Sequence heterogeneity in the duplicate large subunit ribosomal RNA genes of Tetrahymena pyriformis mitochondrial DNA

In the ciliated protozoan, Tetrahymena pyriformis, the mitochondrial large subunit ribosomal RNA (LSU rRNA) is discontinuous, consisting of two discrete RNA species: a 280-nucleotide LSU alpha (constituting the 5'-portion) and a 2315-nucleotide LSU beta (corresponding to the remaining 3'-portion of this rRNA). The T. pyriformis mitochondrial genome contains two copies of the LSU alpha.beta gene complex, and we have previously provided evidence that both copies are transcribed (Heinonen, T. Y. K., Schnare, M. N., Young, P. G., and Gray, M. W. (1987) J. Biol. Chem. 262, 2879-2887). We now report the complete sequences of the two copies of the LSU alpha.beta gene complex. These are not identical, but differ at 5 out of the 2595 positions by single nucleotide substitutions in one sequence relative to the other. In the secondary structure model we propose here, two of these differences are located in base-paired regions of the LSU rRNA; however, they do not interrupt the complementary interactions in these helices. The other three differences occur in single-stranded regions of the secondary structure. The base substitutions documented here are not localized to those regions of LSU rRNA that are the most highly conserved in global phylogenetic comparisons, and therefore it seems unlikely that they are of fundamental functional significance. Whether they might exert more subtle effects on ribosome function remains to be determined.     

The 3''-terminal region of bacterial 23S ribosomal RNA: structure and homology with the 3''-terminal region of eukaryotic 28S rRNA and with chloroplast 4.5s rRNA.

The sequence of the 110 nucleotide fragment located at the 3'-end of E.coli, P.vulgaris and A.punctata 23S rRNAs has been determined. The homology between the E.coli and P.vulgaris fragments is 90%, whereas that between the E.coli and A.punctate fragments is only 60%. The three rRNA fragments have sequences compatible with a secondary structure consisting of two hairpins. Using chemical and enzymatic methods recently developed for the study of the secondary structure of RNA, we demonstrated that one of these hairpins and part of the other are actually present in the three 3'-terminal fragments in solution. This supports the existence of these two hairpins in the intact molecule. Indeed, results obtained upon limited digestion of intact 23S RNA with T1 RNase were in good agreement with the existence of these two hairpins. We observed that the primary structures of the 3'-terminal regions of yeast 26S rRNA and X.laevis 28S rRNA are both compatible with a secondary structure similar to that found at the 3'-end of bacterial 23S rRNAs. Furthermore, both tobacco and wheat chloroplast 4.5S rRNAs can also be folded in a similar way as the 3'-terminal region of bacterial 23S rRNA, the 3'-end of chloroplast 4.5S rRNAs being complementary to the 5'-end of chloroplast 23S rRNA. This strongly reinforces the hypothesis that chloroplast 4.5S rRNA originates from the 3'-end of bacterial 23S rRNA and suggests that this rRNA may be base-paired with the 5'-end of chloroplast 23S rRNA. Invariant oligonucleotides are present at identical positions in the homologous secondary structures of E.coli 23S, yeast 26S, X.laevis 28S and wheat and tobacco 4.5S rRNAs. Surprisingly, the sequences of these oligonucleotides are not all conserved in the 3'-terminal regions of A.punctata or even P.vulgaris 23S rRNAs. Results obtained upon mild methylation of E.coli 50S subunits with dimethylsulfate strongly suggest that these invariant oligonucleotides are involved in RNA tertiary structure or in RNA-protein interactions.     

Unique sequence arrangement of ribosomal genes in the palindromic rDNA molecule of Physarum polycephalum.

R-loop and restriction mapping procedures reveal the organization of coding regions at each end of the giant rDNA palindrome of Physarum polycephalum. A 19S coding region of 2.10 +/- 0.21 kb is located at each end of a very long central spacer (35.64 +/- 2.08 kb). An internal spacer of 1.66 +/- 0.12 kb lies distal to the 19S gene. The 5.8S rRNA coding region is located in this spacer. The 26S gene lies distal to the internal spacer. The 26S gene is unusual among those of eukaryotes in that it consists of 3 coding regions (alpha, beta and gamma) interrupted by 2 intervening sequences. The 26S alpha (most central) coding segment of 2.41 +/- 0.33 kb is separated from the 26S beta segment by an intervening sequence of 0.68 +/- 0.13 kb. The 26S beta segment (0.70 +/- 0.11 kb) is separated from the most distal 26S gamma segment (0.59 +/- 0.14 kb) by an intervening sequence of 1.21 +/- 0.14 kb. The 2 intervening sequences are present in at least 88% of ribsomal genes from active plasmodia, indicating that genes containing these sequences are transcribed. The rDNA termini contain a heterogeneous region which varies in length by +/- 300 base pairs.     

Phylogenetic analysis and evolution of RNase P RNA in proteobacteria.

The secondary structures of the eubacterial RNase P RNAs are being elucidated by a phylogenetic comparative approach. Sequences of genes encoding RNase P RNA from each of the recognized subgroups (alpha, beta, gamma, and delta) of the proteobacteria have now been determined. These sequences allow the refinement, to nearly the base pair level, of the phylogenetic model for RNase P RNA secondary structure. Evolutionary change among the RNase P RNAs was found to occur primarily in four discrete structural domains that are peripheral to a highly conserved core structure. The new sequences were used to examine critically the proposed similarity (C. Guerrier-Takada, N. Lumelsky, and S. Altman, Science 246:1578-1584, 1989) between a portion of RNase P RNA and the "exit site" of the 23S rRNA of Escherichia coli. Phylogenetic comparisons indicate that these sequences are not homologous and that any similarity in the structures is, at best, tenuous.     

Study of a cloned ribosomal operon region coding for 5.8S rRNA in the loach Misgurnus fossilis L

A fragment of the loach (Misgurnus fossilis L.) ribosomal operon containing 5.8S rDNA and adjacent regions of the internal transcribed spacer (ITS-1, and ITS-2) was sequenced. The 5'-terminal sequencing in 5.8S rDNA was corrected by analysing the primary structure of the loach 5.8S rRNA. This RNA was shown to be presented by three types of molecules; one of these was shorter by 4 nucleotides at the 5'-end because of the processing site being shifted in the rRNA precursor. The two other types differed in the 5'-terminal nucleotide (UMP or AMP). In the cloned fragment under study, the sequence of 5.8S rDNA has TMP at the 5'-terminus. The known nucleotide sequences of 5.8S rRNAs were compared in eukaryotes; as a result, conservative regions were revealed at the sites of molecule modification. All the 5.8S rRNAs of the vertebrates studied were found to have coincidences in the localization of nucleotide substitutions and other mutations (inversions and deletions). The authors propose a model for the secondary structure of ITS-1 and ITS-2 in the region of 5.8S rRNA processing.     

Metagenomic retrieval of a ribosomal DNA repeat array from an uncultured marine alveolate

The aim of this study was to explore the use of large-scale sequencing to better describe the genome content of naturally occurring, uncultured protists. We constructed a metagenomic fosmid library from a picoplanktonic assemblage (0.2–3 μm size cells) collected at the Blanes Bay Microbial Observatory (Western Mediterranean). Seven clones contained a small-subunit ribosomal RNA gene (SSU rDNA) affiliating with prasinophytes and uncultured alveolates. One clone (FBB25; 35 kb in size) was completely sequenced and found to be a tandem repeat array (5.5 times) of the rDNA operon, including three rRNA genes (SSU, large-subunit and 5.8S rDNAs) and three spacer regions (internal transcribed spacers 1, 2 and intergenic spacer). The SSU rDNA of FBB25 affiliated with the marine alveolates group I, cluster 1, and was almost identical to sequences retrieved only in marine surveys from a wide geographic and ecological range. Phylogenetic trees using the different rRNA genes showed FBB25 as an independent branch among the main alveolate groups, but their closest affiliation varied between the SSU tree (dinoflagellates) and the large-subunit and 5.8S trees (perkinsids). The spacer regions of FBB25 were particularly short when compared with other eukaryotes, indicating a possible genome streamlining in this picoeukaryote. Finally, not a single polymorphism was found in the rDNA repeat array, suggesting that the high SSU rDNA variability typically found in molecular surveys derives from organismal and not intragenomic diversity. This first report on the rDNA genomic structure of an uncultured marine alveolate improves their phylogenetic position and helps interpreting data generated during picoeukaryotic molecular surveys.     

tRNA genes are found between 16S and 23S rRNA genes in Bacillus subtilis.

There are at least nine, and probably ten, ribosomal RNA gene sets in the genome of Bacillus subtilis. Each gene set contains sequences complementary to 16S, 23S and 5S rRNAs. We have determined the nucleotide sequences of two DNA fragments which each contain 165 base pairs of the 16S rRNA gene, 191 base pairs of the 23S rRNA gene, and the spacer region between them. The smaller space region is 164 base pairs in length and the larger one includes an additional 180 base pairs. The extra nucleotides could be transcribed in tRNAIIe and tRNA Ala sequences. Evidence is also presented for the existence of a second spacer region which also contains tRNAIIe and tRNA Ala sequences. No other tRNAs appear to be encoded in the spacer regions between the 16S and 23S rRNA genes. Whereas the nucleotide sequences corresponding to the 16S rRNA, 23S rRNA and the spacer tRNAs are very similar to those of E. coli, the sequences between these structural genes are very different.     

Composite structure of the chloroplast 23 S ribosomal RNA genes of Chlamydomonas reinhardii: Evolutionary and functional implications

The chloroplast ribosomal unit of Chlamydomonas reinhardii displays two features which are not shared by other chloroplast ribosomal units. These include the presence of an intron in the 23 S ribosomal RNA gene and of two small genes coding for 3 S and 7 S rRNA in the spacer between the 16 S and 23 S rRNA genes (Rochaix & Malnoë, 1978). Sequencing of the 7 S and 3 S rRNAs as well as their genes and neighbouring regions has shown that: (1) the 7 S and 3 S rRNA genes are 282 and 47 base-pairs long, respectively, and are separated by a 23 base-pair A + T-rich spacer. (2) A sequence microheterogeneity exists within the 3 S RNA genes. (3) The sequences of the 7 S and 3 S rRNAs are homologous to the 5′ termini of prokaryotic and other chloroplast 23 S rRNAs, indicating that the C. reinhardii counterparts of 23 S rRNA have a composite structure. (4) The sequences of the 7 S and 3 S rRNAs are related to that of cytoplasmic 5.8 S rRNA, suggesting that these RNAs may perform similar functions in the ribosome. (5) Partial nucleotide sequence complementarity is observed between the 5′ ends of the 7 S and 3 S RNAs on one hand and the 23 S rRNA sequences which flank the ribosomal intron on the other. These data are compatible with the idea that these small rRNAs may play a role in the processing of the 23 S rRNA precursor.     

Complete sequence and structure of ribosomal RNA gene of Heterosporis anguillarum

The ribosomal RNA (rRNA) gene region of the microsporidium Heterosporis anguillarum has been examined. Complete DNA sequence data (4060 bp, GenBank Accession No. AF402839) of the rRNA gene of H. anguillarum are presented for the small subunit gene (SSU rRNA: 1359 bp), the internal transcribed spacer (ITS: 37 bp), and the large subunit gene (LSU rRNA: 2664 bp). The secondary structures of the H. anguillarum SSU and LSU rRNA genes are constructed and described. This is the first complete sequence of an rRNA gene published for a fish-infecting microsporidian species. In the phylogenetic analysis, the sequences, including partial SSU rRNA, ITS, and partial LSU rRNA sequences of the fish-infecting microsporidia, were aligned and analysed. The taxonomic position of H. anguillarum as suggested by Lom et al. (2000; Dis Aquat Org 43:225-231) is confirmed in this paper.     

Organization of ribosomal RNA gene repeats of the mouse.

The organization of the ribosomal RNA (rRNA) genes of the mouse was determined by Southern blot hybridization using cloned rDNA fragments as probes, which could encompass the entire spacer region between two rRNA gene regions. The rRNA genes are organized into tandem repeats of nearly uniform length of about 44 kb. The heterogeneity detected in the nontranscribed spacer appears to be caused by its sequence rather than its length difference. At least three kinds of repetitive sequences are present in the non-transcribed spacer region; two of them are located 13 kb upstream from the 5'-end of 18S RNA gene and the other located 1 to 4 kb downstream from the 3'-end of 28S RNA gene.     

Ribosomal RNA genes of Trypanosoma brucei: mapping the regions specifying the six small ribosomal RNAs

There are six small ribosomal RNAs in trypanosome ribosomes. sRNA3 and sRNA5 of Trypanosoma brucei brucei have been partially sequenced. Sequence homologies indicate that sRNA3 is 5.8S RNA and sRNA5 is 5S RNA of T. b. brucei. The regions specifying these two, and the remaining four small RNAs, have been identified within clones of rRNA genes and in the genome. Five of the small RNAs, 1, 2, 3, 4 and 6, hybridise exclusively within the major rRNA gene repeat. A map of the regions specifying these small RNAs is presented. sRNA3 (5.8S RNA) hybridises to a region corresponding to the transcribed spacer of other eukaryotes. sRNA1 hybridises to a region between sequences specifying the two large subunit RNA molecules of 2.3 kb and 1.8 kb. Sequences specifying sRNAs 2 and 4 are present near the sequence specifying sRNA1, while sRNA6 appears to be specified 3' to the sequence specifying the 1.8-kb RNA sequence. In addition regions of secondary hybridisation for small RNAs 2, 3, 4 and 6 have also been identified. Though sRNA5 (5S RNA) hybridises within the major rRNA repeat, a separate 5S RNA gene repeat with unit size of 760 bp is also present. It is 10 to 20 times more abundant than the major rRNA gene repeat.