Degradation of 18S and 28S ribosomal RNA in the cytoplasm of rat liver cells after inhibition of protein synthesis

Inhibition of protein synthesis (up to 95%) in starved rat liver cells after a single injection of a sublethal dose of cycloheximide (0.3 mg per 100 g of body weight) results in degradation of 18S rRNA during the first 3 hours, whereas the 28S rRNA remains unaffected. However, the increase of 28S rRNA degradation products was observed by the 6th and 12th hours. The rapid decay of 18S rRNA is due to the degradation of this RNA in 40S ribosomal subunits. In contrast to 28S rRNA the specific radioactivity of 18S rRNA is increased by the 6th hour. Presumably the synthesis and processing of 18S rRNA impaired during the 1st hour are recovered partially or completely by this time. A molecular mechanism underlying 18S rRNA degradation in 40S ribosomal subunits is proposed.     

Chromosomal localization of 18S + 28S and 5S ribosomal RNA genes in evolutionarily diverse anuran amphibians

The chromosomal locations of the 18S + 28S and 5S ribosomal RNA genes have been analyzed by in situ hybridization in ten anuran species of different taxonomic positions. The chosen species belong to both primitive and evolved families of the present day Anura. Each examined species has 18S + 28S rRNA genes clustered in one locus per haploid chromosome set: this locus is placed either in an intercalary position or proximal to the centromere, or close to the telomere. The 5S rRNA genes are arranged in clusters which vary in number from one to six per haploid set. The 5S rDNA sites are found in intercalary positions, at the telomeres, and at, or close to, the centromeres. Microchromosomes and small chromosomes in primitive karyotypes have been found to carry 5S rDNA sequences. The results are discussed in relation to ideas on the karyological evolution of Amphibia.     

Wasting of 18 S ribosomal RNA by human myeloma cells cultured in adenosine.

When human myeloma cells are pulsed for one hour with 3H-uridine and chased for six hours in fresh medium containing unlabeled uridine, the processing of 45 S rRNA precursor into the stable 28 S and 18 S rRNA components can be followed. However, when the cells are chased in exogenous adenosine instead of uridine, the accumulation of 18 S rRNA is selectively inhibited. Cells pulsed with 3H-adenosine and chased in the absence of exogenous nucleosides exhibit normal rRNA precursor processing, while cells pulsed simultaneously with 3H-uridine and 3H-adenosine and chased with uridine and adenosine are deficient in labeled 18 S rRNA. Consequently, the inhibition of 18 S rRNA accumulation by adenosine is not an artifact of labeling nor is it relieved by an equal molar concentration of uridine. The wasting of 18 S rRNA in human myeloma cells is similar to that reported to occur in normal lymphocytes during the quiescent state.     

Physical linkage of the 5 S cistrons to the 18 S and 28 S ribosomal RNA cistrons in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, there are approximately 150 cistrons coding for 5 S RNA. These cistrons are interspersed with the cistrons coding for the high molecular weight ribosomal RNAs.     

Primary and secondary structure of rat 28 S ribosomal RNA.

The primary structure of rat (Rattus norvegicus) 28 S rRNA is determined inferred from the sequence of cloned rDNA fragments. The rat 28 S rRNA contains 4802 nucleotides and has an estimated relative molecular mass (Mr, Na-salt) of 1.66 X 10(6). Several regions of high sequence homology with S. cerevisiae 25 S rRNA are present. These regions can be folded in characteristic base-paired structures homologous to those proposed for Saccharomyces and E. coli. The excess of about 1400 nucleotides in the rat 28 S rRNA (as compared to Saccharomyces 25 S rRNA) is accounted for mainly by the presence of eight distinct G+C-rich segments of different length inserted within the regions of high sequence homology. The G+C content of the four insertions, containing more than 200 nucleotides, is in the range of 78 to 85 percent. All G+C-rich segments appear to form strongly base-paired structures. The two largest G+C-rich segments (about 760 and 560 nucleotides, respectively) are located near the 5'-end and in the middle of the 28 S rRNA molecule. These two segments can be folded into long base-paired structures, corresponding to the ones observed previously by electron microscopy of partly denatured 28 S rRNA molecules.     

Turnover of ribosomal 28S and 18S rRNA during rat liver regeneration

The turnover of 28S and 18S rRNA was studied in the course of 12 d after partial hepatectomy, including the proliferative (1st to 5th d) and post-proliferative (6th to 12th d) phases of liver regeneration. Turnover data, as the day-to-day rates of synthesis and degradation of 28S and 18S rRNA, were obtained by employing a suitable experimental procedure for the estimation of the increase of the amount of rRNA in the regenerating liver. It was found that 28S and 18S rRNA are accumulated into the cytoplasm and degraded at identical rates both in the proliferative and post proliferative phases. The turnover of both rRNA moieties is markedly slower during the first 3 d of liver regeneration.     

Chromosome location of the genes for 28S, 18S and 5S ribosomal RNA in Triturus marmoratus (Amphibia Urodela)

The localization of the 28S, 18S and 5S rRNA genes in the mitotic chromosomes, and of the 5S rRNA genes in the lampbrush chromosomes of Triturus marmoratus has been studied by RNA/DNA in situ hybridization. The 28S and 18S genes are located in a subterminal position, and the 5S genes in an intermediate position, on the long arm of mitotic chromosome X. In situ hybridization on lampbrush chromosomes has shown that the 5S genes are located at or near a dense matrix loop landmark. The cytogenetic implications of these findings are briefly discussed.     

Location of the genes coding for 18S and 28S ribosomal RNA in the human genome

³H-rRNA obtained from Xenopus laevis tissue cultured cells, or a ³H-cRNA made from Xenopus ribosomal DNA, was used for heterologous in situ hybridisation with human lymphocyte metaphase chromosomes. Prior to hybridisation, chromosome spreads were stained with Quinacrine and selected cells showing good Q-banding photographed; the same cells were then rephotographed after autoradiography and pairs of photographs for each cell were used to make dual karyotypes. The chromosomes within each karyotype were divided into equal sized segments (approx. 0.7 μ), with a fixed number of segments for each chromosome type. The distribution of silver grains between segments showed that the ³H-RNAs hybridised specifically to the nucleolar organising regions of the D and G group chromosomes with no other sites of localised labelling in the complement. Control experiments showed no localisation, with insignificant labelling, when metaphase spreads were incubated in a mixture containing Xenopus ³H-rRNA and competing cold human (HeLa) rRNA. Filter hybridisation experiments on isolated human DNA showed that the Xenopus derived ³H-RNAs hybridised to a fraction of human DNA which was on the heavy side of the main DNA peak and that these RNAs were competed out in the presence of excess cold human rRNA, confirming the specificity of the heterologous hybridisation. In situ hybridisation experiments were also carried out on cells from individuals with one chromosome pair showing heteromorphism for either a very long stalk (nucleolar constriction) subtending a satellite, or a large satellite. It was shown that the chromosome with the large stalk hybridised four times as much ³H-rRNA as its homologue, whereas differences in the sizes of the subtended satellites did not materially affect hybridisation levels indicating that rDNA is located in the stalks and not the satellites. The amount of ³H-rRNA hybridised differs between chromosomes and individuals; these differences are heritable and rDNA can be detected by in situ hybridisation in all three chromosomes number 21 in cells from Down's patients and in translocated chromosomes conta.ining a nucleolar constriction. Different D and G group chromosomes which hybridised equal amounts of ³H-rRNA participated in rosette associations at metaphase in a random fashion in some individuals and in a non-random fashion in others. In all individuals studied chromosomes with large amounts of rDNA were not found to be preferentially involved in association. It was therefore concluded that the probability of a chromosome being involved in the formation of a common nucleolus is not a simple function of its rDNA content and other possible factors are considered.     

Photo-induced protein cross-linking to 5S RNA and 28-5.8S RNA within rat-liver 60S ribosomal subunits

Rat liver 60S ribosomal subunits were irradiated with 254-nm ultraviolet light (1.26 X 10(4) quanta/subunit), under conditions which preserved their functional activity. Cross-linked RNA-protein complexes were recovered after unreacted proteins had been removed by repeated acetic acid extractions. Proteins linked to the whole rRNA, to 5S RNA and to 28-5.8 S RNAs were identified by two-dimensional gel electrophoresis after RNA hydrolysis by ribonucleases T1 and A. Our results showed that numerous proteins interact with rRNAs (at least ten with 28-5.8 S RNA, eight with 5S RNA and among these three are common to both) and have been discussed in the light of all the available data.     

Nuclear retention of 18S ribosomal RNA by human myeloma cells

Summary Normal quiescent lymphocytes regulate their ribosome content by selectively degrading newly synthesized 18S ribosomal RNA. Unlike actively dividing HeLa cells, lymphocytes retain 18 S ribosomal RNA in the nucleus after synthesis instead of immediately transporting it to the cytoplasm. Subcellular fractionation of the highly differentiated human neoplastic lymphocyte RPMI-8226 reveals that this cell line also retains 18 S ribosomal RNA in the nucleus, a trait not displayed by the less differentiated human lymphoblastoid cell line RPMI-4265. These observations suggest that neoplastic cells can be phenotypically characterized by their ribosomal RNA processing patterns.Operated by Union Carbide Corporation with the Department of Energy     

Location of genes coding for 18S and 28S ribosomal RNA within the genome of Mus musculus

Cytological detection of cistrons coding for 18S and 28S ribosomal RNA (rRNA) within the genome of Mus musculus inbred strain SEC/1ReJ was accomplished using the technique of in situ hybridization. Metaphase chromosome spreads prepared from cultured fetal mouse cells were stained with quinacrine-HCl and photographed. After destaining, they were hybridized to Xenopus laevis tritiated 18S and 28S rRNA, specific activity 7.5 X 10(6) dpm/mug. Silver grains clustered over specific chromosomes were readily apparent after 4 months of autoradiographic exposure. The identity of the labelled chromosomes was established by comparing the autoradiographs to quinacrine photographs showing characteristic fluorescent banding of the chromosomes in each metaphase spread. The 18S and 28S rRNA was found to hybridize to chromosomes 12, 18, and 16. Statistical analysis of the grain distribution over 26 spreads revealed that the three chromosomes were significantly labelled. Grains over these chromosomes were concentrated in an area immediately distal to the centromere, a region which in chromosomes 12 and 18 in this particular strain is the site of a secondary constriction. The relative size of the secondary constrictions, long and thus prominent on chromosome 12, obvious but shorter on 18, and indistinguishable on chromosome 16, correlated with the average number of grains observed over the centromeric region of these chromosomes, 2.5, 1.0, and 0.78, respectively.     

Location of single-stranded and double-stranded regions in rat liver ribosomal 5S RNA and 5.8S RNA.

Rat liver 5S rRNA and 5.8S rRNA were end-labelled with 32P at 5'-end or 3'-end of the polynucleotide chain and partially digested with single-strand specific S1 nuclease and double-strand specific endonuclease from the cobra Naja naja oxiana venom. The parallel use of these two structure-specific enzymes in combination with rapid sequencing technique allowed the exact localization of single-stranded and double-stranded regions in 5S RNA and 5.8 S RNA. The most accessible regions to S1 nuclease in 5S RNA are regions 33-42, 74-78, 102-103 and in 5.8 S RNA 16-20, 26-29, 34-36, 74-80 and a region around 125-130. The cobra venom endonuclease cleaves the following areas in 5S RNA: 7-8, 17-20, 28-30, 49-51, 56-57, 60-64, 69-70, 81-82, 95-97, 106-112. In 5.8S RNA the venom endonuclease cleavage sites are 4-7, 10-13, 21-22, 33-35, 43-45, 51-55, 72-74, 85-87, 98-99, 105-106, 114-115, 132-135. According to these results the tRNA binding sequences proposed by Nishikawa and Takemura [(1974) FEBS Lett. 40, 106-109], in 5S RNA are located in partly single-stranded region, but in 5.8S RNA in double-stranded region.     

2'-O-methylated oligonucleotides in ribosomal 18S and 28S RNA of a mouse hepatoma, MH 134.

Simple two-dimensional thin-layer chromatography was found to be useful for the separation of sugar methylated dinucleotides in RNA. Of the 16 possible sequences of the type Nm-Np, 15 were separated and all the sequences were determined. In a mouse hepatoma, MH 134, the levels of the sugar methylation in the 18S and 28S RNA molecules were 17-18 and 11-12 per 1000 nucleotides, respectively. Thus, 18s RNA contained approximately 35 2'-O-methylated dinucleotides and 28S RNA approximately 60 2'-O-methylated dinucleotides. The pattern of distribution was also distinct between these two molecules. Two 2'-O-methylated trinucleotides were identified in the 28S RNA with the sequences Um-Gm-Up and Um-Gm-psip. A unique 2'-O-methylated tetranucleotide was present also in the 28S RNA, the sequence of which was Am-Gm-Cm-Ap. The 5'-terminal nucleotides of both 18S and 28S RNA were obtained as nucleoside 3',5'-diphosphates (pNp) in the trinucleotide fraction of the RNase T2 digest. The 5'-termimi of 18S and 28S RNA were pUp and pCp, respectively, and found to be almost homogeneous.