Incorporation of ribonucleic acid in vitro into dense ribonucleoprotein-like materials by isolated rat liver nuclei.
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ABSTRACT:
SUBMITTER: Suzuka I
PROVIDER: S-EPMC1185941 | biostudies-other | 1978 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:1. Isolated rat-liver nuclei incorporated [(14)C]UMP into RNA when incubated in the presence of Mg(2+) and all four ribonucleoside triphosphates. The addition of bentonite to the system diminished the breakdown of the newly synthesized RNA. 2. AMP and CMP were incorporated in the absence of the other added triphosphates, and in the presence of deoxyribonuclease. 3. RNA synthesized in the presence of Mg(2+) contained a high proportion of CMP and GMP, and sedimented in the regions of ribosomal RNA and of heavier molecules. About 1% of this RNA hybridized with homologous DNA, and hybrid formation was more effectively inhibited by nuclear RNA than by ribosomal RNA. 4. RNA synthesized in the presence of Mn(2+) plus ammonium sulphate had a composition intermediate between that of ribosomal RNA and of DNA, and about 4% of this RNA formed hybrids with DNA. 5. Less than 2% of the newly synthesized RNA was capable of forming ribonuclease- and deoxyribonuclease-resistant complexes. 6. It was concluded that the newly synthesized RNA arose as a result of an asymmetric process and included both ribosomal and DNA-like species.
| S-EPMC1270224 | biostudies-other
Project description:1. Isolated nuclei from starved rats showed a lowered incorporation of [(14)C]UMP into RNA. 2. The Mg(2+)-dependent incorporation was decreased by 30% after 1 day of starvation, but incorporation in the presence of Mn(2+) and ammonium sulphate decreased only after longer periods of starvation. 3. RNA synthesis by nuclei in the presence of excess of added RNA polymerase was unchanged after 1 day of starvation and was inhibited by 20% after 4 days. 4. The capacity of nuclei to bind actinomycin D was unchanged after 1 day and was decreased by 20% after 4 days of starvation.
| S-EPMC1179608 | biostudies-other
Project description:A cell-free system is described which permits a significant and prolonged synthesis of RNA in isolated rat liver nuclei, under conditions previously demonstrated to support normal nuclear processing and transport of both rRNA and mRNA. The system contains cytosol but not (NH4)2SO4 or other non-physiological components. Evidence is presented for cytosol factors which stimulate ribosomal, and to a lesser degree, non-ribosomal RNA synthesis.
| S-EPMC1165430 | biostudies-other
Project description:1. Nuclei from rat liver incubated with S-adenosyl[methyl-(14)C]methionine incorporated radioactivity into RNA and into lipid and protein. 2. All of the labelled RNA was extracted from the nuclei with trichloroacetic acid at 90 degrees C. 3. The [(14)C]methyl-group incorporation into the hot-trichloroacetic acid extract was 30% inhibited by the addition of actinomycin D (100mug/mg of DNA) or by the omission of CTP, GTP and UTP. 4. Assuming that the main substrate for this triphosphate-dependent methylation was newly synthesized precursor rRNA containing one methyl group/30 uridylate residues, it was calculated that approx. 60% of the [(14)C]UMP incorporated under similar conditions represented precursor rRNA synthesis. 5. In agreement with this, low concentrations of actinomycin D (approx. 1mug/mg of DNA) sufficient to abolish the triphosphate-dependent incorporation of [(14)C]methyl group inhibited 68% of the [(14)C]UMP incorporation. 6. The incorporation of [(14)C]UMP by nuclei from starved animals decreased progressively with increasing periods of starvation, whereas the triphosphate-dependent [(14)C]methyl-group incorporation was not further decreased after 1 day of starvation. 7. This suggests that precursor rRNA synthesis decreased within 1 day whereas other species of RNA were affected only after longer periods of starvation.
| S-EPMC1177074 | biostudies-other
Project description:Initiation of RNA synthesis was studied in an attempt to determine a possible molecular mechanism for age-related biochemical and physiological changes. Initiation of RNA synthesis was determined by incorporation of [gamma-32 P]ATP and of [gamma-32P]GTP into an acid-insoluble product by intact nuclei isolated from livers of Sprague-Dawley CD-strain rats of various ages. When the rats were grouped into young (0.75-9 months) and old (12-30 months) rats, a significant decrease (P less than or equal to 0.001) in incorporation of initiating nucleotides was observed. The rat population was divided into five age groups (0.75-3 months, 4-9 months, 12-18 months, 19-23 months and 30 months) for further analysis of the effect of age on the initiation of RNA synthesis. Analysis of data from these groups indicated a significant trend for an age-related decrease in RNA-synthesis initiation (correlation coefficient = 0.94). Long-term hypophysectomy coupled with minimal hormone-replacement therapy was shown to have a significant effect on the reversal of the age-related decrease in initiation of RNA synthesis. It was observed that initiation of RNA synthesis in nuclei from 19-month-old rats, hypophysectomized at 12 months of age, was closest to that in 3-month-old intact rats and was not significantly different from that in liver nuclei of 0.75-9-month-old intact rats.
| S-EPMC1162536 | biostudies-other
Project description:Cytoplasmic macromolecules were previously identified which regulate both qualitatively and quantitatively the release of messenger-like RNA from isolated nuclei. These macromolecules are now shown to be denatured at 45-50 degrees C and their synthesis is sensitive to pactamycin or cycloheximide. The putative regulatory proteins are essentially quantitatively precipitated with high specificity from the cytosol by streptomycin at a concentration 10-fold higher than that used to precipitate RNA. The nuclear concentration-dependence of RNA transport from successive samples of nuclei strongly suggests that the regulatory factors are recycled. Quantitative changes in the sequences transported at various dilutions of the cytosol suggest that not all the different classes of the putative regulatory macromolecules are present in an effective concentration at any one dilution.
| S-EPMC1172718 | biostudies-other
Project description:1. The loss of nucleic acids and protein from isolated HeLa-cell nuclei was studied. During 4hr. incubation at 37 degrees DNA was conserved, but appreciable amounts of RNA and protein were lost. 2. Two classes of nuclear RNA were distinguished: at least 75% of the RNA was lost from the nuclei relatively slowly through degradation to acid-soluble fragments; the rest of the RNA was lost much more rapidly, not only through degradation to acid-soluble fragments but also through diffusion of RNA out of the nuclei into the incubation medium. 3. The RNA that was preferentially lost was the fraction of nuclear RNA that was rapidly labelled when intact HeLa cells were grown in a medium containing radioactive precursors of RNA. 4. The RNA appearing in the incubation medium was apparently partially degraded and had a sedimentation coefficient of about that of transfer RNA. 5. Both the degradation of RNA and the loss of RNA from the nuclei were sensitive to bivalent cations. Low concentrations of Mg(2+) and Mn(2+) greatly increased the rate of degradation of the rapidly labelled RNA to acid-soluble fragments, and produced a corresponding decrease in the amount of RNA diffusing into the medium. At higher concentrations they suppressed both degradation and diffusion of RNA. The cations Ca(2+), Cu(2+), Zn(2+) and Ni(2+) all progressively inhibited both forms of loss of RNA. 6. Salts of univalent cations produced appreciable effects only at ionic strengths of about 0.2, when degradation to acid-soluble fragments was preferentially inhibited. 7. Both ADP and ATP inhibited loss of RNA at about 30mm. 8. It was concluded that the diffusion of rapidly labelled RNA out of the isolated nuclei was not related to the movement of RNA from nucleus to cytoplasm in vivo, but reflected the ease with which the rapidly labelled RNA detached from the chromatin and the permeability of the membranes of isolated nuclei.
| S-EPMC1187643 | biostudies-other
Project description:1. The effects of various ions on the Mg(2+)- and Mn(2+)/ammonium sulphate-activated RNA polymerase activities of isolated liver nuclei were studied. 2. The Mg(2+)-activated RNA polymerase reaction was inhibited by more than 60% by Cd(2+), SeO(3) (2-), Be(2+), Cu(2+), Co(2+), Ca(2+) and La(3+), all at 1mm concentrations. 3. The Mn(2+)/ammonium sulphate-activated RNA polymerase reaction was strongly inhibited by Hg(2+), Cd(2+), Cu(2+) and Ag(+). The effect of Hg(2+), Cd(2+) and Ag(+) was relieved by cysteine or mercaptoethanol. 4. Inhibition by Cu(2+) was not affected by addition of DNA, and was relieved only partially by EDTA or histidine. 5. No changes of RNA polymerase activities were observed in nuclei isolated from the liver of rats treated with copper albuminate.
| S-EPMC1187501 | biostudies-other
Project description:The reasons underlying reported discrepancies in the effects of ATP, ADP, adenosine 5'-[beta gamma-methylene]triphosphate, AMP + PPi, P-chloromercuribenzoate and F- on RNA efflux from isolated rat liver nuclei and on nuclear envelope nucleoside triphosphatase activity were investigated. The stimulatory effect of ADP was attributed to myokinase activity associated with the nuclei; this activity was eluted on repeated washing with nuclear incubation medium. In the absence of Ca2+ and Mn2+, ATP, adenosine 5'[beta gamma-methylene]triphosphate and AMP +PPi were found to promote release of both DNA and RNA. In the presence of 0.5 mM-Ca2+ and 9.3 mM-Mn2+, only ATP promoted RNA efflux to a significant extent. In the absence of spermidine, Ca2+ and Mn2+, nuclei released large quantities of DNA and RNA into the medium; this effect was promoted by p-chloromereuribenzoate. In the presence of the three cations, however, p-chloromercuribenzoate inhibited RNA efflux. F- caused a slight leakage of DNA from nuclei. The results are discussed in terms of models for the effects of ATP and analogues on RNA efflux and nuclear stability.
| S-EPMC1162541 | biostudies-other
Project description:1. RNA was isolated from crude nuclear preparations and from ribosomes derived from rat brain and liver. Nuclear RNA was obtained by lysis of the nuclei with sodium dodecyl sulphate, followed by denaturation and removal of DNA and protein with hot phenol. 2. Base composition analyses indicated that the cerebral nuclear RNA preparation contained a higher proportion of non-ribosomal RNA than the analogous hepatic preparation. 3. Sucrose-density-gradient analyses revealed a heterogeneous profile for each nuclear RNA preparation, with two major peaks possessing the sedimentation properties of ribosomal RNA (18s and 28s). 4. Template activities of both preparations were widely distributed through the sucrose density gradients. 5. The cerebral nuclear RNA preparation was more active than the hepatic nuclear RNA preparation in promoting amino acid incorporation in cell-free systems from Escherichia coli and rat brain. 6. Cerebral nuclear RNA stimulated amino acid incorporation in a cerebral ribosomal system even in the presence of an excess of purified E. coli transfer RNA. 7. It is concluded that a significant proportion of cerebral nuclear RNA has the characteristics of messenger RNA.
| S-EPMC1198432 | biostudies-other