Characteristics of the DNA polymerase beta-directed unscheduled DNA synthesis in isolated nuclei from adult rat liver.

Isolated nuclei from adult rat liver have been used as a model system to define several characteristics of the unscheduled DNA synthesis supported by DNA polymerase beta. Many of these characteristics have been found to reflect some catalytic properties (pH optimum, divalent cations requirement, dependence on all four deoxyribonucleoside triphosphates, apparent Km for dTTP) as well as sensitivity to various agents (differential inhibitors of eukaryotic DNA polymerases, phosphate, DNA intercalating drugs, chemical or thermal denaturation) commonly regarded as typical of DNA polymerase beta itself. Given the new picture of the enzymology of DNA repair synthesis which has recently emerged, none of the above characteristics seem to be suitable candidates as diagnostic tools of a repair polymerization process.

[Synthesis of RNA and DNA in isolated nuclei of Colpoda cucullus (Protozoi Ciliati)]. / Sintesi di RNA e DNA nei nuclei isolati di Colpoda cucullus (Protozoi Ciliati).

The ciliated protozoan Colpoda cucullus has been cultivated at 27 degrees C with gentle shaking in a baked lettuce infusion supplemented with Klebsiella suspensions. Under these conditions cells had a mean generation time of about 7 hours and could attain densities up to 20,000/ml and 45,000/ml in the log and stationary phase of growth, respectively. Nuclear preparations obtained from exponentially growing cells by the gum arabic-octanol method showed a satisfactory degree of purity and integrity. They consisted primarily of the large macronuclei attached to which the small micronuclei were sometimes visible. Upon incubation at 27 degrees C in conventional reaction mixtures nuclear preparations actively incorporated 3H-UTP and 3H-dTTP into acid-insoluble material. alpha-amanitin caused a 50% inhibition of RNA synthesis whereas aphidicolin did not affect at all DNA synthesis.

Increased levels of rat liver RNA polymerase I(A) and I(B) following the administration of triiodothyronine.

The levels of the transcribing RNA polymerase I(B) in the nucleus and of the non-transcribing RNA polymerase I(A) in the cytoplasm are both approximately doubled 24 h after a single i.p. injection of triiodothyronine into thyroidectomized rats. This suggests that the triiodothyronine-induced stimulation of ribosomal RNA synthesis is associated with an increase in the total RNA polymerase I content of rat liver cells.

Inhibition of isolated rat liver RNApolymerases I and II by aminoacridines.

9-Aminoacridine and 2 derivatives which contain hydroxyalkylic or aminoalkylic side chains in the 9-position totally inhibit the transcription of calf thymus DNA by rat liver RNA polymerases I and II. This inhibitory action does not always appear to be completely related to the ability of aminoacridines to intercalate into the DNA template.

[Influence of temperature on the preferential extraction of RNA polymerase I from hepatic nuclei of the rat]. / Influenza della temperatura sulla estrazione preferenziale di RNA poimerasi I dai nuclei epatici del ratto.

RNA polymerase I has been extracted from rat liver nuclei by three consecutive washings at 0 degrees C with a medium of relatively low ionic strength (0.15 M KCl) containing Mg++ rather than by incubating the organelles at 37 degrees C in the same medium, as originally proposed by Chesterton and Butterworth. The modified technique, which has the advantage of preventing a temperature-mediated conversion of form IB to IA, gives similar yields of RNA polymerase I and retains the capacity of preferentially extracting the enzyme with respect to the other forms of nuclear RNA polymerase.

[Reduced catalytic effectiveness of RNA polymerase I in hepatocytes of rats treated with cycloheximide]. / Ridotta efficienza catalitica della RNA polimerasi I nell'epatocita del ratto trattato con cicloeximide.

Rat liver RNA polymerase I solubilized from isolated nuclei and present in a soluble form in the cytoplasmic fraction has been analyzed by phosphocellulose chromatography 3 hours after the administration of cycloheximide. The antibiotic did not induce any change in the chromatographic properties of both nuclear and cytoplasmic RNA polymerase I. They appeared to remain in the IB and IA forms, characteristic of the transcribing (IB) and non-transscribing (IA) enzyme. While the level of the nuclear enzyme was not modified, the level of the cytoplasmic one appeared significantly increased. These results support previous ones indicating that the cycloheximide-induced inhibition of ribosomal RNA synthesis cannot be merely explained by a decrease in the nuclear or cellular level of RNA polymerase I. The cellular level of RNA polymerase I, taking into account the relative proportion of the enzyme found in nuclei and cytoplasm, appeared to be slightly increased. Cycloheximide administration did not seem to result in the appearance, in intact nuclei, of enzyme molecules in a free form or as blocked transcription complexes. It is concluded that the antibiotic affects the catalytic efficiency rather than the number of RNA polymerase I molecules actually engaged in the transcription of ribosomal cistrone.

Functional state of rat liver RNA polymerase IA and IB.

Phosphocellulose chromatography has been employed to characterize RNA polymerase I present in two different functional states in rat liver cells. The actively transcribing enzyme solubilized from nuclei appears to belong both to the IA and IB classes, whereas the non-transcribing enzyme present in the cytoplasmic fraction has been found to belong only to the IA class. Indirect and direct evidence indicates, however, that in isolated nuclei only the IB form is to be regarded as the physiological form of the enzyme, the IA form arising as a procedural artefact during the extraction process. It may, therefore, be concluded that rat liver IA and IB RNA polymerase are to be strictly regarded as the non-transcribing and transcribing form of the enzyme, respectively.

DNA-dependent RNA polymerase activities in hepatopancreas nuclei from Mytilus galloprovincialis Lamarck.

It has been shown that RNA synthesis in isolated hepatopancreas nuclei from Mytilus galloprovincialis is catalyzed by three DNA-dependent RNA polymerases (I, II and III) which resemble those identified in nuclei from mammalian cells. RNA polymerase I is active at 50 mM (NH4)2SO4, catalyzes the synthesis of GMP-rich ribosomal-like RNA and is completely resistant to the toadstool toxin alpha-amanitin. RNA polymerase II and III are active at higher (NH4)2SO4 concentrations, catalyze the synthesis of DNA-like RNA and are inhibited by very low (0.5-1 microgram/ml) and high (200 microgram/ml) concentrations of alpha-amanitin, respectively. Hepatopancreas nuclei retain considerable RNAase activity. Nuclear RNA polymerase activity may be underestimated since a part of the synthetized RNA is degraded.

Increased RNA polymerase activity in isolated liver nucleoli from thyroidectomized rats treated with triiodothyronine.

In isolated liver nucleoli from thyroidectomized rats the activity of the two RNA polymerase I populations, one of which is active and the other inactive towards the endogenous chromatin template, is greatly enhanced 10 and 24h after a single ip injection triiodothyronine (T3). When the nucleolar enzyme is solubilized and assayed with exogenous DNA as template, it retains, after T3 treatment, the same increase in activity as observed in intact nucleoli. On the contrary, the template availability, as judged by the binding capacity of isolated nucleoli for [3H]actinomycin D, does not appear to be modified by the hormone. These observations support the conclusion that the enhanced nucleolar RNA synthesis following T3 administration is due to an increased activity of the RNA polymerase I itself rather than to a greater availability of ribosomal RNA cistrons. The hormonal stimulation of both nucleolar RNA polymerase activities depends on continuous protein synthesis since it is almost completely abolished by the administration of cycloheximide.

Sequential stimulation of nuclear RNA polymerase activities in livers from thyroidectomized rats treated with triiodothyronine.

A single ip injection of triiodothyronine (T3; 30 mug/100 g BW) to thyroidectomized rats markedly stimulates RNA synthesis in isolated liver nuclei. The increased level of RNA synthesized in vitro by isolated nuclei does not depend on a reduced degradation of the nascent RNA molecules, since ribonuclease activities are not affected by the administration of T3. In addition, our results have confirmed previous findings of Tata et al. that the increase in nucleolar alpha-amanitin-resistant RNA polymerase I activity at low ionic strength always preceded the rise of the nucleoplasmic alpha-amanitin-sensitive RNA polymerase II activity at high ionic strength. Moreover, it has been found that a significant increase in an alpha-amanitin-resistant activity at high ionic strength occurs as early as 10 h after hormone injection. This enzyme, which forms RNA with a U to G ratio significantly higher than that of RNA synthesized by the nucleolar alpha-amanitin-resistant enzyme, is probably nucleoplasmic RNA polymerase III which is though to synthesize 5S and transfer RNAs. The possible role and the mechanism(s) of the early and concomitant increase in nucleolar and nucleoplasmic alpha-aminitin-resistant activities, and of the subsequent rise of RNA polymerase II activity following T3 administration are discussed.

6-(14C)-orotate uptake and rna synthesis by liver from thyroidectomized rats treated with 3,5,3'-triiodo-1-thyronine (t3).

It has been shown that T3 administration to thyroidectomized rats induces a marked increase of the in vivo labelling of liver nuclear RNA by 6-[14C]-orotic acid and, although to a lesser extent, of the radioactivity of the acid-soluble fraction. Therefore, in evaluating the real increase in RNA synthesis induced by T3 administration it has been taken into account that the hormone stimulates the uptake of the labelled precursor by the liver and enhances the specific activity of the nucleoside and nucleotide pool. The greater penetration of orotate into the liver does not depend on the increased RNA synthesis since it is not prevented by the administration of actinomycin D which markedly inhibits the transcriptional process. These findings indicate that the increased uptake of orotate and the stimulation of RNA synthesis represent distinct effects of the thyroid hormone on its target tissue.