Conserved restriction sites within the ribosomal RNA genes of vertebrates

We have mapped the cleavage sites of four restriction enzymes which recognize six-base sequences within the nuclear ribosomal (rRNA) genes of twelve vertebrates, including several placental mammals (Homo sapiens, man; Bos taurus, cow; Equus caballus, horse; Sus scofra, pig; Ovis aries, sheep; Rattus rattus, rat), a marsupial (Didelphis marsupialis, opossum), a bird (Gallus domesticus, chicken), an amphibian (Xenopus laevis), a reptile (Alligator mississipiensis), a bony fish (Cynoscion nebulosus, sea trout), and a cartilagenous fish (Carcharhinus species, requiem shark). These animals represent a span of approx. 400 million years of evolutionary divergence. Our data identify restriction sites in the rRNA genes which are highly conserved among higher vertebrates and therefore are likely to be in functionally important regions. Additionally, the restriction enzyme sites identified will be useful in cloning and sequencing the rRNA genes in any vertebrate. Finally, the consistent size and conserved sequence homology suggests that these rRNA gene segments will be useful as internal controls in hybridization experiments involving other genomic regions in vertebrates.     

RNA:RNA interactions in the large subunit ribosomal RNA of Euglena gracilis

In Euglena gracilis, the cytoplasmic large subunit (LSU) rRNA is composed of 14 discrete small RNA species that must somehow interact in the functional ribosome. We have isolated native complexes of Euglena rRNA and show here that the largest of these complexes contains eight of the 14 LSU rRNA species. Several of these small rRNA species are able to associate in vitro to reform an isolated domain of LSU rRNA structure.     

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     

The European large subunit ribosomal RNA database

The European Large Subunit (LSU) Ribosomal RNA (rRNA) database is accessible via the rRNA WWW Server at URL http://rrna.uia.ac.be/lsu/. It is a curated database that compiles complete or nearly complete LSU rRNA sequences in aligned form, and also incorporates secondary structure information for each sequence. Taxonomic information, literature references and other information about the sequences are also available, and can be searched via the WWW interface.     

The organization of ribosomal genes in vertebrates

The organization of the repeat unit of the ribosomal genes has been determined in 15 different species of vertebrates. The EcoRI and BamHI restriction maps of the rDNA from single individuals of different species of fishes, amphibians, and reptiles have been analysed. Two rDNA clones from Xenopus laevis (representing one complete repeat unit) were used as probes in Southern blots to detect restriction fragments containing ribosomal genes. The results obtained indicate that the transcribed regions are highly conserved in length and sequence inside the same zoological class. These regions are less conserved when species from different classes are compared but a general trend has been observed. In contrast, the length and sequence of the spacer regions are very variable, even within the same zoological class. Different types of heterogeneity have been observed; examples range from a single type of ribosomal repeat unit within a species to the absence of any detectable regular tandem array of units.     

Database on the structure of large ribosomal subunit RNA.

Our database on large ribosomal subunit RNA contained 334 sequences in July, 1995. All sequences in the database are aligned, taking into account secondary structure. The aligned sequences are provided, together with incorporated secondary structure information, in several computer-readable formats. These data can easily be obtained through the World Wide Web. The files in the database are also available via anonymous ftp.     

Database on the structure of large ribosomal subunit RNA.

A database on large ribosomal subunit RNA is made available. It contains 258 sequences. It provides sequence, alignment and secondary structure information in computer-readable formats. Files can be obtained using ftp.     

Database on the structure of large ribosomal subunit RNA.

The latest release of the large ribosomal subunit RNA database contains 429 sequences. All these sequences are aligned, and incorporate secondary structure information. The rRNA WWW Server at URL http://rrna.uia.ac.be/ provides researchers with an easily accessible resource to obtain the data in this database in a number of computer-readable formats. A new query interface has been added to the server. If necessary, the data can also be obtained by anonymous ftp from the same site.     

Database on the structure of large ribosomal subunit RNA.

The rRNA WWW Server at URL http://rrna.uia.ac.be/ now provides a database of 496 large subunit ribosomal RNA sequences. All these sequences are aligned, incorporate secondary structure information, and can be obtained in a number of formats. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available and searchable. If necessary, the data on the server can also be obtained by anonymous ftp.     

Database on the structure of large subunit ribosomal RNA.

The Antwerp database on large subunit ribosomal RNA now contains 607 complete or nearly complete aligned sequences. The alignment incorporates secondary structure information for each sequence. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available. Information from the database can be downloaded or searched on the rRNA WWW Server at URL http://rrna.uia.ac.be/     

RNA chaperone activity of large ribosomal subunit proteins from Escherichia coli

The ribosome is a highly dynamic ribonucleoprotein machine. During assembly and during translation the ribosomal RNAs must routinely be prevented from falling into kinetic folding traps. Stable occupation of these trapped states may be prevented by proteins with RNA chaperone activity. Here, ribosomal proteins from the large (50S) ribosome subunit of Escherichia coli were tested for RNA chaperone activity in an in vitro trans splicing assay. Nearly a third of the 34 large ribosomal subunit proteins displayed RNA chaperone activity. We discuss a possible role of this function during ribosome assembly and during translation.     

Additional Watson-Crick interactions suggest a structural core in large subunit ribosomal RNA

Two new Watson-Crick type interactions in 23S-like ribosomal RNA have been identified by comparative sequence analysis. These interactions, A1269/U2011 and C1270/G2010 (E. coli numbering) along with the previously proposed A1262/U2017 suggest an anti-parallel helical arrangement characteristic of secondary structure in the 1265/2015 region of 23S rRNA. Nested within these three interactions are three universal juxtapositions which in principle allow the formation of an irregular helix containing two additional A-G interactions and a universal A-U pair. Whether or not this extended helix is biologically significant is uncertain. The proponderance of interactions in the 1265/2015 region and its location relative to the known structural domains of 23S rRNA suggest that this region may be part of a central structural core similar to that already known in 16S rRNA.     

Chemical probing of conformation in large RNA molecules. Analysis of 16 S ribosomal RNA using diethylpyrocarbonate

Peattie & Gilbert (1980) have described an accurate and rapid gel method for assessing conformation of individual nucleotides in RNA, based on chemical modification of bases and aniline-induced strand scission. In order to extend this approach to analysis of large RNA molecules, we introduce the use of hybridization of modified RNA with DNA restriction fragments to generate RNA fragments of defined length. In principle, this permits chemical probing of conformation at any position of any RNA molecule for which a cloned DNA coding sequence is available. To illustrate the utility of this method, we use diethylpyrocarbonate to probe the reactivities of adenine residues in Escherichia coli 16 S rRNA under "native" (80 mM-potassium cacodylate (pH 7.0), 20 mM-MgCl2, 300 mM-KCl) and "quasi-secondary" (80 mM-potassium cacodylate (pH 7.0), 1 mM-EDTA) conditions. This study shows that: (1) there is generally good agreement between diethylpyrocarbonate reactivities of adenine residues in naked 16 S rRNA and a secondary structure model based on comparative sequence analysis; of 309 adenine residues probed under native conditions, only four strongly reactive residues are found in helices in the model. (2) Candidates for possible tertiary interactions are identified as adenine residues that are unpaired in the model and unreactive toward diethylpyrocarbonate under native conditions but reactive under quasi-secondary conditions. (3) An unexpectedly stable structure has been identified in the region between positions 109 and 279, where many adenine residues remain unreactive even at 90 degrees C in 80 mM-potassium cacodylate, 1 mM-EDTA. This may correspond to a structural "core" that is important for early events in ribosome assembly.     

The number of large ribosomal RNA genes in Leptospira interrogans and Leptospira biflexa

We determined the number of large ribosomal RNA genes in five strains of Leptospira by hybridization of 15 restriction endonuclease digests of genomic DNA to the [32P]-labeled fragment of 23s rRNA gene. Almost all the restriction gels gave two radioactive bands. The conclusion from these results is that there are at least two rRNA genes in these leptospiral strains. Furthermore, the hybridization patterns of L. icterohaemorrhagiae strains Ictero No. I and RGA are almost identical. The number of rRNA genes and taxonomic relationships of these leptospires were discussed.     

Dinoflagellates in evolution. A molecular phylogenetic analysis of large subunit ribosomal RNA

Summary The sequence of the large subunit ribosomal RNA (LsuRNA) gene of the dinoflagellateProrocentrum micans has been determined. The inferred rRNA sequence [3408 nucleotides (nt)] is presented in its most probable secondary structure based on compensatory mutations, energy, and conservation criteria. No introns have been found but a hidden break is present in the second variable domain, 690 nt from the 5 end, as judged by agarose gel electrophoresis and primer extension experiments.Prorocentrum micans LsuRNA length and G+C content are close to those of ciliates and yeast. The conserved portions of the molecule (1900 nt) have been aligned with corresponding sequences from various eukaryotes, including five protista, one metaphyta, and three metazoa. An extensive phylogenetic study was performed, comparing two phenetic methods (neighbor joining on difference matrix, and Fitch and Margoliash on Knuc values matrix) and one cladistic (parsimony). The three methods led to similar tree topologies, except for the emergence of yeast that groups with ciliates and dinoflagellates when phenetic methods are used, but emerges later in the most parsimonious tree. This discrepancy was checked by statistical analyses on reduced trees (limited to four species) inferred using parsimony and evolutionary parsimony methods. The data support the phenetic tree topologies and a close relationship between dinoflagellates, ciliates, and yeast.