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.     

Amplified DNA of the Novikoff hepatoma nucleolus is arranged in a 7.3-kilobase tandem repeat

We have reported previously the cloning and characterization of a nucleolar-localized 5.8-kilobase (kb) EcoRI fragment that is approximately 50-fold more highly reiterated in Novikoff hepatoma tumor cells than in normal rat liver [Parker, D. L., Busch, H., & Rothblum, L. I. (1981) Biochemistry 20, 762]. In the present study, the arrangement of these 5.8-kb EcoRI segments within the Novikoff hepatoma genome was investigated. Through the use of "indirect" restriction site mapping, partial restriction enzyme digestions, and molecular cloning, we have determined that the 5.8-kb EcoRI fragment and a 1.5-kb EcoRI fragment together constitute a 7.3-kb unit. The 7.3-kb unit is present in the hepatoma genome as a tandem repeat and constitutes the unit of the DNA that has been amplified. Studies on the arrangement of homologous sequences in the normal rat genome indicate that the amplified DNA may have been derived by a rearrangement and amplification of the nontranscribed spacer of the ribosomal DNA (rDNA) repeat.     

Diversity of rat strains and tumor lines of DNA fragments homologous to an amplified 5.8 kilobase ECO R1 fragment of Novikoff hepatoma cells

The nuclear DNA of several different rat strains and rat tumor lines have been analyzed with respect to Eco R1 fragments homologous to the amplified 5.8 kb Eco R1 fragment (fragment A) of Novikoff hepatoma cells. Two Eco R1 fragments, 4.8 and 4.4 kb, which hybridized to the 5.8 kb Eco R1 fragment, were found in all the genomes investigated. Although none of the examined genomes exhibited evidence of the same degree of amplification of fragment A related sequences as that of Novikoff hepatoma, several had Eco R1 fragments of various other sizes which were homologous to fragment A. These results indicate that the family of fragment A homologous sequences consists of two populations, the constant 4.8 and 4.4 kb fragments, and a second group of sequences which varies with respect to size.     

Evidence for discontinuous replication of circular mitochondrial DNA molecules from Novikoff rat ascites hepatoma cells

Double-forked circular molecules of mitochondrial DNA (mtDNA) from rat tissues, indicated by their form and size to be replicative intermediates, are of two structurally distinct classes. Molecules of the first class are totally double stranded. Molecules of the second class are defined by one daughter segment being totally or partially single stranded. Length histograms of daughter segments measuring between 2% and 44% of the total 5-µm molecular contour were constructed from samples of both classes of replicating molecules derived from mtDNA or Novikoff rat ascites hepatoma cells. For single strand-containing molecules, the lengths fell into eight distinct, reproducible groups with mean values separated by 4.1–7.6% of the circular contour length. For totally double stranded molecules, the lengths fell into seven groups, corresponding to seven of the groups found for single strand-containing molecules. These results suggest that along at least 44% of the contour of mtDNA molecules there exist discrete points at which DNA synthesis tends to be arrested. This may indicate that there are pauses in normal mtDNA synthesis. However, as the DNA used in these experiments was isolated from mitochondrial fractions, the findings may indicate that continuation of synthesis beyond specific points on the nucleotide strands requires a factor which is not available after cell disruption.     

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.     

Phosphorylation of proteins of ribosomes and nucleolar preribosomal particles from Novikoff hepatmoa ascites cells

After labeling for two hours in vivo with ³²P-labeled orthophosphate, proteins from cytoplasmic ribosomes and nucleolar preribosomal particles of Novikoff hepatoma ascites cells were analyzed by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Five proteins (B2, B3, B6, B32 and B35P) were phosphorylated in the ribosomes. Approximately 19 proteins were phosphorylated in the nucleolar preribosomal particles; although four of these were ribosomal proteins, they were different from the proteins labeled in the ribosomes. The 15 additional phosphorylated nucleolar preribosomal particle proteins were non-ribosomal. These results suggest that phosphorylation of proteins of the nucleolar preribosomal particles is independent of phosphorylation of the cytoplasmic ribosomal proteins and may be a part of the maturation process of preribosomal particles.     

Effects of 5-azacytidine on nucleolar RNA and the preribosomal particles in Novikoff hepatoma cells.

Examination of nucleolar RNA from cultured Novikoff hepatoma cells treated for 3 hr with 5 x 10-4 M 5-azacytidine shows that significant amounts of analog-substituted 45S RNA are processed to the 32S RNA species, but 28S RNA formation is completely inhibited. Under these conditions of analog treatment 37% of the cytidine residues in the 45S RNA is replaced by 5-azacytidine. During coelectrophoresis of nucleolar RNA from 5-azacytidine-treated and control cells, the analog-substituted 45S RNA and 32S RNA display reduced mobilities compared to the control 45S RNA and 32S RNA. Coelectrophoresis of analog-substituted and control RNA after formaldehyde denaturation shows no differences in electrophoretic mobility between the two RNA samples, suggesting that 5-azacytidine incorporation may alter the secondary structure of the 45S RNA and the 32S RNA. 5-Azacytidine at 5 x 10-4 M severely inhibits protein synthesis in Novikoff cells by 3 hr. After this length of treatment, however, CsCl buoyant density analysis reveals no difference in density of either the 80S or 55S preribosomal ribonucleoprotein particles when compared to normal particles. Also 5-azacytidine treatment does not appear to cause major changes in the polyacrylamide gel electrophoresis patterns of the proteins in the 80S and 55S preribosomal particles. These results together with previous findings suggest that 5-azacytidine's inhibition of rRNA processing is possibly related to its alteration of the structure of the ribosomal precursor RNAs and is not a consequence of a general inhibition of ribosomal protein formation.