Hepatoma Nucleoli Research Articles

Immunolocalization of a Subnucleolar Nucleoprotein Complex Containing RNA Polymerase 1 in Ascites Hepatoma Cells Using Monoclonal Antibodies

We have isolated a discrete subnucleolar macromolecular nucleoprotein complex by direct treatment of Novikoff ascites hepatoma nucleoli by MspI restriction digestion. Using a monoclonal antibody made against the subnucleolar nucleoprotein complex that was shown to inhibit RNA polymerase (pol) 1 activity in vitro, we localized an Mr approximately 55,000 protein subunit which was demonstrated previously by an enzyme-linked immunosorbent assay and Western blotting to share epitopes with the RNA pol 1 moiety of the subnucleolar complex. By indirect immunofluorescence the distribution of the Mr approximately 55,000 component of the subnucleolar nucleoprotein complex was examined at various phases of the cell cycle. At prophase, it was localized in large (approximately 1.5 microns in diameter) ball-like structures associated with the nuclear periphery and nuclear peripheral chromatin, suggesting that these structures might be related to preribosomal elements. After chromatin condensation and the pairing of daughter chromosomes, the large ball-like spheres increased in size and were associated with propidium iodide staining at one side of the nucleus; whereas throughout and especially at the opposite side of the nucleus, smaller, round, punctate structures of approximately 0.5 micron in diameter were visibly labeled that were not associated with propidium iodide staining. At later stages of the cell cycle, these small round structures were again associated with propidium iodide staining, suggesting that they may be related to prenucleolar and/or preribosomal elements which would likely contain the appropriate nucleic acid in association with RNA pol 1 and cofactors of RNA pol 1.

Association of protein C23 with rapidly labeled nucleolar RNA.

The association of nucleolar phosphoprotein C23 with preribosomal ribonucleoprotein (RNP) particles was examined in Novikoff hepatoma nucleoli. RNA was labeled with [3H]uridine for various times in cell suspensions, and RNP particles were extracted from isolated nucleoli and fractionated by sucrose gradient ultracentrifugation. The majority of protein C23 cosedimented with fractions containing rapidly labeled RNA (RL fraction). To determine whether there was a direct association of RNA with protein C23, the RL fraction was exposed to ultraviolet (UV) light (254 nm) for short periods of time. After 2 min of exposure there was a 50% decrease in C23 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, with no significant further decrease at longer times. When UV-treated fractions were subjected to phenol/chloroform extractions, as much as 30% of the labeled RNA was found in the phenol (protein) layer, indicating that RNA became cross-linked to protein. Similarly, there was an increase in protein C23 extracted into the water layer after irradiation. By SDS-PAGE analyses the cross-linked species migrated more slowly than protein C23, appearing as a smear detected either by [3H]uridine radioactivity or by anti-C23 antibody. With anti-C23 antibodies, up to 25% of the labeled RNA was precipitated from the RL fraction. Dot-blot hybridizations, using cloned rDNA fragments as probes, indicated that the RNA in the RL fraction and the immunoprecipitated RNA contained sequences from 18S and 28S ribosomal RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple states of U3 RNA in Novikoff hepatoma nucleoli.

U3 RNA, a capped small nuclear RNA found thus far only in the nucleolus, has been implicated in the processing and/or transport of preribosomal RNA [Busch, H., Reddy, R., Rothblum, L., & Choi, Y. C. (1982) Annu. Rev. Biochem. 51, 617-654]. Tris(hydroxymethyl)aminomethane (Tris) (10 mM, pH 7.0) extracts of Novikoff hepatoma nucleoli, which contained about 80% of total nucleolar U3 RNA, were analyzed by sucrose density gradient centrifugation. Approximately 65% of the U3 RNA was bound to greater than 60S preribosomal ribonucleoprotein (RNP) particles, and about 15% sedimented at less than 20 S. The association between the 65% of U3 RNA that was bound to the preribosomal RNP particles was stable up to 55 degrees C. About 10% of U3 RNA was base paired to preribosomal RNA after deproteinization at 22 degrees C. The base-paired fraction of U3 RNA was released from the preribosomal RNA by heating to 45 degrees C or treating with 4 M urea. These results show that of the total nucleolar U3 RNP, (a) about 55% is bound to preribosomal RNP particles primarily by protein interactions, (b) about 10% is base paired to preribosomal RNA, (c) approximately 15% sedimented slowly and consisted presumably of free U3 RNP particles, and (d) the remaining 20% of U3 RNP was not extractable using 10 mM Tris buffer. On the basis of the different association states of U3 RNP particles, a model is proposed for the binding and dissociation events which take place between U3 RNP and preribosomal RNP particles.

Purification and partial characterization of a 19 KD/pI 4.5 nucleolar phosphoprotein

Two-dimensional PAGE analysis of proteins associated with the slowly sedimenting “fibrillar” structures of HeLa nucleoli revealed a protein with a M r of 19,000 and a pI of 4.5 which was highly labeled both with 32P-orthophosphate and 35S-methionine. The protein was isolated from Novikoff hepatoma nucleoli by extraction in 0.35 M NaCl and 5 mM DTT followed by chromatography in EDTA on DEAE-cellulose and Sephadex G-100. The protein was homogeneous with respect to two-dimensional PAGE, number of tryptic peptides and carboxyl terminal analysis. The protein contained an acidic/basic amino acid ratio of 2.1, 7 residues of methionine, 2 residues of cysteine, a blocked amino terminus and a carboxyl terminal lysylleucine.

Localization of phosphoprotein C23 to nucleolar structures and to the nucleolus organizer regions

After extraction of Novikoff hepatoma nucleoli with 4 M urea/3 M LiCl, phosphoprotein C23 was isolated by DEAE-cellulose and Bio-Rad AG3-X4A column chromatography. Immunization of rabbits with the highly purified protein C23 resulted in the production of a specific antibody as determined by Ouchterlony diffusion analysis. When the immunoperoxidase method was used to localize protein C23 in cells, it was found in ‘fibrillar centers’ (nucleolonemas) in nucleoli. Protein C23 was also demonstrated to be present on the nucleolus organizer regions (NORs) of metaphase chromosomes.

Proteins C23 and B23 are the major nucleolar silver staining proteins

To examine the silver staining proteins of Novikoff hepatoma nucleoli, the nucleolar proteins were separated on two-dimensional polyacrylamide gels with an isoelectric focusing first dimension and an acid-urea gel second dimension. The nucleoli were sequentially extracted with (1) 0.6 M potassium acetate, pH 5.5 and (2) 2 M potassium acetate — 5 M urea — 10 mM Tris, pH 7.5. The silver staining method used for the detection of silver binding proteins in gels was similar to that used to stain the nucleolar granules on microscope slides. Two major silver staining proteins were found which were identified as (molecular weight × 10 −3/pI) proteins C23 (100/5.3) and B23 (37/5.1). These two proteins are the major acidic proteins in Novikoff hepatoma nucleoli.

Nucleolar phosphoprotein phosphatase from novikoff hepatoma and rat liver: Characterization and partial purification

Phosphoprotein phosphatase which dephosphorylates 32P-labeled nucleolar protein substrates was found in nucleoli of Novkoff hepatoma ascites cells and normal rat liver. The activity was extracted in high yield from nucleoli with 0.01 m Bis/Tris (pH 7.0). Low ionic strength was also required for activity: the activity was only 50% of maximum in 0.075 M NaCl. Activity was affected differently by various divalent cations: MgCl 2 had little effect; CaCl 2, MnCl 2 and CoCl 2 above 4 mM inhibited the activity 30–60%; ZnCl 2 above 2 mM completely destroyed the activity. EDTA had no effect, indicating that divalent cations are probably not required. The enzyme activity was enhanced 20% by 5–8 mM dithiothreitol and was inhibited 60% by 7–10 mM N-ehtylmaleimide indicating a requirement for free sulfhydryl groups. The K m of the extracted enzyme for 32P-labeled nucleolar protein was 0.6 mg/ml. The phosphatase was capable of dephosphorylating the major phosphorylated nucleolar proteins C23-24 and B23-24 and also histone H l. The enzyme was purified more than 200-fold on hydroxyapatite followed by DEAE-Sephadex, which resolved the activity into three major components. The activity of enzyme extracted from Novikoff hepatoma nucleoli was approximately 2.5 times greater than from normal liver nucleoli.

Nuclear and nucleolar protein phosphokinases in novikoff hepatoma

1. 1. Two chromosomal protein phosphokinases and their substrates were partially characterized in nuclei and nucleoli of Novikoff hepatoma. 2. 2. Two nucleolar protein knase peaks eluted from a phosphocellulose columns with 0.5 and 0.7 M NaCl. These two peaks of enzymatic activity differed in their divalent cation optima and substrate specificities from the corresponding nuclear enzymes. 3. 3. It was concluded that Novikoff hepatoma nucleoli contain at least two specific protein phosphokinase-substrate systems.

Characterization of a phosphoprotein phosphatase activity in ribonucleoprotein particles from HeLa cell nuclei

Ribonucleoprotein particles containing heterogenous nuclear RNA (hnRNP) have a phosphoprotein phosphatase activity. This activity is optimal at pH 7.5, inhibited by divalent cations and by increasing ionic strength above 200 mM NaCl, stimulated by 2-mercaptoethanol and inhibited by N-ethylmaleimide. It is clearly distinct from non specific alkaline phosphatase and resembles the phosphoprotein phosphatase present in Novikoff hepatoma nucleoli (Olson et al. B.B.R.C. (1976), 70, 717–721). This enzyme may be involved in regulating the phosphorylation level of hnRNP proteins in combination with the protein kinase previously described (Blanchard et al. Eur. J. Biochem. (1977) in press).

Isolation and characterization of rDNA-containing chromatin from nucleoli

Fractions corresponding to ‘euchromatin’ and ‘heterochromatin’ were isolated from Novikoff hepatoma nucleoli by a modification of the DNase II method of Gottesfeld et al. [18, 19]. Nucleolar chromatin [36] was digested with DNase II, and ‘euchromatin’ was obtained as a soluble fraction following differential precipitation of ‘heterochromatin’ with Mg 2+. In this ‘euchromatin’ fraction, the rDNA was enriched approx. 8.5-fold over the unfractionated nucleolar chromatin, as determined by DNA-RNA hybridization. On 8–80% glycerol gradients, the ‘euchromatin’ separated into three discrete subfractions as with euchromatin obtained from the bulk chromatin [18, 19]. Two subfractions which sedimented at 14S and 20S, respectively, were consistently found after DNase II digestion (to 15 min) which contained all histone fractions. All euchromatin subfractions were enriched in rDNA and had a high protein/DNA ratio, a high RNA-DNA ratio, and a high template activity with exogenous RNA polymerase I. Fraction B with a sedimentation of 14S was most enriched in the parameters related to rRNA synthesis. The Mg 2+-precipitable fractions reported to be ‘heterochromatin’ [18, 19] not only contained substantial amounts of ribosomal genes, but also had higher template activities than unsheared chromatin. These results imply (1) nucleolar ‘euchromatin’ has a structural heterogeneity similar to that of the euchromatin containing active single copy sequences; (2) at least a portion of rDNA in ‘euchromatin’ is protected by proteins, including histones, which does not preclude active transcription of rDNA; (3) none of the currently used criteria for ‘euchromatin’ alone defines the chromatin containing rDNA; and (4) an appreciable enrichment of rDNA occurs in isolated euchromatin but not all the rDNA was separated into ‘euchromatin’ by the DNase II method.

Phosphoprotein phosphatase activity of Novikoff hepatoma nucleoli

Nucleoli from Novikoff hepatoma ascites cells contain phosphatase activity that acts upon 32P-labeled nucleolar protein substrates. The activity is optimal near pH 7.0 and is inhibited by increasing concentrations of NaCl. The divalent cations CaCl 2, MnCl 2 and CoCl 2 at 6 mM inhibited phosphatase activity from 30–60%. ZnCl 2 completely inhibited the activity above 2 mM while EDTA and MgCl 2 had little effect. The activity was stimulated by dithiothreitol and inhibited by N-ethylmaleimide indicating a requirement for free sulfhydryl groups.

Studies of phosphorus metabolism by isolated nuclei: X. Nucleolar nucleosidediphosphatase activity

1. 1. Isolated rat liver nucleoli catalyze formation of P i with both ribo- and 2-deoxyribonucleoside diphosphates as substrates. Nucleolar GDPase activity is maximal at pH 7.2 to 7.8 and 40°; it is stimulated maximally by magnesium or manganese. The principal products of nucleolar GDPase are GMP and P i; no evidence has been obtained for a contribution to this activity by polynucleotide phosphorylase. 2. 2. Nucleoli also contain glucose-6-phosphatase and inorganic pyrophosphatase. Although such a content of phosphatases is analogous to that of microsomes, the absence of microsomes in the isolated nucleoli, and the non-identity of the nucleolar phosphatases with microsomal phosphatases, has been established by several means. 3. 3. The results of this work verify previous histochemical demonstrations of nucleoside diphosphatase activity in liver and hepatoma nucleoli.