Characterization of ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two different in vitro systems.

The ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two in vitro systems were studied and the following results were obtained. 1. When the isolated nuclei of regenerating rat liver labeled in vivo with [14C] orotic acid were incubated in medium containing ATP and an energy-regenerating system (medium I) release of labeled 40-S particles was observed. Analysis of these 40-S particles showed that they contained heterogeneous RNA but no 18 S or 28 S ribosomal RNAs and their buoyant density in CsCl was 1.42-1.45 g/cm3, suggesting that they were nuclear informosome-like particles released during incubation. 2. When the same nuclei were incubated in the same medium fortified with dialyzed cytosol, spermidine and yeast RNA (medium II), release of labeled 60-S and 40-S particles was observed. Using CsCl buoyant density gradient centrifugation, two components were found in the labeled ribonucleoprotein particles released from nuclei in this medium. The labeled 60-S particles were found to contain 28-S RNA as the main component and their buoyant density in CsCl was 1.61 g/cm3, suggesting that they were labeled large ribosomal subunits. The labeled 40-S particles contained both 18 S RNA and heterogeneous RNA and they formed two discrete bands in CsCl, at 1.40 and 1.56 g/cm3, suggesting that they contained small ribosomal subunits and nuclear informosome-like particles. 3. These results clearly indicate that addition of dialyzed cytosol, spermidine and low molecular yeast RNA to medium I causes the release of ribosomal subunits or their precursors from isolated nuclei in the in vitro system.     

Phosphorylation of nuclear matrix proteins in isolated regenerating rat liver nuclei

The phosphorylation of nuclear matrix proteins from normal and regenerating rat liver nuclei was examined using an $\text{in vitro}$ system of isolated nuclei and γ-³²P-ATP. Phosphorylation of the nuclear matrix proteins was 2–3 fold higher than that of the total nuclear proteins in normal nuclei. The level of phosphorylation of the matrix proteins was enhanced an additional three fold at a period in liver regeneration (12 hours) just preceding the onset of DNA synthesis.     

Formation of deoxyribonucleoside 5'-monophosphates dependent on DNA synthesis in nuclei isolated from regenerating rat liver

Hydrolysis of deoxyribonucleoside 5'-triphosphate, resulting in deoxyribonucleoside 5'-monophosphate formation dependent on DNA synthesis, was observed in nuclei isolated from regenerating rat liver. The intensity of the hydrolysis in nuclei varied at different times after partial hepatectomy, showing its maximum at 48 h. The rates of DNA synthesis altered corresponding to the intensities of hydrolysis. Proportionality between decrease in DNA synthesis and decrease in dNMP production was also observed in nuclei treated with various inhibitors of DNA synthesis. The formation of dNMP was detected with the four DNA substrates, indicating no involvement of specific dNTPase . Although regenerating nuclei contained a nonspecific dNTPase activity that can cause release of dNMP , this activity was independent of DNA synthesis and not inhibited by inhibitors of DNA synthesis. These results indicated that regenerating liver nuclei had two different activities for dNMP production; one is DNA synthesis-dependent, and the other is a non-specific dNTPase activity. This paper has focused on the former activity.     

Nucleotides and coenzymes in nuclei isolated from rat liver.

In rat-liver nuclei, isolated by the non-aqueous technique, the concentrations and labelling rates of the purine moiety of acid-soluble nucleotides were determined and compared with corresponding data for non-fractionated tissue and nuclei-free cytoplasm. Livers were used from untreated rats, from rats with a highly stimulated synthesis of NAD and from rats following a heavy metabolic load with adenosine. Under all circumstances, the nuclear and cytoplasmic concentrations of nucleotides (e.g. ATP and its dephosphorylated forms, pyridine nucleotides) and of free glucose were practically identical. Specific radioactivities after a pulse with formate also indicated a nucleo-cytoplasmic equilibrium for purine-containing nucleotides. It is concluded that precursor pools for nuclear biosyntheses as well as energy supply for other nuclear activities may be determined by an analysis of the non-fractionated tissue.     

Properties of DNA-methylase reaction in isolated nuclei of normal and regenerating rat liver]

Evidence from comparative determination of DNA radioactivity methylation degree of acidic extraction and chlorophormic deproteination of the samples suggest that the former technique is a more efficient one. The properties of the DNA-methylase reaction in isolated rat liver nuclei were studied. The DNA-methylase activity is found to be considerably stable during incubation of the nuclei at 37 degrees C; a broad pH-optimum in the alkaline region is observed (pH 8.6--9.8); this activity is inhibited by Mn2+, nucleotides, actynomycin and S-adenosyl methionine analogs and is activated by Mg2+; the incorporation of methyl groups into DNA is reversible. The data suggest that the DNA-methylase activities of the nuclei isolated at different stages of regeneration do not show substantial variations. No differences in DNA methylation before and after DNA synthesis in the regenerating nuclei were observed. Inhibition of DNA synthesis in the course of regeneration does not decrease the level of DNA methylation. The interrelationship between methylation and replication of DNA is discussed.     

Partial purification of a ribosomal ribonucleic acid methylase from rat liver nuclei and methylation of undermethylated nuclear ribonucleic acid from regenerating liver of ethionine-treated rat

S-Adenosylmethionine-dependent ribosomal RNA (rRNA) methylase has been purified approx. 90-fold from rat liver nuclei. The partially purified methylase catalyzes the methylation of base and ribose in hypomethylated nuclear rRNA prepared from the regenerating rat liver after treatment with ethionine and adenine. The enzyme has an apparent molecular weight of about 3 x 10(4) and a sedimentation coefficient of 3.0 S. The enzyme is optimally active at pH 9.5 and sensitive to p-chloromercuribenzoate. Thiol-protecting reagents, such as dithiothreitol, are necessary for its activity, and the enzyme requires no divalent cations for its full activity. This enzyme did not efficiently transfer the methyl group to nuclear rRNA from normal rat liver, compared with hypomethylated nuclear rRNA. Methyl groups were mainly incorporated into pre-rRNA larger than 28 S, and the extent of 2'-O-methylation of ribose by this enzyme was greater than that of base methylation in the hypomethylated rRNA. No other nucleic acids, including transfer RNA (tRNA) and microsomal RNA from normal as well as ethionine-treated rat livers, tRNA from Escherichia coli, yeast RNA, and DNA from rat liver and calf thymus, were significantly methylated by this methylase. These results suggest that partially purified rRNA methylase from rat liver nuclei incorporates methyl groups into hypomethylated pre-rRNA from S-adenosylmethionine.     

Formation of tetraploid nuclei in regenerating rat liver]

The proliferation of binucleated cells in the liver of young Wistar rats after partial (2/3) hepatectomy was studied by means of autoradiography and cytophotometry. The analysis of the kinetics of 3H-thymidine labelled cells has shown that both the bi- and mononucleated cells proceed through the mitotic cycle and enter mitosis simultaneously. The nuclei of 2nX2 cells enter prophase simultaneously but fuse during metaphase, so that the subsequent division results in the formation of mononucleated tetraploid cells.     

Effect of an inhibiting factor isolated from rat liver on DNA polymerases in regenerating rat liver.

We partially purified an inhibitory factor (LIFE), isolated from 105,000 g supernatant of a saline adult rat liver homogenate. LIF stopped in vitro cell multiplication by blocking the G1--S transition, and reduced in vivo [3H]thymidine incorporation into liver DNA in two-thirds hepatectomized rats. This reduction in DNA synthesis was observed at 24 hr after hepatectomy, even when the LIF was injected before the beginning of the S phase, 10 hr after hepatectomy, i.e. when DNA polymerase activity had not yet increased. Under these experimental conditions, LIF in vivo treatment prevented alpha DNA polymerase activity from increasing after partial hepatectomy, so that enzyme activity at 24 hr in LIF-treated rats decreased compared to the controls. No direct inhibitory effect of LIF on alpha DNA polymerase was detected. LIF did not affect beta DNA polymerase. These results suggest that LIF plays a part in controlling liver growth.     

Intranucleolar localization of the RNA polymerase A activity in isolated nuclei of regenerating rat liver

Ultrastructural autoradiography was used to visualize RNA polymerase A activity in parenchymal cell nuclei isolated from normal and regenerating (3, 24, 36 and 48 h after partial hepatectomy) rat liver. High resolution autoradiography showed that the activity of RNA polymerase A which was not inhibited by α-amanitin in a concentration of 0.8 μg/ml, was restricted to the nucleolus. Both the distribution pattern and number of grains were similar in control liver and regenerating liver 3 h after hepatectomy. Twentyfour, 36, and 48 h after hepatectomy nucleoli were enlarged and labeling was distinctly increased. In all experimental groups the activity of RNA polymerase A was located within fibrillar components of the nucleolus. The association of enzyme activity with this component was especially distinct in later stages (36 and 48 h) of liver regeneration. These results suggest that the fibrillar component of the nucleolus contains the active template for ribosomal RNA (rRNA) synthesis in rat liver cell nuclei.