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PubMed2002

Sequence diversity of Neisseria meningitidis 16S rRNA genes and use of 16S rRNA gene sequencing as a molecular subtyping tool.

We investigated the diversity of the primary sequences of 16S rRNA genes among Neisseria meningitidis strains (Men) and evaluated the use of this approach as a molecular subtyping tool. We aligned and compared a 1,417-bp fragment of the 16S rRNA gene from 264 Men strains of serogroups A, B, C, and Y (MenA, MenB, MenC, and MenY, respectively) isolated throughout the world over a 30-year period. Thirty-one positions of difference were found among 49 16S types: differences between types ranged from 1 to 14 positions (0.07 to 0.95%). 16S types and serogroups were highly associated; only 3 out 49 16S types were shared by two or more serogroups. We have identified 16S types that are exclusively associated with strains of certain hypervirulent clones: 16S type 5 with MenA subgroup III, 16S type 4 with the MenB electrophoretic type 5 (ET-5) complex, and 16S types 12 and 13 with MenC of the ET-37 complex. For MenC strains, 16S sequencing provided the highest sensitivity and specificity and the best overall association with the outbreak-related versus sporadic isolates when compared with pulsed-field gel electrophoresis, multilocus enzyme electrophoresis, and multilocus sequence typing. We demonstrated for the first time an unexpected diversity among 16S rRNA genes of Men strains, identified 16S types associated with well-defined hypervirulent clones, and showed the potential of this approach to rapidly identify virulent strains associated with outbreaks and/or an increased incidence of sporadic disease.

Bacterial Typing Techniques↗
PubMed1976

Transfer RNA genes between 16S and 23S rRNA genes in rRNA transcription units of E. coli.

We have identified genes for tRNAGLU/2 on the transducing phages o80d3ilvsu7+ (see Ohtsubo et al., 1974) and lambdarifd18 (Kirschbaum and Konrad, 1973), and a gene for tRNAlle/1 on the transducing phage o80rifr (Konrad, Kirschbaum, and Austin, 1973). All these phages have previously been shown to carry genes for rRNA (Ohtsubo et al., 1974; Lindahl et al., 1975; Jaskunas et al., 1975a). We have analyzed the position of these tRNA genes by hybridizing purified RNAs to restriction fragments of the phage DNA. The tRNA genes are located inside the rRNA transcription unit in the spacer region between the 16S and 23S rRNA genes.

Chromosome Mapping↗
PubMed1978

Processing of rRNA by RNAase P: spacer tRNAs are linked to 16S rRNA in an RNAase P RNAase III mutant strain of E. coli.

To determine which enzymes are responsible for the processing cleavages of ribosomal RNA transcripts in Escherichia coli, we constructed a mutant strain lacking RNAase III and containing a thermolabile RNAase P. At the nonpermissive temperature, this strain accumulates a novel "19S" RNA species which contains 17S precursor rRNA sequences covalently linked to tRNA sequences transcribed from the ribosomal RNA spacer region between the 16S and the 23S rRNA cistrons. In vitro treatment of 19S RNA with cell extracts releases tRNA2Glu and other tRNA species. These "spacer" tRNA sequences are apparently not contained with the 18S RNA species found in an RNAase III- RNAase P+ cell. RNAase P-deficient extracts are incapable of cleaving space tRNA from 19S RNA, indicating that RNAase P is required for the release of spacer tRNAs from rRNA transcripts of E. coli cells.

Escherichia coli↗
PubMed1986

Evolutionary change in 5S rRNA secondary structure and a phylogenic tree of 352 5S rRNA species.

The secondary structure models of 5S rRNA have been constructed from the primary structure of 352 5S rRNA species available at present. All the 5S rRNAs examined can take essentially the same secondary structure, however they reveal characteristic differences between eukaryotes, metabacteria (= archaebacteria) and eubacteria. These three types of models can be further subgrouped by minor but characteristic differences. A phylogenic tree of organisms has been constructed using these 5S rRNA sequences by the weighted pairing method (WPG method). The tree reveals that there exist several major groups of eubacteria which seem to have diverged into different directions in the early stages of bacterial evolution. After emergence of eubacteria, metabacteria and eukaryotes separated from each other from their common ancestor. In the eukaryotic evolution, red algae (Rhodophyta) emerged first, and thereafter, thraustocytrids-Proctista, Ascomycota, green plants (green algae and land plants), Basidiomycota, Chromophyta (brown algae, diatoms and golden-yellow algae), slime- and water molds, various protozoans, and animals emerged in this order.

Animals↗
PubMed2006

Phylogenetic reconstruction of the wolf spiders (Araneae: Lycosidae) using sequences from the 12S rRNA, 28S rRNA, and NADH1 genes: implications for classification, biogeography, and the evolution of web building behavior.

Current knowledge of the evolutionary relationships amongst the wolf spiders (Araneae: Lycosidae) is based on assessment of morphological similarity or phylogenetic analysis of a small number of taxa. In order to enhance the current understanding of lycosid relationships, phylogenies of 70 lycosid species were reconstructed by parsimony and Bayesian methods using three molecular markers; the mitochondrial genes 12S rRNA, NADH1, and the nuclear gene 28S rRNA. The resultant trees from the mitochondrial markers were used to assess the current taxonomic status of the Lycosidae and to assess the evolutionary history of sheet-web construction in the group. The results suggest that a number of genera are not monophyletic, including Lycosa, Arctosa, Alopecosa, and Artoria. At the subfamilial level, the status of Pardosinae needs to be re-assessed, and the position of a number of genera within their respective subfamilies is in doubt (e.g., Hippasa and Arctosa in Lycosinae and Xerolycosa, Aulonia and Hygrolycosa in Venoniinae). In addition, a major clade of strictly Australasian taxa may require the creation of a new subfamily. The analysis of sheet-web building in Lycosidae revealed that the interpretation of this trait as an ancestral state relies on two factors: (1) an asymmetrical model favoring the loss of sheet-webs and (2) that the suspended silken tube of Pirata is directly descended from sheet-web building. Paralogous copies of the nuclear 28S rRNA gene were sequenced, confounding the interpretation of the phylogenetic analysis and suggesting that a cautionary approach should be taken to the further use of this gene for lycosid phylogenetic analysis.

Animals↗
PubMed2003

Phylogenetic analyses among octocorals (Cnidaria): mitochondrial and nuclear DNA sequences (lsu-rRNA, 16S and ssu-rRNA, 18S) support two convergent clades of branching gorgonians.

Gorgonian octocorals lack corroborated hypotheses of phylogeny. This study reconstructs genealogical relationships among some octocoral species based on published DNA sequences from the large ribosomal subunit of the mitochondrial RNA (lsu-rRNA, 16S: 524bp and 21 species) and the small subunit of the nuclear RNA (ssu-rRNA, 18S: 1815bp and 13 spp) using information from insertions-deletions (INDELS) and the predicted secondary structure of the lsu-rRNA (16S). There were seven short (3-10bp) INDELS in the 18S with consistent phylogenetic information. The INDELS in the 16S corresponded to informative signature sequences homologous to the G13 helix found in Escherichia coli. We found two main groups of gorgonian octocorals using a maximum parsimony analysis of the two genes. One group corresponds to deep-water taxa including species from the suborders Calcaxonia and Scleraxonia characterized by an enlargement of the G13 helix. The second group has species from Alcyoniina, Holaxonia and again Scleraxonia characterized by insertions in the 18S. Gorgonian corals, branching colonies with a gorgonin-containing flexible multilayered axis (Holaxonia and Calcaxonia), do not form a monophyletic group. These corroborated results from maternally inherited (16S) and biparentally inherited (18S) genes support a hypothesis of independent evolution of branching in the two octocoral clades.

Animals↗
PubMed2000

Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: application of a method called SERF.

Two-thirds of the 54 proteins of the Escherichia coli ribosome interact directly with the rRNAs, but the rRNA binding sites of only a very few proteins are known. We present a method (selection of random RNA fragments; SERF) that can identify the minimal binding region for proteins within ribonucleo-protein complexes such as the ribosome. The power of the method is exemplified with the ribosomal proteins L4 and L6. Binding sequences are identified for both proteins and characterized by phosphorothioate footprinting. Surprisingly, the binding region of L4, a 53-nt rRNA fragment of domain I of 23S rRNA, can simultaneously and independently bind L24, one of the two assembly initiator proteins of the large subunit.

Base Sequence↗
PubMed1986

Identification of the binding site on 5S rRNA for the transcription factor IIIA: proposed structure of a common binding site on 5S rRNA and on the gene.

Transcription factor IIIA interacts specifically with an internal control region of Xenopus 5S ribosomal RNA genes and is also a component, along with 5S rRNA, of a 7S ribonucleoprotein particle present in previtellogenic oocytes. We have determined the region of the 5S rRNA in the 7S ribonucleoprotein complex that is protected by the transcription factor from digestion with the ribonuclease alpha-sarcin. The binding site for factor IIIA extends from nucleotide 64 through nucleotide 116; the protected region includes two CCUGG helices separated by 11 nucleotides. The same helices occur in the factor IIIA binding site in the 5S rRNA gene and may constitute a common structural feature recognized by the protein in the gene and in the gene product.

Animals↗
PubMed1996

Analysis of the binding of Xenopus transcription factor IIIA to oocyte 5 S rRNA and to the 5 S rRNA gene.

Binding of transcription factor IIIA (TFIIIA) to site-specific mutants of Xenopus oocyte 5 S rRNA has been used to identify important recognition elements in the molecule. The putative base triple G75:U76:A100 appears to determine the conformation of the loop E region whose integrity is especially important for binding of the factor. Proximal substitutions in helices IV and V indicate that the proper folding of loop E is also dependent on these structures. Mutations in helix V affect binding of TFIIIA to 5 S rRNA and to the gene similarly and provide evidence that zinc finger 5 makes sequence-specific contact through the major groove of both nucleic acids. Although fingers 1-3 are positioned along helix IV and loop D, mutations in this region, including those that disrupt the tetraloop or close the opening in the major groove of the helix created by the U80:U96 mismatch, have no impact on binding. Substitutions made at stem-loop junctions in the arm of the RNA comprised of helix II-loop B-helix III display minor decreases in affinity for TFIIIA. Despite the alignment of the factor along nearly the entire length of 5 S rRNA, the essential elements for high affinity binding are limited to the central region of the molecule. Analysis of the corresponding mutations in the gene confirm that box C and the intermediate element provide the high affinity sites for binding of the factor to the DNA. Despite the small thermodynamic contribution made by contacts to box A, mutations made in this element can cause substantial changes in the orientation of the carboxyl-terminal fingers along the 5'-end of the internal control region.

Animals↗
PubMed1984

The complete nucleotide sequence of mouse 28S rRNA gene. Implications for the process of size increase of the large subunit rRNA in higher eukaryotes.

We have determined the complete nucleotide sequence (4712 nucleotides) of the mouse 28S rRNA gene. Comparison with all other homologs indicates that the potential for major variations in size during the evolution has been restricted to a unique set of a few sites within a largely conserved secondary structure core. The D (divergent) domains, responsible for the large increase in size of the molecule from procaryotes to higher eukaryotes, represent half the mouse 28S rRNA length. They show a clear potential to form self-contained secondary structures. Their high GC content in vertebrates is correlated with the folding of very long stable stems. Their comparison with the two other vertebrates, xenopus and rat, reveals an history of repeated insertions and deletions. During the evolution of vertebrates, insertion or deletion of new sequence tracts preferentially takes place in the subareas of D domains where the more recently fixed insertions/deletions were located in the ancestor sequence. These D domains appear closely related to the transcribed spacers of rRNA precursor but a sizable fraction displays a much slower rate of sequence variation.

Animals↗
PubMed1988

Mutational study of the rRNA in yeast mitochondria: functional importance of T1696 in the large rRNA gene.

Four intragenic suppressors of a mitochondrial mutation in the 21S rRNA gene have been characterized in S. cerevisiae. The determination of the nature of the nucleotide changes in the suppressor strains showed that a T at position 1696 in the large rRNA gene is essential for correct function of the mitoribosome. The importance of this specific nucleotide and the fact that this mitochondrial mutation can also be suppressed by a mutation in a nuclear gene are in good agreement with a rRNA-r protein interaction in this part of domain IV, which functional importance is demonstrated in vivo by our results.

Base Sequence↗
PubMed2000

Comparison of conserved structural and regulatory domains within divergent 16S rRNA-23S rRNA spacer sequences of cyanobacteria.

PCR amplification of the internal transcribed spacer (ITS) between the 16S rRNA and 23S rRNA genes of the cyanobacterium NOSTOC: PCC 7120 gave three products. Two represented true ITS regions of different sizes, while the third was a heteroduplex. The longer spacer (ITS-L) contained 512 nucleotides and carried tRNA(Ile) and tRNA(Ala) genes, separated by a large stem-loop structure (V2) composed of short tandemly repeated repetitive sequences. Both tRNA genes, and the 5' half of the intervening stem, were absent from the shorter spacer (ITS-S), of length 283 nucleotides, which was otherwise almost completely identical to ITS-L. The two spacer regions of NOSTOC: PCC 7120 were aligned to published ITS sequences of cyanobacteria, the cyanelle of Cyanophora paradoxa and Escherichia coli. Although the ITS regions of cyanobacteria vary in length from 283 to 545 nucleotides and contain either both tRNA(Ile) and tRNA(Ala) genes, only the tRNA(Ile) gene, or neither, there is no correlation between ITS size and coding capacity for tRNAs. Putative secondary structures were determined for the deduced transcripts of the rrn operons of several cyanobacteria and were compared to that of E. coli. Highly conserved motifs important for folding and for maturation of the rRNA transcripts were identified, and regions homologous to bacterial antiterminators (box B-box A) were located. The conserved and variable regions of the cyanobacterial ITS are potential targets of PCR primers and oligonucleotide probes for detection and identification of cyanobacteria at different taxonomic levels.

Anabaena↗
PubMed1992

Structural analysis of three prokaryotic 5S rRNA species and selected 5S rRNA--ribosomal-protein complexes by means of Pb(II)-induced hydrolysis.

Lead ions have been applied to the structural analysis of 5S rRNA from Thermus thermophilus, Bacillus stearothermophilus and Escherichia coli. Based on the distribution of Pb(II)-induced cleavages, some minor modifications of the consensus secondary structure model of 5S rRNA are proposed. They include the possible base pairing between nucleotides at positions 11 and 109, as well as changes in secondary interactions within the helix B region. The 'prokaryotic arm' region is completely resistant to hydrolysis in the three RNA species, suggesting that it is a relatively stable, highly ordered structure. Hydrolysis of E. coli 5S rRNA complexed with ribosomal protein L18 shows, besides the shielding effect of the bound protein, a highly enhanced cleavage between A108 and A109. It supports the concept that the major L18-induced conformational change involves the junction of helices A, B and D.

Autoradiography↗
PubMed1997

Physical localization of rRNA genes by two-colour fluorescent in-situ hybridization and sequence analysis of the 5S rRNA gene in Phalaris coerulescens.

The 18S-5.8S-26S rDNA and 5S rDNA loci have been mapped physically by fluorescent in-situ hybridization to the chromosomes of Phalaris coerulescens. The biotin-labelled heterologous 18S-5.8S-26S rRNA probe (pTa71) detected one locus, which corresponded to the secondary constriction (nucleolar organizer) on the long arm of the satellited chromosome II. The homologous 5S rDNA probe (Bam2.12) detected two pairs of 5S rRNA gene clusters which were localized at two different non-satellited chromosomes, one near the telomere on the short arm of the chromosome I, which is the largest chromosome of the complement, and the other about 42% out on the long arm of the chromosome III. A BamHI fragment containing the 5S rRNA gene, has been isolated and characterized. The 5S rDNA repeat unit is 309 bp in length, consisting of 121 bp highly conserved coding region and 188 bp variable spacer region. The karyotype of Phalaris coerulescens is characterized by the similar size of chromosomes within the group 2, group 3, or group 4. This study represents the first step towards the understanding the genome organization of Phalaris coerulescens and provides reliable markers for chromosome identification in this grass, an important species as a model system for the study of self-incompatibility in grasses.

Base Sequence↗
PubMed2005

Characterization and phylogenetic relationships among microsporidia infecting silkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis.

This study is the first report on the genetic characterization and relationships among different microsporidia infecting the silkworm, Bombyx mori, using inter simple sequence repeat PCR (ISSR-PCR) analysis. Six different microsporidians were distinguished through molecular DNA typing using ISSR-PCR. Thus, ISSR-PCR analysis can be a powerful tool to detect polymorphisms and identify microsporidians, which are difficult to study with microscopy because of their extremely small size. Of the 100 ISSR primers tested, only 28 primers had reproducibility and high polymorphism (93%). A total of 24 ISSR primers produced 55 unique genetic markers, which could be used to differentiate the microsporidians from each other. Among the 28 SSRs tested, the most abundant were (CA)n, (GA)n, and (GT)n repeats. The degree of band sharing was used to evaluate genetic similarity between different microsporidian isolates and to construct a phylogenetic tree using Jaccard's similarity coefficient. The results indicate that the DNA profiles based on ISSR markers can be used as diagnostic tools to identify different microsporidia with considerable accuracy. In addition, the small subunit ribosomal RNA (SSU-rRNA) sequence gene was amplified, cloned, and sequenced from each of the 6 microsporidian isolates. These sequences were compared with 20 other microsporidian SSU-rRNA sequences to develop a phylogenetic tree for the microsporidia isolated from the silkworms. This method was found to be useful in establishing the phylogenetic relationships among the different microsporidians isolated from silkworms. Of the 6 microsporidian isolates, NIK-1s revealed an SSU-rRNA gene sequence similar to Nosema bombycis, indicating that NIK-1s is similar to N. bombycis; the remaining 5 isolates, which differed from each other and from N. bombycis, were considered to be different variants belonging to the species N. bombycis.

Animals↗
PubMed2004

Molecular phylogeny of the nasuta subgroup of Drosophila based on 12S rRNA, 16S rRNA and CoI mitochondrial genes, RAPD and ISSR polymorphisms.

The nasuta subgroup is a cluster of morphologically almost similar forms with a wide range of geographic distribution. During the last three decades nature of inter-relationship among the members has been investigated at different levels of organization. The phylogenetic relationships of the members of the nasuta subgroup of the immigrans species group of Drosophila was made by employing Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats-PCR (ISSR-PCR) polymorphisms, mitochondrial 12S rRNA, 16S rRNA and Cytochrome C Oxidase subunit I (CoI) gene sequences. The phylogenetic tree generated by RAPD analysis is in nearly complete congruence with the classification based on morphophenotypic characters. The 12S and 16S rRNA genes were highly conserved across the nasuta subgroup and revealed only 3 and 4 variable sites respectively, of which only one site was informative. The CoI gene, on the other hand, revealed 57 variable sites of which 25 sites were informative. All the three species of orbital sheen complex were included in a major cluster in the phylogenetic trees derived from mitochondrial gene sequence data consistent with the morphophenotypic classification. The CoI analysis placed two species of frontal sheen complex, D. n. nasuta and D. n. albomicans in two different clades and this is inconsistent with morphological classification. The molecular clock suggested that divergence between the kohkoa complex and the albomicans complex occurred approximately 2.2 MYA, indicating recent evolution of the nasuta subgroup. The higher transition bias in the mitochondrial genes reported in the present study also suggested recent evolution of the nasuta subgroup.

Animals↗
PubMed1983

Chemical modification of adenine residues in mouse 5S rRNA with monoperphthalate: the secondary structure of 5S rRNA.

The chemical modification of adenine residues in mouse 5S rRNA with monoperphthalate was carried out to investigate the higher ordered structure of 5S rRNA. The adenine residues at positions 11, 22 (or/and 23), 49 (or/and 50), 54 (or/and 55), 77, 83, 88, 90 and 100 (or/and 101) were modified. This result further confirmed the secondary structure of 5S rRNA constituted of 5 helices and 5 loops postulated by other chemical modifications.

Animals↗
PubMed2005

Polymorphism and gene conversion of the 16S rRNA genes in the multiple rRNA operons of Vibrio parahaemolyticus.

The genome sequence of a strain of Vibrio parahaemolyticus holds 11 copies of rRNA operons (rrn) with identical 16S rRNA genes (rrs). Conversely, the species type strain contains two rrs classes differing in 10 nucleotide sites within a short segment of 25 bp. Furthermore, we show here that the sequence of this particular segment largely differs between some strains of this species. We also show that of the eleven rrn operons in the species type strain, seven contain one rrs class and four the other, indicating gene conversion. Our results support the hypothesis that the rrs differences observed between strains of this species were caused by lateral transfer of an rrs segment and subsequent conversion.

Base Sequence↗