Surface ultrastructure and cytochemistry of isolated nucleoli from Novikoff hepatoma ascites cells

Surface ultrastructure and cytochemistry of isolated Novikoff hepatoma cell nucleoli were examined using scanning electron microscopy (SEM). Nuclear preparations were examined at 15 sec intervals during the sonication procedure for isolation of nucleoli. In the initial stages of nuclear disruption the nucleoli were attached to large chromatin masses. A compact nucleoprotein nucleolar stalk relatively resistant to shear was observed in association with many nucleoli. Further sonication disrupted these structures and left tightly coiled, helical filaments still attached to the purified nucleoli. These filaments were removed by DNase digestion but were resistant to RNase digestion. The present study provides a new perspective of nucleolar ultrastructure, its surface organization, and its relationships to other nuclear components.     

Negatively charged phosphopeptides of nucleolar nonhistone proteins from Novikoff hepatoma ascites cells.

To characterize the sites phosphorylated by endogenous kinases, phosphopeptides of isolated nucleolar nonhistone proteins were analyzed. Major phosphoprotein bands C23 and B23 were ³²P labeled $\text{in vitro}$ and electrophoretically isolated. Tryptic phosphopeptides were resolved by DEAE-Sephadex chromatography into fractions A, B and C for band C23 and α and β for band B23. Each of these fractions contained phosphoserine, had a distinct amino acid composition of 49–65% glx + asx and 4–11% lys, and had molecular weights of 7–11,000 determined on Sephadex G50. These data indicate that two nucleolar nonhistone proteins have similar phosphorylated regions of high negative charge density.     

Cross-linking of Novikoff ascites hepatoma cytokeratin filaments

We have investigated the structure of solubilized cytokeratins from Novikoff ascites hepatoma using the cleavable cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) in the presence of 6 M urea to effect partial complex melting. By two-dimensional gel electrophoresis, in which the protein cross-links were broken in the second dimension, we have identified two major complexes as a p39-p56 dimer and a (p39-p56)2 tetramer, p39 and p56 being two of the major cytokeratins in Novikoff ascites hepatoma. Experiments investigating possible relationships between the dimer and tetramer employed immunoblots and two monoclonal antibodies which recognized either p56 or p39 cytokeratins. When very low protein concentrations were cross-linked, the dimer was the predominant product. As protein concentration increased, we noted a decrease in dimers and a corresponding increase in tetramers, suggesting that the dimer may be a precursor to the tetramer. In support of the cross-linking experiments, two-dimensional gel electrophoresis using 4 M urea in the first dimension indicated a predominant association of p56 and p39 in the Novikoff ascites hepatoma cytokeratin complexes.     

Alterations in the maturation and structure of ribosomal precursor RNA in Novikoff hepatoma cells induced by 5-fluorocytidine

The effects of 5-fluorocytidine on ribosomal RNA maturation and structure in Novikoff hepatoma cells were investigated. Like other nucleic acid base analogues that are incorporated into RNA, this compound inhibits maturation of the 45S ribosomal RNA precursor. The 45S RNA precursor produced in the presence of 5-fluorocytidine has an abnormal electrophoretic mobility compared with that of the control precursor under nondenaturing conditions, but the two have identical mobilities under denaturing conditions. Under the conditions of these experiments, 5-fluorocytidine inhibited cellular protein synthesis only slightly, whereas equimolar concentrations of 5-azacytidine resulted in nearly 75% inhibition of this process. Despite this difference in the effects of the two analogues as well as the greater chemical lability of the 5-azacytidine, their effects on ribosomal RNA maturation are identical.     

DNA-binding proteins from Novikoff hepatoma cells.

In 0.05 M NaCl, 6-8% of the total soluble proteins from Novikoff hepatoma cells bind rapidly and reversibly to columns containing either heterologous or homologous DNA adsorbed to cellulose. These proteins can be eluted by buffer containing 2.0 M NaCl. 0.5-1% of the total protein exhibits a 7-17-fold preference for rat DNA over Escherichia coli DNA. 1-1.5% of the proteins bind DNA so strongly that elution cannot be effected by 4.0 M NaCl but can be accomplished by deoxyribonuclease I treatment of the columns. DNA-binding proteins eluted by 2.0 M NaCl were labeled with 125I or 131I and characterized by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. These experiments indicate that DNA-binding proteins represent a discrete subset of the total soluble protein. Many similarities were noted between the major components of the homologous and heterologous DNA-binding fractions.     

Biochemical effects of bleomycin A2 on Novikoff hepatoma ascites cells.

Purified nucleolar DNA was markedly degraded at a concentration of 13 mug/ml by bleomycin A2; bleomycin concentrations 20-30 times greater were required to degrade nucleoplasmic DNA. Whole nuclear DNA was degraded to only a small extent at 13 mug/ml but was markedly degraded at higher bleomycin concentrations. Treatment of the various types of DNA with high concentrations of bleomycin A2 produced low molecular weight (approximately 6S) fragments that were no longer sensitive to degradation by bleomycin A2. Hybridization studies demonstrated a loss of ribosomal DNA sequences from nucleolar DNA treated with bleomycin A2 in vitro. Studies on RNA synthesis in Novikoff hepatoma ascites cells in vitro showed there was a decreased uptake of 32Pi into high molecular weight nuclear RNA in the presence of bleomycin A2. These results indicate that nucleolar function is inhibited by a direct effect of bleomycin A2 on nucleolar DNA.