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.     

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.     

Fractionation of nucleoli from auxin-treated soybean hypocotyl into nucleolar chromatin and preribosomal particles

Nucleoli from auxin-treated tissues (Glycine max L. var Wayne or Kaoshiung No. 3) were isolated and purified by Percoll density gradient centrifugation. There was a 2.1-fold increase in RNA and a 2.8-fold increase in protein after a 24-h auxin treatment per unit nucleolar DNA. More than 150 acid-soluble protein spots were associated with the auxin-treated nucleoli on two dimensional (2-D) gel electropherograms.

Nucleoli from auxin-treated tissue were fractionated by suspension in 20 millimolar dithiothreitol at room temperature for 20 minutes into two distinct fractions referred to as the nucleolar chromatin and preribosomal particle fractions. The DNA:RNA:protein ratio of the chromatin fraction was 1:2.5:14. Most of RNA polymerase 1 activity and nucleolar DNA recovered in this fraction. The acid-soluble proteins in the chromatin were resolved into 32 protein spots on 2-D gel electropherogram. The most abundant spots were identified as histones.

The nucleolar preribosomal particle fraction had a DNA:RNA:protein ratio of 1:24:102 and contained only trace amounts of RNA polymerase 1 activity and only 10 per cent of the nucleolar DNA. Acid-soluble proteins associated with these particles were resolved into 78 protein spots; 72 of these (acid-soluble) protein spots corresponded in 2-D gel electrophoresis to 80S cytoplasmic ribosomal proteins. Some 15 protein spots found in 80S ribosomal proteins were absent in the preribosomal particles. It seems reasonable, based on these data, that the enlargement of nucleoli after auxin treatment is primarily due to the large increase in ribosomal proteins and rRNA which accumulate and assemble in the nucleoli in the form of preribosomal particles.

     

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.     

Utp23p is required for dissociation of snR30 small nucleolar RNP from preribosomal particles

Yeast snR30 is an essential box H/ACA small nucleolar RNA (snoRNA) that promotes 18S rRNA processing through forming transient base-pairing interactions with the newly synthesized 35S pre-rRNA. By using a novel tandem RNA affinity selection approach, followed by coimmunoprecipitation and in vivo cross-linking experiments, we demonstrate that in addition to the four H/ACA core proteins, Cbf5p, Nhp2p, Nop10p and Gar1p, a fraction of snR30 specifically associates with the Utp23p and Kri1p nucleolar proteins. Depletion of Utp23p and Kri1p has no effect on the accumulation and recruitment of snR30 to the nascent pre-ribosomes. However, in the absence of Utp23p, the majority of snR30 accumulates in large pre-ribosomal particles. The retained snR30 is not base-paired with the 35S pre-rRNA, indicating that its aberrant tethering to nascent preribosomes is likely mediated by pre-ribosomal protein(s). Thus, Utp23p may promote conformational changes of the pre-ribosome, essential for snR30 release. Neither Utp23p nor Kri1p is required for recruitment of snR30 to the nascent pre-ribosome. On the contrary, depletion of snR30 prevents proper incorporation of both Utp23p and Kri1p into the 90S pre-ribosome containing the 35S pre-rRNA, indicating that snR30 plays a central role in the assembly of functionally active small subunit processome.     

Early nucleolar preribosomal RNA - protein in mammalian cells.

Early steps of ribosomal maturation have been studied by analysis of nucleolar extracts using different extraction procedures. Early 45-S nucleolar RNA is found to be associated with slowly sedimenting elements, distinct from previously described 80-S and 55-S nucleolar preribosomes. This early 45-S RNA has been shown to be of preribosomal type according to the following criteria. (a) When hybridized with nucleolar DNA, competition with rRNA can be observed; (b) its biosynthesis is sensitive to low doses of actinomycin D; (c) it is methylated at an early stage; (d) it contains no linked poly(A) segments. This early 45-S RNA seems to be specifically linked with proteins. The protein content of these early RNA-protein complexes is significantly lower than that for 80-S preribosomes. 45-S RNA sensitivity to nucleolytic activities that can take place in the course of preribosome extraction has been found to be higher in these RNA-protein complexes than in 80-S preribosomes. Identical results were obtained when different mammalian cell species were studied (rat, hamster, or HeLa cells).     

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.     

Cloning and characterization of a highly reiterated 5.8-kilobase pair nucleolar EcoRI DNA fragment found in Novikoff hepatoma ascites cells

The DNA of Novikoff hepatoma ascites cells was found to contain a 3.6-megadalton EcoRI restriction fragment, referred to as EcoRI fragment A (Parker et al., 1979). C0t analyses demonstrated an enrichment of fragment A sequences in Novikoff hepatoma genome relative to normal rat liver DNA. This fragment was cloned in lambda gtWES to determine its molecular structure and sequence organization. The DNA from a positive clone was labeled by nick translation and hybridized to a Southern blot of EcoRI digested Novikoff DNA. Distinct hybrids formed with the region corresponding to fragment A. The greater degree of hybridization to the nucleolar fraction suggested a nucleolar enrichment of fragment A. Fragment A has a PstI site approximately 300 base pairs from one terminus which was used to generate mono-5'-32P-labeled fragments. The larger PStI subfragment, 5500 base pairs, labeled at a single terminus, was used to evolve a restriction enzyme map. The 300 base pair fragment was partially sequenced, revealing the presence of a repetitive sequence "island", TT(GTCT)8(GAAT)5G-. C0t analysis, utilizing the purified clone as a probe, confirmed the enrichment of fragment A sequences in the tumor relative to the normal rat liver control.     

Two-dimensional polyacrylamide gel electrophoresis of chromatin proteins of normal rat liver and Novikoff hepatoma ascites cells

Chromatin was prepared from the citric acid nuclei of normal rat liver and Novikoff hepatoma ascites cells. After sulfuric acid extraction, the dehistonized chromatin was solubilized by digestion with deoxyribonuclease I. The proteins of normal liver and of Novikoff hepatoma chromatin fractions were analyzed by two-dimensional polyacrylamide gel electrophoresis. The liver pattern contained 69 components and the hepatoma pattern contained 84 components. Comparison of the two patterns revealed two dense protein spots migrating in the B region in the liver pattern that were absent from the tumor pattern and two dense protein spots migrating in the C region in the tumor pattern that were absent from the liver pattern.