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PubMed2000

A nested array of rRNA targeted probes for the detection and identification of enterococci by reverse hybridization.

Complete 23S and almost complete 16S rRNA gene sequences were determined for the type strains of the validly described Enterococcus species, Melissococcus pluton and Tetragenococcus halophilus. A comprehensive set of rRNA targeted specific oligonucleotide hybridization probes was designed according to the multiple probe concept. In silico probe design and evaluation was performed using the respective tools of the ARB program package in combination with the ARB databases comprising the currently available 16S as well as 23S rRNA primary structures. The probes were optimized with respect to their application for reverse hybridization in microplate format. The target comprising 16S and 23S rDNA was amplified and labeled by PCR (polymerase chain reaction) using general primers targeting a wide spectrum of bacteria. Alternatively, amplification of two adjacent rDNA fragments of enterococci was performed by using specific primers. In vitro evaluation of the probe set was done including all Enterococcus type strains, and a selection of other representatives of the gram-positive bacteria with a low genomic DNA G+C content. The optimized probe set was used to analyze enriched drinking water samples as well as original samples from waste water treatment plants.

Base Sequence↗
PubMed1993

Small subunit (18S) ribosomal RNA gene divergence in the genus Schistosoma.

An entire 18S rRNA gene sequence from Schistosoma spindale (1990 bases) and partial 18S rRNA gene sequences from S. haematobium (1950 bases) and S. japonicum (1648 bases) have been determined. Together with the previously published sequence of the S. mansoni 18S rRNA gene, these data encompass the 4 recognized Schistosoma species groups. Although Schistosoma 18S rRNA genes are highly conserved, the sequences permit a preliminary molecular phylogeny to be established for the genus. This identifies S. haematobium and S. spindale as sister taxa in a clade with S. mansoni. S. japonicum does not appear to be closely related to this clade. Much of the observed variation occurs within a 'hypervariable' stretch of the gene corresponding to part of the V4 region of 18S rRNA. Despite this variation, the 3 new sequences fit models of 18S rRNA secondary structure predicted from the S. mansoni sequence.

Animals↗
PubMed1994

Mapping RNA regions in eukaryotic ribosomes that are accessible to methidiumpropyl-EDTA.Fe(II) and EDTA.Fe(II).

Methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and EDTA.Fe(II) were used to investigate the structure of Drosophila melanogaster ribosomes. Cleavage reactions were performed on intact ribosomes in cell lysates in vitro and analyzed by primer extension with reverse transcriptase using oligodeoxynucleotide primers. Regions of 18S and 28S ribosomal RNAs (rRNAs) which are accessible to MPE.Fe(II) and EDTA.Fe(II) are located almost exclusively within expansion segments. The accessibility of these regions to cleavage indicates that they are likely exposed on the surface of eukaryotic ribosomes. These results provide information about the overall tertiary structure of rRNA in ribosomes.

Animals↗
PubMed2002

Review of Papillostrongylus Johnston & Mawson, 1939 (Nematoda: Strongyloidea) from wallabies and kangaroos (Marsupialia: Macropodidae) using morphological and molecular techniques, with the description of P. barbatus n. sp.

The strongyloid nematode genus Papillostrongylus Johnston & Mawson, 1939, from kangaroos and wallabies, is reviewed using morphological and molecular methods. P. labiatus Johnston & Mawson, 1939 is re-described from material from the type-host, the black-striped wallaby Macropus dorsalis, from eastern Queensland, Australia, in which it is a relatively common parasite. Additional records from M. parryi and Thylogale thetis are confirmed and considered to represent examples of host-switching. A geographically disjunct population of the nematode species occurs in M. bernardus and Petrogale brachyotis in Arnhem Land, Northern Territory, but assessment of its status requires additional material. Nematodes from M. rufus, M. giganteus, M. fuliginosus and M. robustus from inland regions of Australia, formerly attributed to P. labiatus, are here assigned to a new species, P. barbatus, distinguished by the presence of an external leaf-crown, larger size, by greater spicule length in the male and by a sinuous vagina in the female. Additional hosts of P. barbatus n. sp. are Petrogale assimilis and Pet. lateralis purpureicollis. Sequence analyses of the second internal transcribed spacer of ribosomal DNA (ITS-2) also showed that P. barbatus n. sp. differed at 40 (16.7%) of the 240 alignment positions when compared with P. labiatus. Most of these interspecific sequence differences occurred in loops or bulges of the predicted precursor rRNA secondary structure, or represented partial or total compenstory base pair changes in stems.

Animals↗
PubMed2001

Psyllid endosymbionts exhibit patterns of co-speciation with hosts and destabilizing substitutions in ribosomal RNA.

Eubacterial 16S rDNAs were sequenced from endosymbionts of seven psyllids (Psylloidea) and one whitefly (Aleyrodoidea), to investigate the evolution of endosymbionts and their hosts. Primary endosymbionts from all psyllids formed a highly supported clade, tentatively placed as the sister to whitefly primary endosymbionts, and showing several points of congruence with the host morphological phylogeny. Almost all host taxa yielded an additional eubacterial sequence, related either to known psyllid secondary endosymbionts or to other insect endosymbionts or parasites. The relationships of some secondary endosymbionts also suggested cospeciation with psyllid hosts, or ancient horizontal transfers. All primary endosymbionts, and some secondary endosymbionts, exhibited molecular genetic effects of a long-term, intracellular existence in their biased nucleotide content and decreased stability of rRNA secondary structure.

Animals↗
PubMed2000

The complete sequence of mtDNA genes in idiopathic dilated cardiomyopathy shows novel missense and tRNA mutations.

BACKGROUND: Previous studies have shown that mitochondrial DNA (mtDNA) mutations are often present in patients with myocardial dysfunction. We sought to assess the prevalence and significance of heart mtDNA sequence changes in patients with idiopathic dilated cardiomyopathy (DCM). METHODS AND RESULTS: DNA sequence of all the transfer ribonucleic acid (tRNA), ribosomal RNA (rRNA), and structural genes in cardiac mtDNA of 28 patients with DCM was determined and compared with a control group that had no evidence of heart disease. An increased number of point mutations were found in DCM cardiac mtDNA when compared with controls. Both novel and previously reported mutations were found in mitochondrial tRNA and structural genes. One of these mutations was heteroplasmic and resulted in changing a highly conserved nucleotide in tRNAArg. Novel, heteroplasmic mtDNA mutations (n = 4) specifying changes in moderate to highly conserved amino acid residues were found in COII, COIII, ND5, and cytb. These novel mtDNA mutations were found only in patients with severe reduction in mitochondrial enzyme activities. CONCLUSIONS: Our results indicate that a high incidence of mtDNA nucleotide sequence changes in both tRNA and structural genes are present in DCM. Five heteroplasmic mutations were detected that both changed evolutionarily conserved residues (which may impair the function of proteins or tRNAs) and were associated with specific enzymatic defects. These mutations could play an important role in the pathogenesis of cardiomyopathy.

Adolescent↗
PubMed1990

Architecture of ribosomal RNA: constraints on the sequence of "tetra-loops".

The four-base loops that cap many double-helical structures in rRNA (the so-called "tetra-loops") exhibit highly invariant to highly variable sequences depending upon their location in the molecule. However, in the vast majority of these cases the sequence of a tetra-loop is independent of its location and conforms to one of three general motifs, GNRA, UNCG, and (more rarely) CUUG. For the most frequently varying of the 16S rRNA tetra-loops, that at position 83 (Escherichia coli numbering), the three sequences CUUG, UUCG, and GCAA account for almost all examples encountered, and each of them has independently arisen at least a dozen times. The closing base pair of tetra-loop hairpins reflects the loop sequence, tending to be C.G for UUCG loops and G.C for CUUG loops.

Bacteria↗
PubMed1991

Ribosomal DNA: molecular evolution and phylogenetic inference.

Ribosomal DNA (rDNA) sequences have been aligned and compared in a number of living organisms, and this approach has provided a wealth of information about phylogenetic relationships. Studies of rDNA sequences have been used to infer phylogenetic history across a very broad spectrum, from studies among the basal lineages of life to relationships among closely related species and populations. The reasons for the systematic versatility of rDNA include the numerous rates of evolution among different regions of rDNA (both among and within genes), the presence of many copies of most rDNA sequences per genome, and the pattern of concerted evolution that occurs among repeated copies. These features facilitate the analysis of rDNA by direct RNA sequencing, DNA sequencing (either by cloning or amplification), and restriction enzyme methodologies. Constraints imposed by secondary structure of rRNA and concerted evolution need to be considered in phylogenetic analyses, but these constraints do not appear to impede seriously the usefulness of rDNA. An analysis of aligned sequences of the four nuclear and two mitochondrial rRNA genes identified regions of these genes that are likely to be useful to address phylogenetic problems over a wide range of levels of divergence. In general, the small subunit nuclear sequences appear to be best for elucidating Precambrian divergences, the large subunit nuclear sequences for Paleozoic and Mesozoic divergences, and the organellar sequences of both subunits for Cenozoic divergences. Primer sequences were designed for use in amplifying the entire nuclear rDNA array in 15 sections by use of the polymerase chain reaction; these "universal" primers complement previously described primers for the mitochondrial rRNA genes. Pairs of primers can be selected in conjunction with the analysis of divergence of the rRNA genes to address systematic problems throughout the hierarchy of life.

Animals↗
PubMed2004

The ambush hypothesis: hidden stop codons prevent off-frame gene reading.

Coding sequences lack stop codons, but many stops appear off-frame. Off-frame stops (stops in -1 and +1 shifted reading frames, termed hidden stops) terminate frame-shifted translation, potentially decreasing energy, and resource waste on nonfunctional proteins. Benefits may include reduced waste elimination costs and avoidance of potentially cytotoxic frame-shifted products. Our "ambush" hypothesis suggests that hidden stops are sometimes selected for. Codons of many amino acids can contribute to hidden stops, depending on the synonymous position state and adjacent codons. In vertebrate mitochondria, 31.75% of all amino acid combinations can form hidden stops. Codons with more potential to form hidden stops have greater usage frequency and bias in their favor among synonymous codons. Among primates, predicted mitochondrial rRNA secondary structure stability correlates negatively with the number of hidden stops in the mitochondrial genome. The taxonomic distribution of genetic codes suggests that +1 frameshifts might be more frequent than -1 frameshifts. This is confirmed by analyses of primate mitochondrial genomes: species with unstable rRNAs have more +1 stops, but the correlation is weak for -1 stops. High hidden stop density seems to be an adaptation in species with slippage prone ribosomes (unstable rRNAs). Hidden stops may thus compensate for reduced efficiency of some parts of the biosynthetic machinery. Some experimental data confirm our hypothesis: gene expression increases with the experimentally manipulated number of stops in the promoter region of a gene, suggesting biotechnological applications.

Animals↗
PubMed2004

Comparison of tetracycline and tigecycline binding to ribosomes mapped by dimethylsulphate and drug-directed Fe2+ cleavage of 16S rRNA.

OBJECTIVES: The new antibiotic tigecycline (9-t-butylglycylamido-minocycline; GAR-936) overcomes most of the known tetracycline resistance mechanisms. Here we analyse its mode of antibiotic action by probing 70S ribosomes of Escherichia coli with dimethylsulphate (DMS) and Fe(2+)-mediated cleavage to identify binding sites of tetracycline and tigecycline. METHODS: Fe(2+)-mediated cleavage makes use of the ability of Fe2+ to replace the Mg2+ ion complexed with tetracyclines. After addition of H2O2, Fe2+ generates short-lived, highly reactive hydroxyl radicals that can cleave RNA close to the tetracycline binding sites. RESULTS: We identified three prominent Fe(2+)-mediated cleavage sites in helices 29 and 34, and in the internal loop of helix 31 of 16S rRNA in the presence of tetracycline or tigecycline. Qualitatively, these sites are modified identically by both antibiotics, but quantitative differences observed in the cleavage intensities indicate that the drugs bind in slightly different orientations. These results are supported by DMS modification, mutational analysis of 16S rRNA and structural modelling of tigecycline at a tetracycline-binding site in the 30S ribosomal subunit. CONCLUSIONS: Both derivatives bind to identical or overlapping sites and probably share the same mode of antibiotic action. The fact that tigecycline overcomes most of the known tetracycline resistance mechanisms is interpreted as a result of steric hindrance due to the large substituent at position 9.

Base Sequence↗
PubMed2003

Pseudogenes contribute to the extreme diversity of nuclear ribosomal DNA in the hard coral Acropora.

One characteristic of Indo-Pacific Acropora spp. is extremely high diversity in rDNA sequences at both the species and individual levels. In order to test the hypothesis that pseudogenes may contribute to this diversity, three kinds of analyses were conducted. First, for A. millepora (the species containing the most diverse suite of rDNA types), RT-PCR was used to determine which 5.8S rDNA types are expressed. Second, as previous studies have indicated that interspecific hybridization has occurred in the genus Acropora and silencing of rDNA loci via nucleolar dominance has been shown in some cases to involve methylation, patterns of variation were examined at methylation-susceptible sites. Third, patterns of substitution at conserved sites (including those that are likely to contribute to secondary structure in rRNA) in the 5.8S rDNA were examined. These analyses consistently indicated that one rDNA sequence type present in a broad range of Indo-Pacific Acropora species is likely to consist predominantly of pseudogenes. Patterns of variation also suggest that species may differ with respect to which rDNA sequence types have been silenced and which are active. These pseudogenes are likely to have arisen as a consequence of the introduction of highly divergent rDNA types into single genomes by interspecific hybridization events, and we attribute the extreme rDNA diversity characteristic of many Acropora species to both the independent evolution of these silenced rDNA types and to the suppressive effects of high sequence diversity on homogenization processes acting on functional loci.

Animals↗
PubMed1991

Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts.

We describe four monoclonal antibodies (MAB) which specifically recognize double-stranded RNA (dsRNA) together with their use in new methods for detecting and characterizing dsRNA in unfractionated nucleic acid extracts. The specificity of the antibodies was analyzed using a panel of 27 different synthetic and naturally occurring nucleic acids. All four antibodies reacted in a highly specific manner with long dsRNA helices, irrespective of their sequence; no binding to single-stranded RNA homopolymers or to DNA or RNA-DNA hybrids was observed. The apparent affinity of the antibodies to short (less than or equal to 11 bp) RNA helices was very low in all test systems used: only background levels of binding were obtained on single-stranded RNA species which contain double-helical secondary structures (e.g. rRNA, tRNA, viroid RNA). A sandwich ELISA and a dsRNA-immunoblotting procedure have been established which allow detection and characterization of dsRNA by MAB even in the presence of a large excess of other nucleic acids. In combination with temperature-gradient gelelectrophoresis (TGGE) not only the molecular weights but also the highly characteristic Tm-values of conformational transitions of individual dsRNA species could be determined by immunoblotting. An example of the general use of these methods for the detection of plant virus infections is demonstrated with groundnut rosette virus (GRV) dsRNAs. We were able to estimate the dsRNA content of infected leaves, identify the dsRNA species present in crude extracts and to determine the Tm- values of GRV dsRNA-3.

Animals↗
PubMed1992

Exon sequences distant from the splice junction are required for efficient self-splicing of the Tetrahymena IVS.

The presence of a natural rRNA secondary structure element immediately preceding the 5' splice site of the Tetrahymena IVS can inhibit self-splicing by competing with base pairing between the 5' exon and the guide sequence of the IVS (P1). Formation of this alternative hairpin is preferred in short precursor RNAs, and results in loss of G-addition to the 5' splice site. Pre-rRNAs which contain longer exons of ribosomal sequence, however, splice rapidly. As many as 146 nucleotides of the 5' exon and 86 nucleotides of the 3' exon are required for efficient self-splicing of Tetrahymena precursors. The presence of nucleotides distant from the 5' splice site apparently alters the equilibrium between the alternative hairpins, and promotes formation of active precursors. This effect is dependent on the specific sequences of the ribosomal pre-RNA, since point mutations within this region reduce the rate of splicing as much as 50-fold. This system provides an opportunity to study the way in which long-range interactions can influence splice site selection in a highly structured RNA.

Animals↗
PubMed1993

The ribosomal database project.

The Ribosomal Database Project (RDP) is a curated database that offers ribosome data along with related programs and services. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software packages for handling, analyzing and displaying alignments and trees. The data are available via ftp and electronic mail. Certain analytic services are also provided by the electronic mail server.

Databases, Factual↗
PubMed1994

The Ribosomal Database Project.

The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services, and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (rdp.life.uiuc.edu), electronic mail (server/rdp.life.uiuc.edu) and gopher (rdpgopher.life.uiuc.edu). The electronic mail server also provides ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for chimeric nature of newly sequenced rRNAs, and automated alignment.

Animals↗
PubMed1996

Effects of antisense DNA against the alpha-sarcin stem-loop structure of the ribosomal 23S rRNA.

Antisense DNAs complementary against various sequences of the alpha-sarcin domain (C2646-G2674) of 23S rRNA from Escherichia coli were hybridized to naked 23S rRNA as well as to 70S ribosomes. Saturation levels of up to 0.4 per 70S ribosome were found, the identical fraction was susceptible to the attack of the RNase alpha-sarcin. The hybridization was specific as demonstrated with RNase H digestion, sequencing the resulting fragments and blockage of the action of alpha-sarcin. The RNase alpha-sarcin seems to approach its cleavage site from the 3' half of the loop of the alpha-sarcin domain. Hybridization is efficiently achieved at 37 degrees C and can extend at least into the 3' strand of the stem of the alpha-sarcin domain. However, the inhibition of alpha-sarcin activity is observed at 30 degrees C but not at 37 degrees C. For a significant inhibition of poly(Phe) synthesis the temperature had to be lowered to 25 degrees C. The results imply that the alpha-sarcin domain changes its conformation during protein synthesis and that the conformational changes may include a melting of the stem of the alpha-sarcin domain.

Base Sequence↗
PubMed1997

Chromatin structure and methylation of rat rRNA genes studied by formaldehyde fixation and psoralen cross-linking.

By using formaldehyde cross-linking of histones to DNA and gel retardation assays we show that formaldehyde fixation, similar to previously established psoralen photocross-linking, discriminates between nucleosome- packed (inactive) and nucleosome-free (active) fractions of ribosomal RNA genes. By both cross-linking techniques we were able to purify fragments from agarose gels, corresponding to coding, enhancer and promoter sequences of rRNA genes, which were further investigated with respect to DNA methylation. This approach allows us to analyse independently and in detail methylation patterns of active and inactive rRNA gene copies by the combination of Hpa II and Msp I restriction enzymes. We found CpG methylation mainly present in enhancer and promoter regions of inactive rRNA gene copies. The methylation of one single Hpa II site, located in the promoter region, showed particularly strong correlation with the transcriptional activity.

Animals↗