Electron microscopic and biochemical characteristics of nuclei and nucleoli isolated from rat liver

Rat liver nuclei were freed of cytoplasmic contamination by washing with Triton-X-100 and subsequent centrifugation through 2.2 M sucrose. Electron microscopic examination showed that the outer membranes of the nuclei had been removed, but that the nuclei otherwise resembled the nuclei of intact liver. Morphological studies, chemical estimations of DNA, RNA, and protein and the estimation of cytoplasmic "marker" enzymes suggested that contamination of nuclei by cytoplasmic components was limited. These nuclei were obtained in yields of about 70% and were suitable for the isolation of nucleoli. Nucleoli were isolated by the breaking of the nuclei by ultrasound and subsequent differential centrifugation. In ultrastructural appearance, the isolated nucleoli resembled nucleoli in intact tissue. However, at high magnifications the "granular" component of isolated nucleoli appeared to consist of tightly twisted fibers. The nucleoli could be obtained in yields of at least 30%, and the values for the chemical composition of the isolated nucleoli agreed with values previously reported.     

Ultrastructural and biochemical studies of the isolated fibrillar component of nucleoli from Novikoff hepatoma ascites cells

Previously we have presented evidence for a direct relationship between post-mitotic new membrane formation and changes in the electrical membrane characteristics during cytokinesis of Xenopus eggs [1, 2]. In the present study the phenomena underlying the hyperpolarization of the electrical membrane potential during cytokinesis were investigated. Total Na⁺ and K⁺ contents at the onset of the first and second cleavage were measured independently by flame spectophotometry and by means of ion-selective electrodes. Total Cl^? content was measured by the latter method only. The water content was determined from the difference between wet weight and dry weight. ³H₂O-influx experiments yielded an independent estimate of the water content (0.737 μl/egg), a rate constant for the influx of 1.412 × 10^?3 sec,^?1 and a low water permeability of 1.87 × 10^?5 cm sec^?1. They furthermore revealed the absence of intracellular water compartmentation. The Na⁺, K⁺ and Cl^? concentrations remained constant during first cleavage at 58.6, 87.3 and 62.6 mM/l cell water, respectively. The membrane potential (E_m), the membrane resistance (R_m), and the intracellular ion activities of Na⁺, K⁺ and Cl^? (a_Naⁱ, a_Kⁱ and a_Clⁱ) were measured simultaneously and continuously during cleavage, using conventional glass microelectrodes and ion-selective microelectrodes. a_Naⁱ showed an increase from 19.4 to 22.4 mM concomitant with the hyperpolarization of E_m and the decline of R_m. a_Kⁱ and a_Clⁱ remained constant at 51.4 and 53.1 mM, respectively. From the calculated activity coefficients it was concluded that all Cl^? ions were free, whereas 30% of the K⁺ ions and 60% of the Na⁺ ions were bound. The influence of changes in the ionic composition of the medium on E_m was analysed in the uncleaved egg, in normally cleaving eggs, and in eggs cleaving outside the vitelline membrane. The latter conditions leads to exposure of the whole area of newly formed membrane to the medium. The cell membrane of the uncleaved egg exhibits no permselectivity. In normally cleaving eggs the relative permeability $\text{P}{}_{\mathrm{Na}}\text{P}{}_{\mathrm{K}}$ was 0.73, while in eggs cleaving outside the vitelline membrane it was 0.19. It was concluded that the changes of E_m during cytokinesis are due to the insertion of a part of the newly formed membrane into the cell surface. The K⁺ permeability of this new membrane is at least five times greater that that of the pre-existing membrane. The possible role of the hyperpolarization of E_m in the regulation of the cell cycle is briefly discussed.     

Comparative studies of the effects of galactosamine and actinomycin D on nuclear ribonucleoprotein particles from rat liver

Nuclear ribonucleoprotein particles of 75S were obtained from rat liver nuclei after mild sonication and isotonic salt extraction only when the preparation was carried out in the presence of a cytosolic ribonuclease inhibitor. Particles of 38S were isolated in the absence of inhibitor. The 38S nuclear ribonucleoprotein (nRNP) particles showed a protein/RNA ratio of 8, and a buoyant density of 1.39 g/ml in cesium chloride solution. They were further characterized by the pattern of their proteins on sodium dodecylsulfate (SDS)-acrylamide gel electrophoresis. Incorporation of [³H]cytidine into nuclear RNA was reduced to approx. 20% of controls 3 and 6 h after administration of galactosamine or actinomycin D. However, when [³H]cytidine was administered 30 min prior to the drugs a decrease of radioactivity in 38S nRNP particles to 43 and 81% of controls was found after 3 h. The yield of 38S particles 3 h after galactosamine or actinomycin D dropped to 41% and 78% of controls, and after 6 h to 43 and 70%, respectively. Six hours after galactosamine or actinomycin D treatment, the protein to RNA ratio increased to 13.3 and 9.1. No significant changes in protein patterns 3 h after treatment with galactosamine or actinomycin D were observed. Possible mechanisms, such as impaired transport of 38S nRNP particles after actinomycin D treatment or increased loss of particles due to a defective nuclear membrane after galactosamine administration are discussed.     

A comparative study of the protein components of ribonucleoprotein particles isolated from rat liver and hepatoma nuclei

To solve the mechanism for the complete cessation of DNA synthesis in Tetrahymena cells involved in the amino acid starvation, the nature of DNA polymerase activity was investigated in crude enzyme preparations or in toluene-permeabilized specimens. In crude enzyme preparations from growing cells, ³H-TTP incorporation into acid-insoluble products showed little dependency on exogenous DNA template, while incorporation increased markedly in the presence of ATP. These characteristics were very similar to those of replicative DNA synthesis in permeabilized Escherichia coli.Variations of DNA and RNA polymerase activities following transfer of exponentially growing Tetrahymena cells to amino acid-deprived medium showed that in the crude enzyme preparations DNA polymerase activity dropped sharply within 3 h after the transfer and practically no activity was detected thereafter, whereas RNA polymerase activity did not disappear in the same preparations. Such enzyme kinetics coincided well with the kinetics of in vivo synthesis of the corresponding nucleic acid.The cessation of DNA synthesis in the amino acid-starved cells may be due not to the activation of DNase or a soluble polymerase inhibitor, nor to the deficiency of each kind of deoxyribonucleoside triphosphate or magnesium ion or ATP generation system. It follows from this that the cessation of DNA polymerase activity in the starved cells may be due to the deficiency of DNA polymerase or its associated factor(s) as a reflection of short life-span of such a protein.     

Studies on the preparation and properties of ribonucleoprotein particles from rat liver nuclei

Ribonucleoprotein particles of 38 S were extracted from rat liver nuclei with isotonic salt buffer under concomitant sonication. The fate of the endogeneous nuclear RNAases assayed with poly(A), high molecular weight yeast RNA and rapidly labeled hnRNA was followed during the preparation of 38-S nuclear ribonucleoprotein (nRNP) particles. Essentially all the RNAase activity could be removed from the particle preparation. The effect of synthetic RNAase inhibitors on the nRNP particles was studied. Upon extraction of nuclei with 0.14 M NaCl, approximately 38% of the total nuclear radioactivity was found in the 38-S nRNP particles. By two successive extractions of the remaining chromatin with either isotonic or 0.22 and 0.3 M NaCl, an additional 25 and 9% of rapidly labeled hnRNA of 38 S particle were dissociated from chromatin, respectively. The chromatin components, DNA, nonhistone proteins, histones and RNA were determined after successive salt extractions. Particularly alterations in the nonhistone proteins and RNA were found. The protein patterns upon SDS-acrylamide gel electrophoresis of the salt-extracted chromatin preparations were compared with those of the 38-S nRNP particles. Particularly proteins in the molecular weight range of 32 000-43 000 were dissociated from chromatin after treatment with 0.22 or 0.3 M NaCl.     

Protein A24 lyase activity in nucleoli of thioacetamide-treated rat liver releases histone 2A and ubiquitin from conjugated protein A24

Isolated liver nucleoli from rats treated for 3 days with thioacetamide contained an enzyme activity which specifically degraded conjugate protein A24. Two-dimensional polyacrylamide gel electrophoresis indicated that the amount of protein A24 in chromatin decreased during incubation at 37 degrees C for 60 min with these nucleoli. Concomitantly, a marked increase was found in the content of free ubiquitin, the nonhistone component of protein A24. Incubation of 3H-labeled protein A24 with the thioacetamide-treated liver nucleoli resulted in the linear release of 3H-labeled histone 2A and 3H-free ubiquitin in the presence of phenylmethanesulfonyl fluoride (PMSF) for 2 h. Pretreatment of the nucleoli with trypsin or by heating at 80 degrees C for 10 min inhibited their ability to cleave protein A24. Protein A24 lyase catalyzes the reaction: protein A24 leads to histone 2A plus ubiquitin.     

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.