Intermolecular hybridization of 5S rRNA with 18S rRNA: identification of a 5'-terminally-located nucleotide sequence in mouse 5S rRNA which base-pairs with two specific complementary sequences in 18S rRNA.

Eukaryotic 5S rRNA hybridizes specifically with 18S rRNA in vitro to form a stable intermolecular RNA:RNA hybrid. We have used 5S rRNA/18S rRNA fragment hybridization studies coupled with ribonuclease digestion and primer extension/chain termination analysis of 5S rRNA:18S rRNA hybrids to more completely map those mouse 5S rRNA and 18S rRNA sequences responsible for duplex formation. Fragment hybridization analysis has defined a 5'-terminal region of 5S rRNA (nucleotides 6-27) which base-pairs with two independent sequences in 18S rRNA designated Regions 1 (nucleotides 1157-1180) and 2 (nucleotides 1324-1339). Ribonuclease digestion of isolated 5S rRNA:18S rRNA hybrids with both single-strand- and double-strand-specific nucleases supports the involvement of this 5'-terminal 5S rRNA sequence in 18S rRNA hybridization. Primer extension/chain termination analysis of isolated 5S rRNA:18S rRNA hybrids confirms the base-pairing of 5S rRNA to the designated Regions 1 and 2 of 18S rRNA. Using these results, 5S rRNA:18S rRNA intermolecular hybrid structures are proposed. Comparative sequence analysis revealed the conservation of these hybrid structures in higher eukaryotes and the same but smaller core hybrid structures in lower eukaryotes and prokaryotes. This suggests that the 5S rRNA:16S/18S rRNA hybrids have been conserved in evolution for ribosome function.     

RNase L-independent specific 28S rRNA cleavage in murine coronavirus-infected cells

We characterized a novel 28S rRNA cleavage in cells infected with the murine coronavirus mouse hepatitis virus (MHV). The 28S rRNA cleavage occurred as early as 4 h postinfection (p.i.) in MHV-infected DBT cells, with the appearance of subsequent cleavage products and a decrease in the amount of intact 28S rRNA with increasing times of infection; almost all of the intact 28S rRNA disappeared by 24 h p.i. In contrast, no specific 18S rRNA cleavage was detected in infected cells. MHV-induced 28S rRNA cleavage was detected in all MHV-susceptible cell lines and all MHV strains tested. MHV replication was required for the 28S rRNA cleavage, and mature cytoplasmic 28S rRNA underwent cleavage. In certain combination of cells and viruses, pretreatment of virus-infected cells with interferon activates a cellular endoribonuclease, RNase L, that causes rRNA degradation. No interferon was detected in the inoculum used for MHV infection. Addition of anti-interferon antibody to MHV-infected cells did not inhibit 28S rRNA cleavage. Furthermore, 28S rRNA cleavage occurred in an MHV-infected mouse embryonic fibroblast cell line derived from RNase L knockout mice. Thus, MHV-induced 28S rRNA cleavage was independent of the activation of RNase L. MHV-induced 28S rRNA cleavage was also different from apoptosis-related rRNA degradation, which usually occurs concomitantly with DNA fragmentation. In MHV-infected 17Cl-1 cells, 28S rRNA cleavage preceded DNA fragmentation by at least 18 h. Blockage of apoptosis in MHV-infected 17Cl-1 cells by treatment with a caspase inhibitor did not block 28S rRNA cleavage. Furthermore, MHV-induced 28S rRNA cleavage occurred in MHV-infected DBT cells that do not show apoptotic signs, including activation of caspase-3 and DNA fragmentation. Thus, MHV-induced 28S rRNA cleavage appeared to differ from any rRNA degradation mechanism described previously.     

Accelerated rates of ribosomal RNA synthesis during growth of contracting heart cells in culture

Contractile activity of neonatal cardiac myocytes stimulated hypertrophic growth as compared with nonbeating cells that were depolarized with 50 mM KCl. Growth of contracting myocytes was associated with an increased rRNA content as measured by the total RNA/DNA ratio. The fractional rates of rRNA synthesis (K8) and rRNA degradation were determined in contracting and nonbeating myocytes to assess their relative contributions in increasing rRNA content during growth. The values for K8 were calculated from the specific radioactivity of 3'-[3H]UMP in 18 and 28 S rRNA after purification by hybridization to cloned rDNA. The cellular [3H]UTP pool served as the precursor for rRNA synthesis in myocytes that were labeled with 50 microM [3H]uridine. K8 values for 18 and 28 S rRNA in contracting myocytes were accelerated by 59 and 53%, respectively, after 3 days as compared with nonbeating myocytes. Calculations of the rate of cellular rRNA synthesis, which took into account the increased content of myocyte rRNA, revealed that synthesis of both 18 and 28 S rRNA was accelerated 2-fold after 2 days of contraction. The derived values for degradation of 18 and 28 S rRNA were increased marginally in contracting myocytes, but cellular rRNA degradation rates averaged 57% higher. The difference between cellular rates of rRNA synthesis and degradation in contracting myocytes accounted for the 30% increase in rRNA content. These data demonstrated that increased rRNA content in contracting myocytes resulted from acceleration of the fractional rate of rRNA synthesis.     

The complete nucleotide sequence of a 23-S rRNA gene from tobacco chloroplasts

The nucleotide sequence of a tobacco chloroplast 23-S rRNA gene, including the spacer between it and the 4.5-S rRNA gene, has been determined. The 23-S rRNA coding region is 2804-base-pairs long. A comparison with the 23-S rRNA sequence of Escherichia coli reveals strong homology and further shows a similarity between the chloroplast 4.5-S rRNA and the 3'-terminal region of E. coli 23-S rRNA. However, the 101-base-pair spacer sequence between the 23-S and 4.5-S rRNA genes has little homology with E. coli 23-S rRNA.     

Analysis of 23S rRNA genes in metagenomes - a case study from the Global Ocean Sampling Expedition

As an evolutionary marker, 23S ribosomal RNA (rRNA) offers more diagnostic sequence stretches and greater sequence variation than 16S rRNA. However, 23S rRNA is still not as widely used. Based on 80 metagenome samples from the Global Ocean Sampling (GOS) Expedition, the usefulness and taxonomic resolution of 23S rRNA were compared to those of 16S rRNA. Since 23S rRNA is approximately twice as large as 16S rRNA, twice as many 23S rRNA gene fragments were retrieved from the GOS reads than 16S rRNA gene fragments, with 23S rRNA gene fragments being generally about 100 bp longer. Datasets for 16S and 23S rRNA sequences revealed similar relative abundances for major marine bacterial and archaeal taxa. However, 16S rRNA sequences had a better taxonomic resolution due to their significantly larger reference database.Reevaluation of the specificity of previously published PCR amplification primers and group specific fluorescence in situ hybridization probes on this metagenomic set of non-amplified 23S rRNA sequences revealed that out of 16 primers investigated, only two had more than 90% target group coverage. Evaluations of two probes, BET42a and GAM42a, were in accordance with previous evaluations, with a discrepancy in the target group coverage of the GAM42a probe when evaluated against the GOS metagenomic dataset.     

5S rRNA sequences of myxobacteria and radioresistant bacteria and implications for eubacterial evolution

5S rRNA sequences were determined for the myxobacteria Cystobacter fuscus, Myxococcus coralloides, Sorangium cellulosum, and Nannocystis exedens and for the radioresistant bacteria Deinococcus radiodurans and Deinococcus radiophilus. A dendrogram was constructed by using weighted pairwise grouping based on these and all other previously known eubacterial 5S rRNA sequences, and this dendrogram showed differences as well as similarities compared with results derived from 16S rRNA analyses. In the dendrogram, Deinococcus 5S rRNA sequences clustered with 5S rRNA sequences of the genus Thermus, as suggested by the results of 16S rRNA analyses. However, in contrast to the 16S rRNA results, the Deinococcus-Thermus cluster divided the 5S rRNA sequences of the alpha subdivision of the class Proteobacteria from the 5S rRNA sequences of the beta and gamma subgroups of the Proteobacteria. The myxobacterial 5S rRNA sequence data failed to confirm the existence of a delta subgroup of the class Proteobacteria, which was suggested by the results of 16S rRNA analyses.     

向日葵rRNA基因克隆及其染色体定位研究

该研究以内葵杂3号三交种为材料,采用同源序列法克隆了5SrRNA和18SrRNA基因并进行了序列测定,测得片段长度分别为515bp和1808bp。以5SrRNA、18SrRNA和45SrRNA基因为探针,分别与内葵杂3号三交种染色体进行荧光原位杂交（FISH）分析。结果表明：45SrRNA和18SrRNA基因均得到3对杂交信号且位点分布相同,分别位于第3对和第10对染色体及第2对随体染色体的短臂末端;5SrRNA基因的信号位点共有2对,分布在第7对和第10对染色体短臂端部。     

Discordant 16S and 23S rRNA gene phylogenies for the genus Helicobacter: implications for phylogenetic inference and systematics

Analysis of 16S rRNA gene sequences has become the primary method for determining prokaryotic phylogeny. Phylogeny is currently the basis for prokaryotic systematics. Therefore, the validity of 16S rRNA gene-based phylogenetic analyses is of fundamental importance for prokaryotic systematics. Discrepancies between 16S rRNA gene analyses and DNA-DNA hybridization and phenotypic analyses have been noted in the genus Helicobacter. To clarify these discrepancies, we sequenced the 23S rRNA genes for 55 helicobacter strains representing 41 taxa (>2,700 bases per sequence). Phylogenetic-tree construction using neighbor-joining, parsimony, and maximum likelihood methods for 23S rRNA gene sequence data yielded stable trees which were consistent with other phenotypic and genotypic methods. The 16S rRNA gene sequence-derived trees were discordant with the 23S rRNA gene trees and other data. Discrepant 16S rRNA gene sequence data for the helicobacters are consistent with the horizontal transfer of 16S rRNA gene fragments and the creation of mosaic molecules with loss of phylogenetic information. These results suggest that taxonomic decisions must be supported by other phylogenetically informative macromolecules, such as the 23S rRNA gene, when 16S rRNA gene-derived phylogeny is discordant with other credible phenotypic and genotypic methods. This study found Wolinella succinogenes to branch with the unsheathed-flagellum cluster of helicobacters by 23S rRNA gene analyses and whole-genome comparisons. This study also found intervening sequences (IVSs) in the 23S rRNA genes of strains of 12 Helicobacter species. IVSs were found in helices 10, 25, and 45, as well as between helices 31' and 27'. Simultaneous insertion of IVSs at three sites was found in H. mesocricetorum.     

Ribosomal RNA metabolism during renal hypertrophy. Evidence of decreased degradation of newly synthesized ribosomal RNA

The degradation rates of kidney rRNA labeled before UNI or sham are unchanged 5 days after the operations (t one-and-a half, 88 h). Therefore, there is no contribution from pre-existing rRNA to the increased amount of rRNA in the stimulated kidney. After labeling with L-(methyl-3H)methionine, the kinetics of incorporation into rRNA precursors, 10-60 min and at the postoperative times of 4, 16, 36, and 96 h. The specific activity of cytoplasmic rRNA after 1-h labeling with L-(methyl-3H)methionine increased occured at 4 or 96 h. Since (a) the rate of degradation of rRNA, (b) the kinetics of incorporation and processing of rRNA precursors, and (c) the rate of RNA synthesis appear unchanged after UNI, the accretion of rRNA must involve decreased degradation of newly synthesized rRNA.     

Ribosomal RNA genes in Mycoplasma

Using Southern blotting analysis with labelled mycoplasmal ribosomal RNA as probe, two fragments (1 Kb and 5 Kb) were detected in an EcoR I digest of Mycoplasma capricolum DNA. This analysis revealed that the 5 Kb fragment carries both 16S rRNA sequences and the entire 23S rRNA gene of this mycoplasma. The 1 Kb fragment contains 16S rRNA sequences only. The 5 Kb EcoR I fragment has been cloned and used to characterize the structure of rRNA cistrons in various Mycoplasma strains. These experiments clearly demonstrate a substantial homology of Mycoplasma capricolum rRNA sequences with the E. coli rRNA cistron on one hand, and with Mycoplasma mycoides subsp. capri and Acholeplasma laidlawii on the other hand. This analysis also reveals two rRNA cistrons in Mycoplasma mycoides subsp. capri and Acholeplasma laidlawii whereas one rRNA cistron is present in Mycoplasma capricolum.     

Nucleotide sequence of the single ribosomal RNA operon of pea chloroplast DNA

The nucleotide sequence of an 8 kbp region of pea ( Pisum sativum L.) chloroplast DNA containing the rRNA operon and putative promoter sites has been determined and compared to the corresponding sequences from maize, tobacco and the liverwort Marchantia polymorpha . The chloroplast DNA species of all vascular plants investigated, with the exception of a few legumes including pea, and of Marchantia contain an inverted repeat with an rRNA operon. The pea rRNA operon is the first sequenced rRNA operon from a plant with only one copy of the rRNA genes per molecule of chloroplast DNA. The organization of the operon is the same as for maize, tobacco and Marchantia . i.e. tRNA-Val gene/16S rRNA gene/spacer with intron-containing genes for tRNA-Ile and tRNA-Ala/23S rRNA gene/4.5S rRNA gene/5S rRNA gene. Current evidence suggests that the tRNA-Val gene may not be contranscribed with the other genes. For pea 16S, 23S, 4.5S and 5S rRNA have 1488, 2813, 105 and 121 nucleotides, respectively. The homologies of the entire operon (the tRNA-Val gene - 5S rRNA region) to those from tobacco, maize and Marchantia are 88, 82 and 79%, respectively. The corresponding homologies for tobacco/maize, tobacco/ Marchantia and maize/ Marchantia have similar values. The 16S and 23S rRNA genes from pea are more than 90% homologous to those from the 3 other species. We conclude that the fact that pea only has one set of rRNA genes per molecule of chloroplast DNA is apparently not correlated with any significant difference between the pea operon and the rRNA operons from tobacco, maize and Marchantia .     

Post-transcriptional regulation of ribosome accumulation during myoblast differentiation

The synthesis, accumulation and stability of rRNA were examined in embryonic quail myoblasts differentiating in cell culture. Quail myoblasts initially divide rapidly in culture, and accumulate 28S and 18S rRNA and ribosomes at a rate which maintains a constant ribosome content during cell division. After these myoblasts fuse, cell division ceases and ribosomes accumulate in fibers, but at a reduced rate which is only one fourth that in dividing myoblasts. Measurements of rRNA stability by 3H-methyl-methionine pulse-chase analysis show that 28S and 18S rRNA formed by fibers turn over with half-lives of 45 hr, and rRNA formed by myoblasts remains stable until fusion and then also turns over in fibers. Turnover of rRNA in fibers accounts for only half the reduction in ribosome accumulation following myoblast fusion. Measurements of the incorporation of 3H-adenosine into rRNA and ATP pools show that the rates of synthesis of rRNA precursor do not decrease after myoblast fuse, but half the rRNA molecules synthesized by fibers are degraded during processing. Degradation of rRNA during processing reduces the rate of formation of 28S and 18S rRNA, and together with rRNA turnover quantitatively accounts for the reduced rate of ribosome accumulation in fibers.     

Bacterial ribosomal RNA in pieces

The exact knowledge on the ribosomal RNA (rRNA) structure is an important prerequisite for work with rRNA sequences in bioinformatic analyses and in experimental research. Most available rRNA sequences of bacteria are based on gene sequences and on similarity analyses using Escherichia coli rRNA as a standard. Therefore, it is often overlooked that many bacteria harbour mature rRNA 'in pieces'. In some cases, the processing steps during the fragmentation lead to the removal of rRNA segments that are usually found in the ribosome. In this review, the current knowledge on the mechanisms of rRNA fragmentation and on the occurrence of fragmented rRNA in bacteria is summarized, and the physiological implications of this phenomenon are discussed.     

Primary and secondary structures of Tetrahymena and aphid 5.8S rRNAs: structural features of 5.8S rRNA which interacts with the 28S rRNA containing the hidden break

The Tetrahymena 5.8S rRNA is 154 nucleotides long, the shortest so far reported except for the split 5.8S rRNAs of Diptera (m5.8S plus 2S rRNA). In this molecule several nucleotides are deleted in the helix e (GC-rich stem) region. Upon constructing the secondary structure in accordance with "burp-gun" model, the Tetrahymena 5.8S rRNA forms a wide-open "muzzle" of the terminal regions due to both extra nucleotides and several unpaired bases. The aphid 5.8S rRNA consists of 161 nucleotides and can form stable helices in both terminal and helix e regions. As a whole, the secondary structure of Tetrahymena 5.8S rRNA resembles that of Bombyx 5.8S molecule while the aphid 5.8S rRNA shares several structural features with the HeLa 5.8S molecule. Likely, the 5.8S rRNA attached to the 28S rRNA with the hidden break differs in structure from those interacting with the 28S partners without the break. Nucleotide sequences of 5.8S rRNA in insects as well as in protozoans are not so conservative evolutionarily as in vertebrates.     

Monitoring precursor 16S rRNAs of Acinetobacter spp. in activated sludge wastewater treatment systems

Recently, Cangelosi and Brabant used oligonucleotide probes targeting the precursor 16S rRNA of Escherichia coli to demonstrate that the levels of precursor rRNA were more sensitive to changes in growth phase than the levels of total rRNA (G. A. Cangelosi and W. H. Brabant, J. Bacteriol. 179:4457-4463, 1997). In order to measure changes in the levels of precursor rRNA in activated sludge systems, we designed oligonucleotide probes targeting the 3' region of the precursor 16S rRNA of Acinetobacter spp. We used these probes to monitor changes in the level of precursor 16S rRNA during batch growth of Acinetobacter spp. in Luria-Bertani (LB) medium, filtered wastewater, and in lab- and full-scale wastewater treatment systems. Consistent with the previous reports for E. coli, results obtained with membrane hybridizations and fluorescence in situ hybridizations with Acinetobacter calcoaceticus grown in LB medium showed a more substantial and faster increase in precursor 16S rRNA levels compared to the increase in total 16S rRNA levels during exponential growth. Diluting an overnight culture of A. calcoaceticus grown in LB medium with filtered wastewater resulted in a pattern of precursor 16S rRNA levels that appeared to follow diauxic growth. In addition, fluorescence in situ hybridizations with oligonucleotide probes targeting total 16S rRNA and precursor 16S rRNA showed that individual cells of A. calcoaceticus expressed highly variable levels of precursor 16S rRNA when adapting from LB medium to filtered sewage. Precursor 16S rRNA levels of Acinetobacter spp. transiently increased when activated sludge was mixed with influent wastewater in lab- and full-scale wastewater treatment systems. These results suggest that Acinetobacter spp. experience a change in growth activity within wastewater treatment systems.     

Characterization of Vibrio fischeri rRNA operons and subcloning of a ribosomal DNA promoter.

Analysis of rRNA genes in Vibrio fischeri indicates the presence of eight rRNA gene sets in this organism. It was found that the genes for 5S rRNA, 16S rRNA, and 23S rRNA are organized in operons in the following order: 5' end 16S rRNA 23S RNA 5S rRNA 3' end. Although the operons are homologous, they are not identical with regard to cleavage sites for various restriction endonucleases. A DNA library was constructed, and three ribosomal DNA clones were obtained. One of these clones contained an entire rRNA operon and was used as a source for subcloning. The promoter region which leads to plasmid instability was successfully subcloned into pHG165. The terminator region was subcloned into pBR322.     

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.