Quantitative analysis of rat liver nucleolar and nucleoplasmic ribosomal ribonucleic acids.

rRNA from detergent-purified nuclei was fractionated quantitatively, by two independent methods, into nucleolar and nucleoplasmic RNA fractions. The two RNA fractions were analysed by urea/agar-gel electrophoresis and the amount of pre-rRNA (precursor of rRNA) and rRNA components was determined. The rRNA constitutes 35% of total nuclear RNA, of which two-thirds are in nucleolar RNA and one-third in nucleoplasmic RNA. The identified pre-rRNA components (45 S, 41 S, 39 S, 36 S, 32 S and 21 S) are confined to the nucleolus and constitute about 70% of its rRNA. The remaining 30% are represented by 28 S and 18 S rRNA, in a molar ratio of 1.4. The bulk of rRNA in nucleoplasmic RNA is represented by 28 S and 18 S rRNA in a molar ratio close to 1.0. Part of the mature rRNA species in nucleoplasmic RNA originate from ribosomes attached to the outer nuclear membrane, which resist detergent treatment. The absolute amount of nuclear pre-rRNA and rRNA components was evaluated. The amount of 32 S and 21 S pre-rRNA (2.9 x 10(4) and 2.5 x 10(4) molecules per nucleus respectively) is 2-3-fold higher than that of 45 S, 41 S and 36 S pre-rRNA.     

Extensive homologies between the methylated nucleotide sequences in several vertebrate ribosomal ribonucleic acids.

The methylated nucleotide sequences in the rRNA molecules of the following vertebrate cultured cells were compared: human (HeLa); hamster (BHK/C13); mouse (L); chick-embryo fibroblast; Xenopus laevis kidney. In each species the combined 18S, 28S and 5.8S molecules possess approx. 110-115 methyl groups, and the methylated oligonucleotides released after complete digestion of the rRNA by T1 ribonuclease encompass several hundred nucleotides. "Fingerprints" of the three mammalian methyl-labelled 18S rRNA species were qualitatively indistinguishable. "Fingerprints" of digests of 28S rRNA of hamster and mouse L-cells were extremely similar to those of HeLa cells, differing in one and three methylated oligonucleotides respectively. "Fingerprints" of methyl-labelled rRNA from chick and Xenopus strongly resembled those of mammals in most respects, but differed in several oligonucleotides in both 18S and 28S rRNA. At least some of the differences between "fingerprints" appear to be due to single base changes or to the presence or absence of methyl groups at particular points in the primary sequence. The findings strongly suggest that the methylated-nucleotide sequences are at least 95% homologous between the rRNA molecules of the two most distantly related vertebrates compared, man and Xenopus laevis.     

Quantitative alterations in the nucleolar and nucleoplasmic ribosomal ribonucleic acids in regenerating rat liver

A quantitative analysis of the nuclear pre-rRNA (precursor to rRNA) and rRNA in normal and 12h-regenerating rat liver was carried out, and the absolute amounts of the identified pre-rRNA and rRNA species in the nucleolus and nucleoplasm were determined. Characteristic changes in the pre-rRNA and rRNA pool sizes in regenerating liver are found which reveal alternations in both pre-rRNA processing and nucleocytoplasmic transition of ribosomes.     

Fidelity of ribosomal ribonucleic acid synthesis by nucleoli and nucleolar chromatin.

Isolated nucleoli, nucleolar chromatin, and nucleolar DNA were used as templates for DNA synthesis in appropriately supplemented systems in which RNA polymerases other than RNA polymerase I were blocked by alpha-amanitin. With the aid of nucleotide analysis, DNA-RNA hybridization, and homochromatography fingerprinting, it was found that isolated nucleoli and nucleolar chromatin serve primarily as templates for synthesis of rRNA. However, the products formed with purified nucleolar DNA as a template do not contain the specific rRNA oligonucleotides nor are they appreciably hybridized to the rDNA region on cesium chloride gradients. These results indicate that whole nucleoli and nucleolar chromatin contain control mechanisms that restrict readouts by RNA polymerase I of nucleolar DNA to rDNA.     

The circular dichroism of ribosomal ribonucleic acids.

1. The c.d. (circular dichroism) of Drosophila melanogaster rRNA (42% G+C) and of G+C-rich fragments (78% G+C) obtained by partial hydrolysis of rabbit L-rRNA (the largest RNA species isolated from the large subribosomal particle) were measured and found to differ substantially. 2. To interpret these spectra a relation between c.d. of bihelical RNA and % G+C was derived, namely delta epsilonfG = AFG2+bfG+c, where deltaepsilonfG is the c.d. of RNA characterized by a mole fraction, fG, of guanine nucleotides and a, b and c are constants. 3. A frame of reference was established by studying the c.d. of a range of rRNA species, including S-rRNA (the RNA species isolated from the smaller subribosomal particle) and L-rRNA of Escherichia coli. 4. It was found for the rRNA species studied that 0.60+/-0.05 of residues appear to form bihelical secondary structure. 5. A higher helical content, 0.66+/-0.05, was found for the G+C-rich fragment of L-rRNA. The difference in the c.d. of rabbit L-rRNA and of D. melanogaster rRNA is attributable to the dependence of c.d. of the bihelical parts on %G+C. 6. The minimum in c.d. at 295 nm increases with increasing %G+C. The c.d. of rRNA was compared with that of the parent subparticle in this region of the spectrum, where high precision may be attained.     

Biosynthesis of a hypermodified nucleotide in Saccharomyces carlsbergensis 17S and HeLa-cell 18S ribosomal ribonucleic acid.

The biosynthesis of a hypermodified nucleotide, similar to or identical with 3-(3-amino-3-carboxypropyl)-1-methylpseudouridine monophosphate, present in Saccharomyces carlsbergensis 17S and HeLa-cell 18S rRNA, was investigated with respect to the sequence of reactions required for synthesis and their timing in ribosome maturation. In both yeast and HeLa cells methylation precedes attachment of the 3-amino-3-carboxypropyl group. In yeast the methylated precursor nucleotide was tentatively characterized as 1-methylpseudouridine. This precursor nucleotide was demonstrated in both 37S and most of the cytoplasmic 18S pre-rRNA (rRNA precursor) molecules. The synthesis of the hypermodified nucleotide is completed just before the final cleavage of 18S pre-rRNA to give 17S rRNA, so that the final addition of the 3-amino-3-carboxypropyl group is a cytoplasmic event. Comparable experiments with HeLa cells indicated that formation of 1-methylpseudouridine occurs at the level of 45S RNA and addition of the 3-amino-3-carboxypropyl group occurs in the cytoplasm on newly synthesized 18S RNA.     

23S ribosomal ribonucleic acid of macrolide-producing streptomycetes contains methylated adenine.

Coresistance to macrolide, lincosamide, and streptogramin B-type (MLS) antibiotics by a common biochemical mechanism characterizes clinically resistant pathogens. Of 10 streptomycetes tested for resistance to macrolide, lincosamide, and streptogramin B-type antibiotics, only 1, Streptomyces erythreus, the organism used for production of erythromycin, was found resistant to all three classes; moreover, it was the only streptomycete in the series tested found to contain N6-dimethyladenine (m62A) in 23S ribosomal ribonucleic acid, the structural alteration of ribosomal ribonucleic acid associated with clinical resistance. Of the seven streptomycetes tested for the presence of m62A and N6-methyladenine (m6A), two, S. fradiae and S. cirratus, which produce the macrolide antibiotics tylosin and cirramycin, respectively, were found to contain m6A, but not m62A. The remaining strains tested, including strains which produce lincomycin and streptogramins, contained neither m6A nor m62A.     

Evidence on the conformation of HeLa-cell 5.8S ribosomal ribonucleic acid from the reaction of specific cytidine residues with sodium bisulphite.

The reaction of HeLa-cell 5.8S rRNA with NaHSO3 under conditions in which exposed cytidine residues are deaminated to uridine was studied. It was possible to estimate the reactivities of most of the 46 cytidine residues in the nucleotide sequence by comparing 'fingerprints' of the bisulphite-treated RNA with those of untreated RNA. The findings were consistent with the main features of the secondary-structure model for mammalian 5.85S rRNA proposed by Nazar, Sitz, & Busch [J. Biol. Chem (1975) 250, 8591--8597]. Five out of six regions that are depicted in the model as single-stranded loops contain cytidine residues that are reactive towards bisulphite at 25 degrees C (the other loop contains no cytidine). The cytidine residue nearest to the 3'-terminus is also reactive. Several cytidines residues that are internally located within proposed double-helical regions show little or no reactivity towards bisulphite, but the cytidine residues of several C.G pairs at the ends of helical regions show some reactivity, and one of the proposed loops appears to contain six nucleotides, rather than the minimum of four suggested by the primary structure. Two cytidine residues that are thought to be 'looped out' by small helix imperfections also show some reactivity.     

Synthesis and maturation of ribosomal ribonucleic acids in isolated HeLa cell nuclei. A tracer study on the topology of the 45S precursor of ribosomal ribonucleic acids

The synthesis and processing of RNA by isolated HeLa cell nuclei was studied at low ionic strength in the presence of alpha-amanitin. The RNA polymerase reaction, with endogenous template and enzyme, rapidly reaches a plateau dependent on the amount of nuclei. Evidence is presented that incorporation of [(3)H]UMP proceeds only in growing RNA chains, whereas initiation of new RNA chains is arrested. The product formed contains all the main components of the 45S pre-rRNA (precursor of rRNA) maturation pathway (45S, 32S and 20S pre-rRNA; 28S and 18S rRNA). Most of the labelled material is in the mature rRNA components and their immediate precursors, even at very short times of incubation (2min). Small, but definite, 5S and 4S RNA peaks are also observed. At shorter incubation times a substantial amount of [(3)H]UMP is incorporated into RNA molecules in the 24S and 10-16S zones. This RNA material is considered to represent the non-conserved segments of 45S pre-rRNA in the process of nucleolytic degradation. A model for the tracer study of the topology of 45S pre-rRNA, on arrest of rRNA initiation, is discussed. The experimental evidence obtained supports the following structure of 45S pre-rRNA: 5'-end-28S rRNA unit-18S rRNA unit-nonconserved segment-3'-end.