Polyadenylated RNA complementary to repetitive DNA in mouse L-cells.

Complementary DNA, synthesized with L-cell polyadenylated RNA as template, renatured with total L-cell DNA to about 70%. About 30% complementary to unique sequence DNA and another 10 and 30% corresponded to sequences about 20- and 500-fold repetitive. Complementary DNA was fractionated after partial hybridization with total polyadenylated RNA to obtain preparations enriched or impoverished in complements of the most frequent polyadenylated RNA. Renaturation of these complementary DNA fractions with L-cell DNA revealed that most frequent RNAs are transcribed from repetitive DNA sequences, Complementary DNA, density labeled with bromodeoxyuridine, was fractionated by renaturation with L-cell DNA to yield fractions enriched in repetitive and unique sequence DNA. The denisty labeled complementary DNA was purified by equilibrium centrifiguation in an alkaline Cs2SO4 gradient. The complementary DNA representing mainly repetitive DNA sequences hybridized preferentially to frequent polyadenylated RNA.     

Studies on ribonucleic acid metabolism using nuclear columns. Release of rapidly labeled RNA from rat liver nuclei.

A method is described to study the effect of successively changing incubation conditions on the release of rapidly labeled RNA from isolated nuclei. Nuclear columns containing immobilized rat liver nuclei isolated after in vivo application of labeled orotic acid are perfused with different non-radioactive media. Within the course of one perfusion, the rate of RNA release can be repeatedly altered by variation of temperature, acidity and concentrations of nucleoside triphosphates, complexing agents, sodium chloride and manganese chloride. RNA release can be started and stopped, indicating that the reaction does not result from damage to nuclei. During 60 min perfusion the same product, labeled ribonucleoprotein (sigma = 1.43 g/cm3 in CsCl), is released. High release rates depend on the ratio of nucleoside triphosphate to divalent cation concentration, not on the concentration of the agents per se. Ribonucleoside and deoxyribonucleoside triphosphates exert the same effect as ATP. The SH reagents iodoacetamide and iodoacetate only slightly affect the ATP-induced reaction. In contrast, p-chloromercuribenzoate, after an initial stimulation, causes inhibition of RNA release.     

Fractionation of nucleic acids with special regard to rapidly labeled RNA.

Fractionation of nucleic acids with potassium acetate is useful for studying the synthesis of ribosomal RNA and messenger RNA. By quantitative removal of 4 and 5 s RNA and of DNA, the detection of messenger RNA is facilitated.     

Characterization of RNA molecules restricted to the nucleus in mouse L-cells

RNA molecules which are restricted to the nucleus in mouse L-cells were characterized by the technique of RNA/DNA hybridization. Competition of cytoplasmic RNA with labeled nuclear RNA of various sizes revealed that the RNA restricted to the cell nucleus is heterogeneous in size. Competition for sites on fractions of mouse DNA of various base compositions indicated that this unstable RNA is also heterogeneous in base composition. Fractionation of nuclei into three subfractions failed to separate the uniquely nuclear RNA from the precursors of cytoplasmic RNA. The significance of the selective transport of RNA from the nucleus to the cytoplasm and its importance in the control of gene activity in eucaryotic cells is discussed.     

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)     

Isolation and characterization of a 125-kilodalton rapidly labeled nucleolar phosphoprotein

A 125-kilodalton (kDa) phosphoprotein was isolated from nucleoli of Novikoff hepatoma cells in the presence of various inhibitors of proteases, alkaline phosphatase, and RNase. This protein was the most highly phosphorylated protein found thus far in the nucleolus. The half-life of [32P]phosphate in the 125-kDa phosphoprotein was approximately 60 min. Amino acid analysis of the protein showed it had a high serine content (15.5 mol %), a high glutamine plus glutamic acid content (15.5 mol %), and a high lysine content (10.3 mol %). Phosphoserine was the only phosphorylated amino acid identified. After alkaline hydrolysis of the 32P-labeled protein, ribonucleotides were found which accounted for approximately 8.5% of the [32P]phosphate. After cytidine 3',5'-[32P]diphosphate ([32P]pCp) labeling by RNA ligase, several oligoribonucleotide sequences were purified including GGGCOH and GGGGCOH. The binding of oligonucleotides to peptides was stable under denaturing fractionation conditions including 6 M urea treatment and incubation at 100 degrees C for 10 min in sodium dodecyl sulfate and beta-mercaptoethanol. Furthermore, when nucleotide-peptide complex was treated with ribonuclease T2 followed by snake venom phosphodiesterase, the junctional nucleotide pCp was released. These results suggest that one or more ribonucleotides are covalently bound to the 125-kDa phosphoprotein.