Evolution according to large ribosomal subunit RNA

Evolutionary trees were constructed, by distance methods, from an alignment of 225 complete large subunit (LSU) rRNA sequences, representing Eucarya, Archaea, Bacteria, plastids, and mitochondria. A comparison was made with trees based on sets of small subunit (SSU) rRNA sequences. Trees constructed on the set of 172 species and organelles for which the sequences of both molecules are known had a very similar topology, at least with respect to the divergence order of large taxa such as the eukaryotic kingdoms and the bacterial divisions. However, since there are more than ten times as many SSU as LSU rRNA sequences, it is possible to select many SSU rRNA sequence sets of equivalent size but different species composition. The topologies of these trees showed considerable differences according to the particular species set selected.The effect of the dataset and of different distance correction methods on tree topology was tested for both LSU and SSU rRNA by repetitive random sampling of a single species from each large taxon. The impact of the species set on the topology of the resulting consensus trees is much lower using LSU than using SSU rRNA. This might imply that LSU rRNA is a better molecule for studying wide-range relationships. The mitochondria behave clearly as a monophyletic group, clustering with the Proteobacteria. Gram-positive bacteria appear as two distinct groups, which are found clustered together in very few cases. Archaea behave as if monophyletic in most cases, but with a low confidence.Abbreviations LSU rRNA large subunit ribosomal RNA - SSU rRNA small subunit ribosomal RNA - JC Jukes and Cantor - JN Jin and Nei Correspondence to: R. De Wachter     

Comparison of in vivo and in vitro ribosomal RNA synthesis in nucleolar mutants ofXenopus laevis

Summary Cells of embryos carrying a lethal nucleolar mutation have been maintained in vitro for extended periods of time. Normally these mutants live only 9 to 12 days after fertilization but their cells in culture will survive for more than 3 months. The extent of ribosomal RNA (rRNA) synthesis was determined in primary cultures prepared from normal embryos and nucleolar mutants having different numbers of ribosomal RNA genes. We found that the accumulation of radioactivity into rRNA for normal and mutant embryos was similar in vivo and in vitro. In primary cultures of normal embryos which have two nucleoli per cell and mutant embryos which have only one nucleolus per cell, the incorporation of radio-activity into rRNA was similar even though the normal cells have twice as many rRNA genes. Thus the mechanism which regulates dosage compensation of the rRNA genes operates both in vivo and in vitro. This work was supported by Grant GB38651 from the National Science Foundation.     

Nol9 is a novel polynucleotide 5'-kinase involved in ribosomal RNA processing

In a cell, an enormous amount of energy is channelled into the biogenesis of ribosomal RNAs (rRNAs). In a multistep process involving a large variety of ribosomal and non-ribosomal proteins, mature rRNAs are generated from a long polycistronic precursor. Here, we show that the non-ribosomal protein Nol9 is a polynucleotide 5'-kinase that sediments primarily with the pre-60S ribosomal particles in HeLa nuclear extracts. Depletion of Nol9 leads to a severe impairment of ribosome biogenesis. In particular, the polynucleotide kinase activity of Nol9 is required for efficient generation of the 5.8S and 28S rRNAs from the 32S precursor. Upon Nol9 knockdown, we also observe a specific maturation defect at the 5' end of the predominant 5.8S short-form rRNA (5.8S(S)), possibly due to the Nol9 requirement for 5'>3' exonucleolytic trimming. In contrast, the endonuclease-dependent generation of the 5'-extended, minor 5.8S long-form rRNA (5.8S(L)) is largely unaffected. This is the first report of a nucleolar polynucleotide kinase with a role in rRNA processing.     

Nucleolar localization of human methionyl-tRNA synthetase and its role in ribosomal RNA synthesis

Human aminoacyl-tRNA synthetases (ARSs) are normally located in cytoplasm and are involved in protein synthesis. In the present work, we found that human methionyl-tRNA synthetase (MRS) was translocated to nucleolus in proliferative cells, but disappeared in quiescent cells. The nucleolar localization of MRS was triggered by various growth factors such as insulin, PDGF, and EGF. The presence of MRS in nucleoli depended on the integrity of RNA and the activity of RNA polymerase I in the nucleolus. The ribosomal RNA synthesis was specifically decreased by the treatment of anti-MRS antibody as determined by nuclear run-on assay and immunostaining with anti-Br antibody after incorporating Br-UTP into nascent RNA. Thus, human MRS plays a role in the biogenesis of rRNA in nucleoli, while it is catalytically involved in protein synthesis in cytoplasm.     

Control of ribosomal RNA synthesis by hematopoietic transcription factors

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The interaction between streptomycin and ribosomal RNA

The present study shows that a mutation in the 530 loop of 16S rRNA impairs the binding of streptomycin to the bacterial ribosome, thereby restricting the misreading effect of the drug. Previous reports demonstrated that proteins S4, S5 and S12 as well as the 915 region of 16S rRNA are involved in the binding of streptomycin, and indicated that the drug not only interacts with the 30S subunit but also with the 50S subunit. The relationship between the target of streptomycin and its known interference with the proofreading control of translational accuracy is examined in light of these results.     

RNA Pol II preferentially regulates ribosomal protein expression by trapping disassociated subunits

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Localization of mitochondrial ribosomal RNA on the chromatoid bodies of marine planarian polyclad embryos

Electron-dense cytoplasmic structures, referred to as chromatoid bodies, are observed in the somatic stem cells, called neoblasts, and germline cells in adult planarians. Although it has been revealed that the chromatoid bodies morphologically resemble germline granules in Drosophila and Xenopus embryos, what essential role it plays in the planarian has remained unclear. In the present study, to examine whether chromatoid bodies in planarian embryos are responsible for germline formation, the presence and behavior of chromatoid bodies during embryogenesis were examined. Mitochondrial large ribosomal RNA and mitochondrial small ribosomal RNA were used as candidate markers for components of the chromatoid body. Starting from the fertilized egg, extramitochondrial signals of both RNA (mtrRNA) were observed. At the ultrastructural level, mtrRNA were localized on the surface of the chromatoid bodies. At subsequent stages, the signals of mtrRNA were observed in certain restricted blastomeres that contribute to the formation of larval structures. The signals gradually decreased from the gastrula stage. These results suggest that the chromatoid bodies associated with mtrRNA in embryogenesis are not germline granules. The chromatoid bodies of blastomeres may be concerned with the toti- or pluripotency and cell differentiation as proposed in adult planarian neoblasts.     

The adenosine wedge: A new structural motif in ribosomal RNA

Here, we present a new recurrent RNA arrangement, the so-called adenosine wedge (A-wedge), which is found in three places of the ribosomal RNA in both ribosomal subunits. The arrangement has a hierarchical structure, consisting of elements previously described as recurrent motifs, namely, the along-groove packing motif, the A-minor and the hook-turn. Within the A-wedge, these elements are involved in different types of cause–effect relationships, providing together for the particular tertiary structure of the motif.     

Post-maturation of the plastid ribosomal RNA in the plant kingdom

Summary The in vivo fragmentation of the plastid rRNA from plants situated at different places in the evolutionary scale, with the exception ofAlgae, was analysed by electrophoresis using fully denaturing conditions. This fragmentation corresponds to an in vivo post-maturation. It exists only in some bacteria and is not random. Five main groups of fragments with the following real molecular weights (Mr) are found in 23 S:ca 0.9 × 10⁶; 0.7 × 10⁶; 0.45 × 10⁶; 0.35 × 10⁶ and 0.15 × 10⁶. The existence of a large fragment (Mr = 0.9 × 10⁶) corresponds to a primitive type of fragmentation found in some archaic plants. Dicotyledons and several other groups have the same pattern of 23 S fragmentation, often comprising all the fragments mentioned above, whilstGraminaceae (Monocotyledons) constitute a special group with a very predominant 0.35 × 10⁶ dalton fragment and the absence of the 0.45 × 10⁶ dalton fragment. The plastid 16 S rRNA in all plants studied here has aMr of 0.54 × 10⁶ which is smaller than the 16 S ofEscbericbia coli taken as reference (0.56 × 10⁶ dalton).     

Questionable 16S ribosomal RNA gene annotations are frequent in completed microbial genomes

According to recent reports, many ribosomal RNA gene annotations are still questionable, and the use of inappropriate tools for annotation has been blamed. However, we believe that the abundant 16S rRNA partial sequence in the databases, mainly created by culture-independent PCR methods, is another main cause of the ambiguous annotations of 16S rRNA. To examine the current status of 16S rRNA gene annotations in complete microbial genomes, we used as a criterion the conserved anti-SD sequence, located at the 3′ end of the 16S rRNA gene, which is commonly overlooked by culture-independent PCR methods. In our large survey, 859 16S rRNA gene sequences from 252 different species of the microbial complete genomes were inspected. 67 species (234 genes) were detected with ambiguous annotations. The common anti-SD sequence and other conserved 16S rRNA sequence features could be detected in the downstream-intergenic regions for almost every questionable sequence, indicating that many of the 16S rRNA genes were annotated incorrectly. Furthermore, we found that more than 91.5% of the 93,716 sequences of the available 16S rRNA in the main databases are partial sequences. We also performed BLAST analysis for every questionable rRNA sequence, and most of the best hits in the analysis were rRNA partial sequences. This result indicates that partial sequences are prevalent in the databases, and that these sequences have significantly affected the accuracy of microbial genomic annotation. We suggest that the annotation of 16S rRNA genes in newly complete microbial genomes must be done in more detail, and that revision of questionable rRNA annotations should commence as soon as possible.     

Conservation of ribosomal RNA gene arrangement in the mitochondrial DNA of angiosperms

In six different angiosperms, mitochondrial genes for 18S and 5S rRNAs were found to be closely linked but distant from mitochondrial 26S rRNA genes.This is paper no. 5 in the series Organization and Expression of the Mitochondrial Genome of Plants     

Structural motifs in ribosomal RNAs: implications for RNA design and genomics

The various motifs of RNA molecules are closely related to their structural and functional properties. To better understand the nature and distributions of such structural motifs (i.e., paired and unpaired bases in stems, junctions, hairpin loops, bulges, and internal loops) and uncover characteristic features, we analyze the large 16S and 23S ribosomal RNAs of Escherichia coli. We find that the paired and unpaired bases in structural motifs have characteristic distribution shapes and ranges; for example, the frequency distribution of paired bases in stems declines linearly with the number of bases, whereas that for unpaired bases in junctions has a pronounced peak. Significantly, our survey reveals that the ratio of total (over the entire molecule) unpaired to paired bases (0.75) and the fraction of bases in stems (0.6), junctions (0.16), hairpin loops (0.12), and bulges/internal loops (0.12) are shared by 16S and 23S ribosomal RNAs, suggesting that natural RNAs may maintain certain proportions of bases in various motifs to ensure structural integrity. These findings may help in the design of novel RNAs and in the search (via constraints) for RNA-coding motifs in genomes, problems of intense current focus.     

Rapid and specific detection of pathogenicleptospira species by amplification of ribosomal sequences

We have developed an assay for the detection of pathogenicLeptospira that is based on the polymerase chain reaction. With the combination of agarose gel electrophoresis and blotting, pathogenicLeptospira can be discriminated specifically from nonpathogenicLeptospira and other bacterial species. This method, based on the amplification of 16S ribosomal RNA sequences, is able to detect 10 leptospiral cells/mL in cattle urine samples and 100 leptospiral cells/mL in pig urine samples. Using this assay leptospires were detected in urine samples from cattle that were experimentally infected withLeptospira interrogate serovarhardjo type hardjobovis.     

16S rRNA序列分析法在医学微生物鉴定中的应用

１６Ｓ ｒＲＮＡ序列分析作为微生物系统分类的主要依据已得到了广泛认同,随着微生物核糖体数据库的日益完善,该技术成为细菌分类和鉴定的一个有力工具。本文概述了 １６５ ｒＲＮＡ序列分析法的技术步骤以及该技术在医学微生物研究中的应用,总结了目前文献报导的各种致病微生物种属特异性 １６５ ｒＲＮＡ引物和探针序列,同时分析了该技术在应用中存在的一些问题。     

小鲵科线粒体16S rRNA基因序列分析及其系统发育

To study the phylogeny of Hynobiidae, we amplified DNA fragments of 470 bp 16S ribosomal RNA (16S rRNA) gene on mitochondrial DNA from Ranodon sibiricus and Ranodon tsinpaensis. PCR products were cloned into PMD18 T vector after purification. These sequences were determined and deposited in the GenBank (accession numbers: AY373459 for Ranodon sibiricus, AY372534 for Ranodon tsinpaensis). By comparing the nucleotide differences of 16S ribosomal RNA sequences among Liua shihi, Pseudohynobius flavomaculatus and Batrachuperus genus from GenBank database, we analyzed the divergences and base substitution among these sequences with the MEGA software. The molecular results support that B. tibetanus, B. pinchonii and B. karlschmidti are classified into three valid species. Liua shihi has closer phylogenetic relationships to Ranodon tsinpaensis than to other species. More our results reveal that Pseudohynobius flavomaculatus is not a synonym of Ranodon tsinpaensis. [Acta Zoologica Sinica 50 (3) : 464 - 469,2004].     

Systematics of the ascomycetous yeasts assessed from ribosomal RNA sequence divergence

Extent of divergence in partial nucleotide sequences from large and small subunit ribosomal RNAs was used to estimate genetic relationships among ascomycetous yeasts and yeastlike fungi. The comparisons showed four phylogenetically distinct groups comprised of the following taxa: Group 1. The budding yeastsSaccharomyces, Saccharomycopsis, Debaryomyces, Metschnikowia, Saturnospora, andLipomyces, and the yeastlike generaAscoidea, Cephaloascus, Dipodascus, Dipodascopsis, andGalactomyces; Group 2.Eremascus, Emericella andCeratocystis; Group 3.Taphrina andProtomyces; Group 4.Schizosaccharomyces. Because of the genetic relationships indicated by sequence analysis, Group 1 taxa are retained in the order Endomycetales, andSchizosaccharomyces is retained in the Schizosaccharomycetales Prillinger et al. ex Kurtzman.