content from 20 to 3% induces a fall in content and specific activity of RNA polymerase I and II in liver.
| S-EPMC1165505 | biostudies-other
Project description:1. The subunits alpha and beta of Halobacterium cutirubrum DNA-dependent RNA polymerase have been purified to electrophoretic homogeneity. Both have mol.wt. 18000 and they are required in equimolar amounts for optimum activity. 2. The instability of the complete enzyme, alphabeta, in the absence of salt is due to the rapid inactivation of the beta subunit in these conditions. 3. Nearest-neighbour analysis of the product formed on poly[d(A-T)] as template shows that the enzyme copies the latter accurately. 4. The enzyme initiates new chains with purine nucleoside triphosphates exclusively. 5. The product obtained in the standard assay conditions contains some high mol.wt. (>16S) material, but consists primarily of short chains, of average length 70-80 nucleotide units. 6. The template specificity of the complete enzyme has been studied at high and low ionic strength. Its extreme dependence on salt concentration is unrelated to the gross overall base composition of the DNA used. 7. T(7) DNA is transcribed asymmetrically and the enzyme selectively copies the T(7) ;early' genes. 8. Preliminary amino acid analyses of alpha and beta subunits show that their overall content of acidic, basic and neutral amino acids does not differ appreciably from that of Escherichia coli RNA polymerase.
| S-EPMC1178560 | biostudies-other
Project description:It is shown that tri-iodothyronine injected intravenously into thyroidectomized rats induces an early and transient activation of rat liver RNA polymerase II which could be demonstrated to occur 40-80 min after hormonal treatment. There was a simultaneous increase in the concentration of acidic proteins bound to chromatin.
| S-EPMC1165713 | biostudies-other
Project description:1. The stimulations of DNA-dependent RNA polymerase in isolated rat-liver nuclei by thyroid hormone, human growth hormone and testosterone are compared. 2. Single or multiple administrations of growth-promoting doses of tri-iodo-l-thyronine, human growth hormone and testosterone stimulate the Mg(2+)-activated RNA-polymerase reaction in nuclei from thyroidectomized, hypophysectomized and castrated rats respectively. The magnitude of stimulation was proportional to the degree of enhancement of liver growth by each hormone. After a single injection, the latent period preceding the stimulation was 1, 2 and 10hr. for growth hormone, testosterone and tri-iodothyronine respectively. The time-course of stimulation of enzyme activity and the synthesis of rapidly labelled nuclear RNA in vivo were also different for each hormone. 3. Growth hormone administration failed to stimulate the Mn(2+)/ammonium sulphate-activated RNA-polymerase reaction. Thyroid hormone and testosterone, however, stimulated it but the effect was less pronounced and occurred several hours later than that observed for the Mg(2+)-activated RNA-polymerase reaction. 4. In combination experiments, hypophysectomized or the thyroidectomized rats were given growth hormone or tri-iodothyronine in a single or repeated doses at levels that produced the maximum stimulation of Mg(2+)-activated RNA-polymerase activity. Taking into account the different latent period for each hormone, a single administration of the second hormone caused an additional stimulation of the enzyme activity. Similar additive effects were observed in thyroidectomized-castrated rats after treatment with tri-iodothyronine and testosterone. The magnitude of the additional stimulation caused by the administration of the second hormone was compatible with the capacity of that hormone to promote liver growth in rats deprived of it. 5. It is concluded that, although these hormones have some similar effects, the regulation of nuclear RNA synthesis may be mediated via different routes for each hormone.
| S-EPMC1264885 | biostudies-other
Project description:To investigate the possibility that the pattern of multiple DNA-dependent RNA polymerases of an animal cell exerts a controlling influence on its nature, the activities of these enzymes were compared in differentiated rat liver and in a rapidly growing minimal-deviation rat hepatoma cell line by using established techniques of enzyme extraction, separation and determination. Relative to the DNA content of the tissues, RNA polymerase activities of forms AI, AII and B were approx. ninefold, twofold and twofold higher respectively in the cell line than in the liver. Tests indicated that these results could not be explained by differences in extraction efficiency or by the presence of unbound inhibitors or stimulators of polymerase activity in the final enzyme preparations. New forms of the enzyme were not detected in either tissue. The significance of these findings with respect to the possible role of multiple RNA polymerases in the control of cellular activities is discussed.
| S-EPMC1178425 | biostudies-other
Project description:1. The Widnell & Tata (1966) assay method for Mg(2+)-activated DNA-dependent RNA polymerase was used for initial-velocity determinations of rat liver nuclear RNA polymerase. One unit (U) of RNA polymerase was defined as that amount of enzyme required for 1 mmol of [(3)H]GMP incorporation/min at 37 degrees C. 2. Colony fed rats were found to have a mean RNA polymerase activity of 65.9muU/mg of DNA and 18h-starved rats had a mean activity of 53.2muU/mg of DNA. Longer periods of starvation did not significantly decrease RNA polymerase activity further. 3. Rats that had been starved for 18h were used for all feeding experiments. Complete and tryptophan-deficient amino acid mixtures were given by stomach tube and the animals were killed 15-120min later. The response of RNA polymerase to the feeding with the complete amino acid mixture was rapid and almost linear over the first hour of feeding, resulting in a doubling of activity. The activity was still elevated above the starvation value at 120min after feeding. The tryptophan-deficient amino acid mixture produced a much less vigorous response about 45min after the feeding, and the activity had returned to the starvation value by 120min after the feeding. 4. The response of RNA polymerase to the feeding with the complete amino acid mixture was shown to occur within a period of less than 5min to about 10min after the feeding. 5. Pretreatment of the animals with puromycin or cycloheximide was found to abolish the 15min RNA polymerase response to the feeding with the complete amino acid mixture, but the activity of the controls was unaffected. 6. The characteristics of the RNA polymerase from 18h-starved animals and animals fed with the complete or incomplete amino acid mixtures for 1h were examined. The effects of Mg(2+) ions, pH, actinomycin D and nucleoside triphosphate omissions were determined. The [Mg(2+)]- and pH-activity profiles of the RNA polymerase from the animal fed with the complete mixture appeared to differ from those of the enzyme from the other groups, but this difference is probably not significant. 7. [5-(3)H]Orotic acid incorporation by rat liver nuclei in vivo was shown to be affected by the amino acid mixtures in a similar manner to the RNA polymerase. 8. The tryptophan concentrations of plasma and liver were determined up to 120 min after feeding with the amino acid mixtures. Feeding with the complete mixture produced a rapid increase in free tryptophan concentrations in both plasma and liver, but feeding with the incomplete mixture did not alter the plasma concentration. The liver tryptophan concentration increased at about 45min after feeding with the tryptophan-deficient diet. 9. There was a good correlation between the liver tryptophan concentration and RNA polymerase activity in all groups of animals. 10. It was concluded that the rat liver nucleus responded to an increase in amino acid supply by increased synthesis of RNA as a result of synthesis of RNA polymerase de novo. The correlation of tryptophan concentration and RNA polymerase activity appears to reflect the general amino acid concentration required to support hepatic protein synthesis and to produce new RNA polymerase. This new polymerase appears to differ from the basal RNA polymerase by its rapid synthesis and destruction, which may be a means of regulating RNA synthesis by the amino acid concentration in the liver.
| S-EPMC1179585 | biostudies-other
Project description:RNA (cRNA) was synthesized in vitro on a template of rat liver DNA and its hybridization with rat liver DNA was studied by using the nitrocellulose-filter method. Sonication of the DNA diminished its apparent capacity to hybridize with RNA by about 50%. This is not due to cross-linkage of DNA molecules, because it could be shown that less than 2% of the sonicated DNA was cross-linked. The effect is due instead to the small size of the sonicated DNA molecules. Below a single-stranded molecular weight of 5x10(5) the DNA showed a progressive loss of capacity to hybridize with decrease in molecular weight. Evidence is presented suggesting that the apparently diminished capacity of the DNA to hybridize is due to loss of hybridized DNA from the membrane filters. When cRNA at concentrations of up to 25mug/ml is annealed with sonicated total DNA, an apparent hybridization saturation value is found at which about 2.5% of the DNA is hybridized with RNA. Increasing the cRNA concentration tenfold brought about the hybridization of a second component of the DNA approximately equal in amount to the first. The renaturation of rat liver DNA was studied by measuring the fall in the extinction at 260nm and two different components of renaturation were observed within the reiterated fraction of DNA. By hybridizing cRNA with different fractions of rat DNA the two components of the hybridization curve are shown to correspond to the two components of the renaturation curve. The conclusion is drawn that at a cRNA concentration of 250mug/ml most of the reiterated fraction of rat liver DNA is hybridized after annealing for 16h under standard conditions (0.30m-sodium chloride-30mm-sodium citrate at 65 degrees C). Even with such a high cRNA concentration little or no hybridization of the slowly renaturing DNA fraction occurs. It is suggested that the most highly reiterated DNA component is poorly transcribed in vitro.
| S-EPMC1179592 | biostudies-other
Project description:1. DNA-dependent RNA polymerase was purified 150-fold from crude extracts of the extreme halophile Halobacterium cutirubrum. 2. The enzyme requires the presence of native DNA and all four nucleoside triphosphates to incorporate (14)C-labelled nucleoside triphosphate into an acid-insoluble ribonuclease-sensitive product. 3. It has an absolute requirement for both Mn(2+) and Mg(2+). 4. The polymerase requires a high salt concentration for stability, but is markedly inhibited by univalent cations. 5. Its molecular weight is very low compared with that of Escherichia coli RNA polymerase.
| S-EPMC1176638 | biostudies-other
Project description:A rapid procedure involving DNA-cellulose chromatography followed either by sedimentation in a high-salt glycerol gradient or by gel filtration is described for the complete purification of Escherichia coli DNA-dependent RNA polymerase.
| S-EPMC1177684 | biostudies-other
Project description:Rat liver mitochondria isolated in sucrose-N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [(3)H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1mug of actinomycin D/ml, 1mug of acriflavine/ml and 0.5mug of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose-EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose-TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP>>GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5'- or 2(3')-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2-3nmol/mg of protein. The [(3)H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20-21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.
| S-EPMC1173765 | biostudies-other