Rat Liver Nucleoli Research Articles

Nucleolar DNA-dependent RNA polymerase from rat liver. 1. Purification and subunit structure.

DNA-dependent RNA polymerase I (or A) was purified from rat liver nucleoli. DNA was effectively removed from the solubilized enzyme with a defined concentration of polyethyleneglycol. The enzyme was purified further with successive DEAE-Sephadex and phosphocellulose column chromatography followed by glycerol gradient centrifugation. The procedure yielded an electrophoretically homogeneous enzyme with a specific activity 400 times that of the nucleolar extracts. The recovery of the activity was approximately 20%. The RNA polymerase I eluted as a single peak from DEAE-Sephadex was separated into two distinct peaks by a phosphocellulose column. The first peak eluting at about 0.12 M ammonium sulfate was designated as RNA polymerase IA and the second peak eluting at about 0.18 M as RNA polymerase IB. In normal rat liver nucleoli IA enzyme comprised approximately 20% of the total RNA polymerase I activity and the IB enzyme comprised approximately 80%. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, enzyme IB contained five subunits with molecular weights of 195000 (a), 130000 (b), 65000 (c), 40000 (d), and 19000 (e) at nearly equimolar amounts. The calculated molecular weight of the enzyme (449000) agreed well with that predicted from the sedimentation coefficient of the enzyme. Enzyme IA contained identical subunits except that subunit c was absent. Preliminary studies could not demonstrate any significant differences in template specificity between IA and IB enzyme.

Incorporation of amino acids by rat liver nucleoli in vitro

Isolated rat liver nucleoli are shown to incorporate radioactive amino acids into proteins. The characteristics of this nucleolar protein-synthesizine system indicated that the incorporation observed was not due to cytoplasmic or bacterial contamination. The products synthesized by isolated nucleoli have been analyzed by polyacrylamide gel electrophoresis and subsequent autoradiography; this revealed a few radioactive bands suggesting that in vitro only a few proteins are labelled. The results presented are discussed in view of a possible nucleolar protein synthesis.

Effect of the ionic environment on the transcriptional activity of rat liver nucleoli

The localization of various RNA polymerase activities in the nucleoplasm and the nucleolus was investigated in isolated nuclei and in isolated nucleoli by means of a combination of cell fractionation, biochemical analysis and ultrastructural autoradiography. This resulted in four chief conclusions. 1. 1. The two main RNA polymerase activities can be localized clearly in the interphase nucleus: at low ionic strength, the activity is restricted to the nucleolus whereas at high ionic strength the activity is found in both nucleolar and nucleoplasmic regions. 2. 2. The preservation of the ultrastructure of the nuclei at high ionic strength is rather poor so that the physiological meaning of the activity revealed is questionable. 3. 3. Contrary to other reports, the nucleolar activity is significantly enhanced by increasing ammonium sulphate concentration in the presence of Mn 2+. 4. 4. This increase is probably related to progressive revealing of RNA polymerase B activity within the nucleolus.

Isolation and electrophoretic analysis of nucleoli, phenol-soluble nuclear proteins, and outer cyst walls from Acanthamoeba castellanii during encystation initiation.

A technique is described for isolating nuceoli from Acanthamoeba castellanii. Nuclei isolated by a modification of the technique of F. J. Chlapowski and R. N. Band (1971) are sonicated in a surcrose-Tris-MgSO4-KC1-Triton X-100 buffer and centrifuged on a linear sucrose gradient extending from 1.3 M to 1.5 M with a 2.6 M cushion, at 41000 rpm for 90 min. The only apparent contaminants in the nucleolar preparation are outer cyst walls. A procedure is described for the isolation of chemically pure outer cyst walls, and a comparison of the proteins with the nucleolar preparation reveals that outer cyst walls represent negligible contaminants. The ultrastructure of these isolated nucleoli examined with transmission electron microscopy is found to be identical with that of nucleoli from whole cells, fixed in an identical manner. The 50 nucleolar proteins separated by SDS gel electrophoresis have been examined throughout the growth cycle of Acanthamoeba and into the strat of induced encystment, at which time 10 protein bands disappear, 11 bands are observed to decrease, and 8 are seen to increase in concentration. Phenol-soluble proteins are extracted from the nucleolus which correspond to 29 of the 50 nucleolar proteins, with 17 of these proteins corresponding to nucleolar proteins that change at the onset of encystment. Thes nucleolar proteins are also compared with those of rat liver nucleoli by gel electrophoresis, resulting in the observation that extremely few protein homologies exist between the two. Numerous quantitative and qualitative changes in the gel pattern of phenol-soluble nuclear proteins during early and late log phase growth and the onset of stationary phase were also observed.

The effects of some carcinogenic nitroso compounds on the rat liver nucleolus.

The hepatocyte nucleoli of rats undergo a variety of specific and nonspecific alterations after the administration of 8 nitroso compounds and 5 corresponding non-nitroso compounds. After the oral administration of dimethylnitrosamine and diethylnitrosamine, and unusual segregation of the nucleolus was encountered in the cells with karyorrhexis. Most characteristic of the segregation are the scattered ribosome-like granules at the outermost layer of the altered nucleolus. Frequently microspherules with halos 150 nm in diameter are found in all rats treated with nitroso compounds, which have been reported to be carcinogenic in the organs other than the liver. With the exception of butylurea and butylamine no such specific microspherules were found in the nucleoli after the administration of corresponding non-nitroso compounds.

Purification of Form AI and AII DNA-Dependent RNA Polymerases from Rat-Liver Nucleoli using Low-Ionic-Strength Extraction Conditions

Recent findings have confirmed the role of form A DNA-dependent polymerase activity as that which is responsible for the transcription of the ribosomal RNA-coding genes. Unfortunately, the form A enzymes have proved to be very labile and difficult to work with, especially under high ionic strength conditions. We have, therefore, investigated a method for the purification of the form AI and AII enzymes from rat liver using mild low-ionic-strength conditions. Since preparations from whole nuclei were found to be grossly contaminated with protein having similar properties, the enzymes are extracted from nucleoli. Forms AI and AII are separated on a phosphocellulose column, purified by further ion-exchange chromatography, and by sedimentation through a glycerol gradient. The purified enzymes each migrate as a single band on native polyacrylamide gels and have the expected characteristics of form A RNA polymerase. Sedimentation rates through glycerol gradients indicate that they both have a similar size to that of Escherichia coli RNA polymerase (Mr about 500,000). The purified enzymes are free of DNase and RNase. A method is also described for the purification of form B from the nucleoplasm remaining after isolation of nucleoli. The presence of form C activity was not detected.

Methylation of ribosomal-precursor RNA, synthesized in vitro, by isolated rat-liver nucleoli.

Nucleoli isolated from rat liver were incubated for synthesis of RNA in vitro in the presence or absence of S-adenosyl [3H] methionine. The results obtained indicate that neither the rate of RNA synthesis not the processing of pre-ribosomal RNA was changed if methylation was allowed to take place. The methylation process acts on the RNA most recently synthesized, rather than on the bulk of the RNA already present in the nucleoli. The reaction seems to occur faithfully both quantitatively and qualitatively. It is calculated that 104 mol methyl groups were incorporated per mol of newly synthesized 45-S RNA. Methylation of the ribose rather than the bases predominated. The pattern of alkali-stable oligonucleotides of RNA methylated in vitro was analyzed and found to correspond closely to that of ribosomal RNA labelled in vivo.

Synthesis of RNA molecules larger than 45 S by isolated rat-liver nucleoli.

Nucleoli, isolated from rat liver, synthesize in vitro high-molecular-weight RNA, the base composition and sedimentation pattern of which resembles that of ribosomal precursor RNA. In addition, RNA molecules larger than 45 S have been found. In this paper experiments are described which indicate that these large RNA molecules represent geniune transcription products and are not aggregates arising under the experimental conditions employed. This was established by comparing different extraction methods, by sedimentation analysis of the RNA after denaturation with formamide and by pulse-chase experiments. Hybridisation-competition studies showed that 45-S RNA competes with those rapidly molecules to about 80-90%, thus providing evidence for the presence of ribosomal precursor RNA sequences in those long transcription products. Intact nuclei are able to synthesize in the presence of Mg2+ and alpha-amanitin RNA molecules larger than 45 S too, provided that the RNAase activity is suppressed effectively by the addition of cytoplasmic RNAase inhibitor. The significance of these results is discussed with respect to the initial transcript of the rDNA genes in rat liver nucleoli.

Comparative effect of heparin on RNA synthesis of isolated rat-liver nucleoli and purified RNA polymerase A.

The polyanion heparin has been employed to study the interaction of rat liver DNA-dependent RNA polymerase A and its template under various conditions. Heparin very efficiently inhibits polymerase molecules, which are not bound to DNA or are associated with the template in a loose, i.e. non-specific fashion. Purified nucleoli, isloated from rat liver nuclei, contain RNA polymerase A in abundant quantities of which only a portion is bound in a transcriptional complex. Excess enzyme, which is contained in the nucleolus in a quasi free form, can be transferred to an exogenously added template and can be completely inhibited by the prior addition of heparin. The enzyme contained in a transcriptional complex, however, initiated in vivo and completing these RNA chains in vitro, is fully resistant to heparin. In contrast to these results it has been found that RNA polymerase A extracted from nuclei and purified by various chromatographic steps does not form heparin-resistant complexes, even after the enzyme has been bound to the DNA template. Moreover it has been found that purified RNA polymerase A transcribes truly native DNA extremely poorly, indicating that the enzyme is highly deficient in the act of initiation on duplex DNA. It is therefore questionable whether the interaction of the purified enzyme and isolated DNA represents binding to true initiation complexes as is observed in the intact nucleolus.

Presence of two protein kinases in highly purified rat liver nucleoli

Nucleoli, highly purified as shown by the absence of nucleoplasmic DNA-dependent RNA polymerase, contain two chromatographically separable protein kinases. Nucleoplasm (obtained in the preparation of nucleoli) also contain the same two protein kinases. The proportion of these protein kinases to total protein in these respective fractions indicates that there is no subnuclear localization of either of these enzymes as was demonstrated for the three DNA-dependent RNA polymerases.

Characterization of an endoribonuclease in rat-liver nucleoli.

Synthetic RNAs, i.e. [3H]poly(U) and [14C]poly(A), and natural RNAs, i.e. 16-S and 23-S Escherichia coli ribosomal [14C]RNA and MS2 coliphage [3H]RNA, were incompletely hydrolyzed by a rat liver nucleolar enzyme as measured by solubilization of radioactivity following ethanol precipitation. Sucrose density gradient analysis of these RNA substrates following incubation with isolated rat liver nucleoli at 36 °C for 15 min demonstrated extensive shifts in their sedimentation coefficients. These data were taken as evidence that the nueleolar enzyme is an endoribonuclease. The enzyme has been solubilized and partially purified. The enzyme has no effect on intact double-stranded reovirus RNA. It cleaves 45-S HeLa precursor rRNA through a series of intermediate fragments of decreasing size to limit 4-S end products. Among other characteristics, this nucleolar endoribonuclease prefers single to multi-stranded RNA and is inhibited by DNAase.

Studies on the phosphorylation of nucleolar proteins: Comparison of the phosphorylation patterns of nuclear and nucleolar proteins labeled in vivo and in vitro

Evidence has been accumulated which suggests that the control of transcription may involve the phosphorylation of nuclear proteins, in particular nonhistone proteins [l-3]. It was therefore interesting to compare the phosphoproteins of nuclear and nucleolar chromatins which show remarkable differences in template activity. In the preceding paper [4] we demonstrated the presence of a protein kinase activity in purified rat liver nucleoli. In this study the proteins of whole nuclei and isolated nucleoli were fractionated into saline-soluble and chromosomal proteins and the distribution of phosphoproteins was analyzed. Since up to now very little is known about the specificity of the phosphorylation reactions in vitro we compared the distribution of nuclear and nucleolar proteins labelled both in vivo and in vitro. [ 101.

Ribosome precursor particles in the nucleolus of rat liver. Easily extractable nucleolar 60-S ribonucleoprotein particles and their relation to cytoplasmic large ribosomal subunits.

Ribonucleoprotein (RNA · protein) particles extracted from isolated nucleoli of rat liver with the aid of DNAase and detergents sediment at approximately 60 S (termed nucleolar 60-S RNA · protein). These particles contained an extra RNA species besides 28-S RNA. The former RNA, when extracted with dodecylsulfate and phenol at room temperature, migrates in polyacrylamide-agarose gel a little slower than the 28-S RNA of cytoplasmic large ribosomal subunit (termed 30-S RNA). The nucleolar 60-S RNA · protein contained 5-S RNA as a low-molecular-weight counterpart. When RNA was extracted at high temperature (65°C), the 30-S RNA was converted to 28-S RNA with a concomitant release of low-molecular-weight RNA's. These findings suggest that these low-molecular-weight RNA's are hydrogen-bonded to 28-S RNA. The protein moiety of the nucleolar 60-S RNA · protein was compared with that of large ribosomal subunits on polyacrylamide gel with a split-gel technique. The proteins of nucleolar 60-S RNA · protein gave approximately 20 bands, many of which were identical in the mobility to the proteins of large ribosomal subunits. However, at least two bands (bands 7 and 15) were not detected in the gel pattern of cytoplasmic large ribosomal subunits. On the other hand, there were protein bands (bands a ∼ k) of large ribosomal subunits which were not detectable in the proteins of nucleolar 60-S particles. Nucleolar 60-S particles were composed of three RNA · protein species having different buoyant densities. The · values of nucleolar 60-S RNA · protein were significantly lower than that of the cytoplasmic large ribosomal subunits, showing that the nucleolar 60-S RNA · protein has a larger protein content than cytoplasmic large ribosomal subunits. It is concluded from these results that when the nucleolar 60-S RNA · protein is transferred from the nucleolus into cytoplasm to form a mature large ribosomal subunit, (a) 30-S RNA is converted to 28-S RNA with a concomitant release of low-molecular-weight RNA's, (b) at least two protein species are removed from the nucleolar 60-S RNA · protein and at least several other proteins are added to it, but (c) the overall protein content decreases during the course of these protein exchanges.

Studies on the processing of 45-S RNA in rat liver nucleolus with specific reference to 29.5-S RNA

Nucleolar RNAs prepared from rat liver labelled with [ 14C]- or [ 3H]orotic acid with cold and hot sodium dodecyl sulphate-phenol extraction, were analysed by means of polyacrylamide gel electrophoresis and the following results have been obtained. 1. 1. In nucleolar RNAs which are prepared with cold sodium dodecyl sulphatephenol extraction not only from normal, but also from thioacetamide-treated and regenerating rat livers, 29.5-S RNA is found in addition to 45-, 37-, 36-, 32-, 28-, 21.5- and 18-S RNAs, whereas in those with hot sodium dodecyl sulphate-phenol extracttion, 29.5-S RNA is not observed in all cases. Nucleolar 28-S RNA and cytoplasmic 28-S RNA of rat liver co-migrate in acrylamide gel electrophoresis and do not change their electrophoretic mobilities with hot sodium dodecyl sulphate-phenol extraction or heat treatment, indicating that the presence of 29.5-S RNA is not due to the conformational change of 28-S RNA. The 37-S minor component is very unstable in the case of normal rat liver, but stable in the case of thioacetamide-treated rat liver. 29.5-S and 37-S RNAs are thought to be newly found RNAs using the cold sodium dodecyl sulphate-phenol extraction method. 2. 2. Taking the kinetics of labelling, the results of actinomycin D chase experiment and the molecular weights of the nucleolar RNA components together, may indicate that 29.5-S RNA is the metabolic intermediate between 32-S and 28-S RNAs, and 37-S RNA is suggested to be the direct precursor of 36-S RNA. The following scheme for the processing of 45-S RNA in rat liver nucleoli is proposed: ▪

Conversion of rat liver nucleolar 29.5-S RNA to 28-S RNA in vitro

By employing nucleolar RNA, especially 28-S and 29.5-S RNAs, isolated from thioacetamide-treated rat liver nucleoli which were labelled with [ 14C]- or [ 3H]orotic acid, the conversion of 29.5-S RNA to 28-S RNA was investigated and the following results have been obtained. 1. 1. Nucleolar 29.5-S RNA disappears with incubation for 13 min, in the sodium acetate buffer (pH 7.0) above 42 °C, in sodium dodecyl sulphate-phenol above 37 °C and in 6 M urea above 25 °C. The slow cooling after heat treatment of 29.5-S RNA does not result in the reappearance of the RNA, suggesting the disappearance to be an irreversible process. The disappearance caused by heat treatment is inhibited by divalent metal ions (Mg 2+, Ca 2+, Ba 2+, Co 2+ and Mn 2+). 2. 2. With incubation in 10 mM sodium acetate buffer (pH 7.0) at 45 °C for 13 min, nucleolar 28-S RNA decreases to 60–75 %. The decrease is accompanied by the appearance of two low molecular weight RNAs (5.3-S and 6.7-S RNAs) in addition to the heterogeneous RNAs distributed from the 28-S to 12-S regions on acrylamide gel electrophoresis. 3. 3. Under the same conditions as above, the conversion of 29.5-S to 28-S RNAs occurs, which accompanies the release of a specific x component with a molecular weight of 180 000 corresponding to the difference in the molecular weight between 29.5-S and 28-S RNAs, although the heterogeneous RNAs distributed from the 28-S to 12-S regions are also observed. The x component has a similar specific activity to that of 29.5-S RNA after labelling periods of 2 and 6 h. The possible mechanism of the conversion of 29.5-S RNA to 28-S RNA is discussed.

Pre-ribosomal RNA synthesis in isolated rat-liver nucleoli.

Isolated rat liver nucleoli represent a simple system to study transcription and processing of ribosomal RNA in vitro. These organelles retain their ability to synthesize ribosomal precursor RNA in vitro as measured by the incorporation of nucleotides into RNA. The incorporation of labelled nucleotides is not affected by α‐amanitin or rifampicin AF‐013. Low concentrations of ammonium sulfate (60 mM) stimulate the nucleolar synthetic activity considerably. The (A + U)/(G + C) ratio of nucleolar RNA synthesized in vitro is 0.5–0.55 and resembles that of ribosomal precursor RNA. Discrete peaks of high‐molecular‐weight RNA, widely distributed as to size, appear in analytical sucrose gradients. Molecules with sedimentation coefficients up to about 80 S have been obtained a surprising result in view of the fact that, in general, only 45‐S precursors are found in vivo. The amount of rapidly sedimenting RNA increases with prolonged incubation. It is uncertain whether or not the presence of ribosomal RNA precursor molecules with sedimentation coefficients greater than 45‐S reflects a normal transcription mechanism in the living cell which remains undetected due to rapid processing. It is also possible that isolated nucleoli may have lost some specific factors which in vivo cause the RNA polymerase to terminate the RNA chains at the end of the 45‐S transcription unit.

Comparison of nucleolar proteins of normal rat liver and Novikoff hepatoma ascites cells by two-dimensional polyacrylamide gel electrophoresis.

A two-dimensional polyacrylamide gel electrophoresis technique is presented for separation of acid-extracted proteins of isolated nucleoli of normal rat liver and Novikoff hepatoma ascites cells. About 100 distinct protein spots were resolved. Comparison of the patterns for normal liver and Novikoff hepatoma revealed nine spots with markedly different intensities in the two patterns. Two spots were found that were unique to the normal liver pattern, and one spot was found that was unique to the hepatoma pattern.

Nucleoprotein methylation in salivary gland chromosomes of Sciara coprophila: correlation with DNA synthesis.

Abstract Methylation and ethylation of chromosomal nucleoproteins have been demonstrated in the salivary glands of Sciara coprophila larvae. A difference in grain distribution pattern was observed between chromosomes incubated in 3 H-methyl-methionine and 3 H-ethyl-ethionine. Like DNA synthesis, methylation could only be demonstrated at certain developmental stages, and during some of these a close correlation was observed between labelling patterns following 3 H-methyl-methionine and 3 H-thymidine in separated gland pairs. No such correlation was observed with RNA synthesis. Ethylation of chromosomal non-histone protein could not be correlated with nucleic acid biosynthesis. Feeding ethionine during larval development produced similar nucleolar abnormalities to those previously described in rat liver nucleoli.

The rapid turnover of RNA polymerase of rat liver nucleolus, and of its messenger RNA.

Turnover rates of the components of systems for RNA synthesis of rat-liver nucleus, nucleolus, and nucleoplasm were investigated. Cycloheximide administered in vivo selectively diminished nucleolar RNA synthesis in vitro. In contrast to the relatively stable nucleoplasmic RNA polymerase, nucleolar RNA polymerase (polymerase I) from rat liver decays rapidly upon cycloheximide administration, following pseudo-first order kinetics with a half-life of about 1.3 hr. Cycloheximide elicits this effect not through direct interaction with nucleolar RNA polymerase itself, nor by alteration of template function, but rather by inhibition of de novo synthesis of one or more of the protein components of nucleolar RNA polymerase. Similarly, when actinomycin-D was administered in vivo to inhibit RNA synthesis, the rate of decay of nucleolar RNA polymerase, assayed in the presence of exogenous poly d(A-T) template, was similar to that observed after cycloheximide administration. Thus, the messenger RNA(s) that codes for one or more of the catalytically essential polypeptide components of this enzyme turn over very rapidly with a half-life considerably shorter than 1.3 hr. The rapidity of turnover of both the enzyme protein and its messenger RNA(s) renders nucleolar RNA polymerase highly responsive to altered transcriptional, translational, or post-translational modulation. The marked differences in turnover rates of nucleolar and nucleoplasmic RNA polymerases indicate that at least certain of the protomeric components of nucleolar RNA polymerase I are distinct from those of nucleoplasmic RNA polymerases II and III.

A factor stimulating the transcription by nucleolar RNA polymerase in the nucleolus of rat liver

The flow-through(FT) fraction of DEAE-Sephadex chromatography of RNA polymerase solubilized from isolated nucleoli of rat liver specifically stimulated transcription by nucleolar RNA polymerase. The effect was observed only with native DNA used as template but not with heat-denatured DNA. The activity was trypsin-sensitive but relatively heat stable. Although the fraction contained DNase activity as revealed by the alteration of sedimentation properties of SV40 DNA, evidence is presented that the stimulatory activity is not ascribed to this DNase activity.