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The loss of rapidly labelled ribonucleic acid from isolated HeLa cell nuclei.

ABSTRACT: 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.

SUBMITTER: Watts JW

PROVIDER: S-EPMC1187643 | biostudies-other | 1969 Mar

REPOSITORIES: biostudies-other

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Project description:1. Rapidly labelled RNA from Escherichia coli K 12 was characterized by hybridization to denatured E. coli DNA on cellulose nitrate membrane filters. The experiments were designed to show that, if sufficient denatured DNA is offered in a single challenge, practically all the rapidly labelled RNA will hybridize. With the technique employed, 75-80% hybridization efficiency could be obtained as a maximum. Even if an excess of DNA sites were offered, this value could not be improved upon in any single challenge of rapidly labelled RNA with denatured E. coli DNA. 2. It was confirmed that the hybridization technique can separate the rapidly labelled RNA into two fractions. One of these (30% of the total) was efficiently hybridized with the low DNA/RNA ratio (10:1, w/w) used in tests. The other fraction (70% of the total) was hybridized to DNA at low efficiencies with the DNA/RNA ratio 10:1, and was hybridized progressively more effectively as the amount of denatured DNA was increased. A practical maximum of 80% hybridization of all the rapidly labelled RNA was first achieved at a DNA/RNA ratio 210:1 (+/-10:1). This fraction was fully representative of the rapidly labelled RNA with regard to kind and relative amount of materials hybridized. 3. In competition experiments, where additions were made of unlabelled RNA prepared from E. coli DNA, DNA-dependent RNA polymerase (EC 2.7.7.6) and nucleoside 5'-triphosphates, the rapidly labelled RNA fraction hybridized at a low (10:1) DNA/RNA ratio was shown to be competitive with a product from genes other than those responsible for ribosomal RNA synthesis and thus was presumably messenger RNA. At higher DNA/rapidly labelled RNA ratios (200:1), competition with added unlabelled E. coli ribosomal RNA (without messenger RNA contaminants) lowered the hybridization of the rapidly labelled RNA from its 80% maximum to 23%. This proportion of rapidly labelled RNA was not competitive with E. coli ribosomal RNA even when the latter was in large excess. The ribosomal RNA would also not compete with the 23% rapidly labelled RNA bound to DNA at low DNA/RNA ratios. It was thus demonstrated that the major part of E. coli rapidly labelled RNA (70%) is ribosomal RNA, presumably a precursor to the RNA in mature ribosomes. 4. These studies have shown that, when earlier workers used low DNA/RNA ratios (about 10:1) in the assay of messenger RNA in bacterial rapidly labelled RNA, a reasonable estimate of this fraction was achieved. Criticisms that individual messenger RNA species may be synthesized from single DNA sites in E. coli at rates that lead to low efficiencies of messenger RNA binding at low DNA/RNA ratios are refuted. In accordance with earlier results, estimations of the messenger RNA content of E. coli in both rapidly labelled and randomly labelled RNA show that this fraction is 1.8-1.9% of the total RNA. This shows that, if any messenger RNA of relatively long life exists in E. coli, it does not contribute a measurable weight to that of rapidly labelled messenger RNA.

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The loss of rapidly labelled ribonucleic acid from isolated HeLa cell nuclei.

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