RNA aptamers directed against release factor 1 from Thermus thermophilus

An in vitro selection/amplification (SELEX) was used to generate RNA aptamers that specifically bind Thermus thermophilus release factor 1 (RF1). From 31 isolated clones, two groups of aptamers with invariable sequences 5'-ACCU-3' and 5'-GAAAGC-3' were isolated. Chemical and enzymatic probing of the structure indicate that in both groups the invariable sequences are located in single-stranded regions of hairpin structures. Complex formations between RF1 and aptamers of both groups were identified by electrophoretic shift assay and chemical footprinting. Deletion of the invariable sequences did not effect the secondary structure of the aptamers but abolished their binding to RF1. RNA motifs matching the invariable sequences of the aptamers are present as consensus sequences in the peptidyl transferase center of 23S rRNAs. T. thermophilus RF1 recognizes UAG stop codons in an Escherichia coli in vitro translation system. Aptamers from both groups inhibited this RF1 activity.     

Chromosome location of the ribosomal genes in Triturus vulgaris meridionalis (Amphibia Urodela)

In Triturus vulgaris meridionalis, the 18S + 28S rDNA sequences have been shown to be located in a number of additional chromosomal sites besides the nucleolus organizing region. The additional ribosomal sites have been found to vary as to their number and chromosomal location in different individuals of the species.—The data presented in this study concern the chromosomal distribution of the ribosomal sequences as analyzed by in situ hybridization technique in two individuals as well as in their offspring. The evidence obtained by this analysis indicates quite clearly that all 18S + 28S rRNA sites present in each individual genome are inherited according to simple mendelian principles.Abbreviations rRNA ribosomal RNA - NOR nucleolus organizer region - rDNA DNA coding for 18S+28S rRNA plus the intervening spacers - SSC 0.15M Sodium chloride, 0.015 M Sodium citrate, pH 7 - RNase ribonuclease     

Cytologische Lokalisation von 5S und 18/25S RNA Genorten in Mitose-Chromosomen von Vicia faba

Homologous in situ hybridization with tritiated 4S, 5S and 18/25S RNA from root tip meristems of Vicia faba has been used to study the pattern of distribution of DNA sequences coding for these RNAs in the diploid nuclei. 5S RNA hybridizes to two regions of the satellites of the pair of satellited chromosomes. The sites differ in the level of in situ hybridization implicating different degrees of redundancy. 18/25S RNA hybridization is concentrated to the secondary constriction of these satellite chromosomes. Both, 5S and 18/25S ribosomal RNA gene sites are located on the same pair of chromosomes, but obviously the sequences are not contiguous. An association of 5S RNA cistrons with heterochromatin is assumed. Additional RNA gene sites as well as 4S RNA gene sites are not detectable.     

Maximum likelihood estimation of the heterogeneity of substitution rate among nucleotide sites

This paper presents a maximum likelihood approach to estimating the variation of substitution rate among nucleotide sites. We assume that the rate varies among sites according to an invariant+gamma distribution, which has two parameters: the gamma parameter alpha and the proportion of invariable sites theta. Theoretical treatments on three, four, and five sequences have been conducted, and computer program have been developed. It is shown that rho = (1 + theta alpha)/(1 + alpha) is a good measure for the rate heterogeneity among sites. Extensive simulations show that (1) if the proportion of invariable sites is negligible, i.e., theta = 0, the gamma parameter alpha can be satisfactorily estimated, even with three sequences; (2) if the proportion of invariable sites is not negligible, the heterogeneity rho can still be suitably estimated with four or more sequences; and (3) the distances estimated by the proposed method are almost unbiased and are robust against violation of the assumption of the invariant + gamma distribution.      

Ribosomal proteins in halobacteria

The amino acid sequences of 16 ribosomal proteins from archaebacterium Halobacterium marismortui have been determined by a direct protein chemical method. In addition, amino acid sequences of three proteins, S11, S18, and L25, have been established by DNA sequencing of their genes as well as by protein sequencing. Comparison of their sequences with those of ribosomal proteins from other organisms revealed that proteins S14, S16, S19, and L25 are related to both eukaryotic and eubacterial ribosomal proteins, being more homologous to eukaryotic than eubacterial counterparts, and proteins S12, S15, and L16 are related to only eukaryotic ribosomal proteins. Furthermore, some proteins are found to be similar to only eubacterial proteins, whereas other proteins show no homology to any other known ribosomal proteins. Comparisons of amino acid compositions between halophilic and nonhalophilic ribosomal proteins revealed that halophilic proteins gain aspartic and glutamic acid residues and significantly lose lysine and arginine residues. In addition, halophilic proteins seem to lose isoleucine as compared with Escherichia coli ribosomal proteins.     

Assessment of fungal diversity in deep-sea sediments by multiple primer approach

Increasing evidence of the fungal diversity in deep-sea sediments has come from amplification of environmental DNA with fungal specific or eukaryote primer sets. In order to assess the fungal diversity in deep-sea sediments of the Central Indian Basin (CIB) at ~5,000 m depth, we amplified sediment DNA with four different primer sets. These were fungal-specific primer pair ITS1F/ITS4 (internal transcribed spacers), universal 18S rDNA primers NS1/NS2, Euk18S-42F/Euk18S-1492R and Euk18S-555F/Euk18S-1269R. One environmental library was constructed with each of the primer pairs, and 48 clones were sequenced per library. These sequences resulted in 8 fungal Operational Taxonomic Units (OTUs) with ITS and 19 OTUs with 18S rDNA primer sets respectively by taking into account the 2% sequence divergence cut-off for species delineation. These OTUs belonged to 20 distinct fungal genera of the phyla Ascomycota and Basidiomycota. Seven sequences were found to be divergent by 79–97% from the known sequences of the existing database and may be novel. A majority of the sequences clustered with known sequences of the existing taxa. The phylogenetic affiliation of a few fungal sequences with known environmental sequences from marine and hypersaline habitat suggests their autochthonous nature or adaptation to marine habitat. The amplification of sequences belonging to Exobasidiomycetes and Cystobasidiomycetes from deep-sea is being reported for the first time in this study. Amplification of fungal sequences with eukaryotic as well as fungal specific primers indicates that among eukaryotes, fungi appear to be a dominant group in the sampling site of the CIB.     

Identifying evolutionary trees and substitution parameters for the general Markov model with invariable sites

The general Markov plus invariable sites (GM+I) model of biological sequence evolution is a two-class model in which an unknown proportion of sites are not allowed to change, while the remainder undergo substitutions according to a Markov process on a tree. For statistical use it is important to know if the model is identifiable; can both the tree topology and the numerical parameters be determined from a joint distribution describing sequences only at the leaves of the tree? We establish that for generic parameters both the tree and all numerical parameter values can be recovered, up to clearly understood issues of 'label swapping'. The method of analysis is algebraic, using phylogenetic invariants to study the variety defined by the model. Simple rational formulas, expressed in terms of determinantal ratios, are found for recovering numerical parameters describing the invariable sites.     

Separating real motifs from their artifacts

The typical output of many computational methods to identify binding sites is a long list of motifs containing some real motifs (those most likely to correspond to the actual binding sites) along with a large number of random variations of these. We present a statistical method to separate real motifs from their artifacts. This produces a short list of high quality motifs that is sufficient to explain the over-representation of all motifs in the given sequences. Using synthetic data sets, we show that the output of our method is very accurate. On various sets of upstream sequences in S. cerevisiae, our program identifies several known binding sites, as well as a number of significant novel motifs.     

Correlation between the substitution rate and rate variation among sites in protein evolution.

It is well known that the rate of amino acid substitution varies among different proteins and among different sites of a protein. It is, however, unclear whether the extent of rate variation among sites of a protein and the mean substitution rate of the protein are correlated. We used two approaches to analyze orthologous protein sequences of 51 nuclear genes of vertebrates and 13 mitochondrial genes of mammals. In the first approach, no assumptions of the distribution of the rate variation among sites were made, and in the second approach, the gamma distribution was assumed. Through both approaches, we found a negative correlation between the extent of among-site rate variation and the average substitution rate of a protein. That is, slowly evolving proteins tend to have a high level of rate variation among sites, and vice versa. We found this observation consistent with a simple model of the neutral theory where most sites are either invariable or neutral. We conclude that the correlation is a general feature of protein evolution and discuss its implications in statistical tests of positive Darwinian selection and molecular time estimation of deep divergences.     

Group I introns within the nuclear-encoded small-subunit rRNA gene of three green algae.

Four group I introns from the nuclear-encoded (18S) rRNA genes of three chlorophycean green algae are described; two are in Dunaliella parva, and one each is in D. salina and Characium saccatum. The introns within the gene in the latter two organisms are located at the sites equivalent to the 5' and 3' introns of D. parva, respectively. All four introns lack open reading frames and are relatively small, 381-447 bp. Both primary- and secondary-structural features place these introns within subgroup IC1 described by Michel and Westhof. Phylogenetic relationships of the three intron-containing taxa and their relatives, as inferred from comparisons of 18S rDNA sequences, suggest that inheritance of the introns along with the gene can account for their present distribution. The discovery of these four introns, in addition to two others known to exist in other chlorophycean green algae, suggests that group I introns within the 18S rRNA gene may be relatively common in the green algae.     

Partial small subunit (18S) rRNA gene sequences from fish parasites of the families Lepocreadiidae and Fellodistomidae (Digenea) and their use in phylogenetic analyses

Digeneans of the families Lepocreadiidae and Fellodistomidae are common parasites of shallow and deep-water marine fishes. Worms recovered from these contrasting environments may be indistinguishable or very similar morphologically and little is known concerning their inter-relationships. To investigate the systematic status of these forms, partial sequences from a small subunit 18S rRNA gene were generated from four species of lepocreadiids (Lepidapedon elongatum, L. gaevskayae, L. rachion and Opechona bacillaris) and two fellodistomids (Fellodistomum fellis and Steringophorus agnotus). Approximately 1,637 bases were sequenced for L. elongatum, L. rachion and F. fellis. A comparison of these sequences identified regions which varied between the two families. As a result, two regions were identified as containing potential phylogenetically informative sites and sequenced from the remaining species. Analyses of these regions by both maximum parsimony and distance methods showed that nucleic acid sequence data can be used to resolve the two families. The results indicate that 18S rRNA sequences are likely to be more satisfactory at resolving higher levels of phylogeny, i.e. at the family level, than at the generic level due to the similarity shown between the sequences of congeners.     

Rapidly evolving lineages impede the resolution of phylogenetic relationships among Clitellata (Annelida)

The phylogenetic relationships of the Clitellata were investigated using a data set with published and new complete or partial 18S rRNA and mtCOI gene sequences of 13 and 49 taxa representing 8 and 14 families, respectively. Three different alignments were considered for 18S, and the possible influence of departures from rate constancy among sites was evaluated by analyses using a Gamma model of rate heterogeneity. Maximum-likelihood estimates of the shape parameter alpha of the Gamma distribution were very low, whatever the alignment or the gene considered, suggesting that phylogenetic reconstructions taking into account the rate heterogeneity among sites are likely to be the most reliable. Analyzed separately, the two genes did not resolve the relationships among the Clitellata, but the consensus tree was congruent with the morphology-based relationships. Our data suggest the inclusion of the Euhirudinea, Acanthobdellida, and Branchiobdellida in the Oligochaeta and suggest the Lumbriculidae as the link between both assemblages. Although separate analyses of both genes, as well as different alignments for the 18S rRNA sequences, yielded conflicting results concerning the phylogenetic position of leeches and leech-like worms vis-à-vis the Oligochaeta, subsequent analyses using the Gamma model greatly reduced the observed inconsistencies. Our analyses show that among the Clitellata, the leeches and the leech-like and gutless worms represent significantly faster evolving lineages. It is suggested that the observed higher mutation rates may be explained by the fact that these lineages contain almost exclusively commensal and/or parasitic taxa.     

Molecular characterisation of the Theileria buffeli/orientalis group

Benign bovine Theileria parasites known as either Theileria buffeli, Theileria orientalis or Theileria sergenti are classified on basis of their morphology, vector specificity, pathogenicity and 18S small subunit ribosomal RNA or major piroplasm protein (MPSP) sequences. Since most isolates have been characterized on only some of these criteria and the existing confusion in nomenclature, an analysis was performed on eight different isolates to combine 18S rRNA data with MPSP data and the results were compared with available biological parameters. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach for both genes was used in combination with reverse line blot hybridisation for the 18S rRNA gene. Both MPSP and 18S rRNA genes were cloned and sequenced from parasites displaying aberrant MPSP RFLP profiles. Phylogeny based on published and determined 18S rRNA and MPSP sequences did correlate within the same isolate but there was no obvious correlation between molecular and biological data. Based on these findings, we suggest that the appropriate name for all these parasites is Theileria buffeli. A more specific nomenclature should be assigned when new molecular markers may become available.     

Deep-level diagnostic value of the rDNA-ITS region

The similarity of certain reported angiosperm rDNA internal transcribed spacer (ITS) region sequences to those of green algae prompted our analysis of the deep-level phylogenetic signal in the highly conserved but short 5.8S and hypervariable ITS2 sequences. We found that 5.8S sequences yield phylogenetic trees similar to but less well supported than those generated by a ca. 10-fold longer alignment from rDNA-18S sequences, as well as independent evidence. We attribute this result to our finding that, compared to 18S, the 5.8S has a higher proportion of sites subject to vary and greater among-site substitution rate homogeneity. We also determined that our phylogenetic results are not likely affected by intramolecular compensatory mutation to maintain RNA secondary structure nor by evident systematic biases in base composition. Despite historical homology, there appears to be no ITS2 primary sequence similarity shared sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that groups, however, share sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that ITS region sequences can diagnose organismal origins and phylogenetic relationships at many phylogenetic levels and provide a useful paradigm for molecular evolutionary study.      

Conditionally Neutral Phylogenetic Markers of Major Taxa: A New Aspect of the Evolution of Macromolecules

The current phase of molecular phylogenetics can be named the 18S rRNA gene era, which is now approaching the end. To date, almost all phyla of metazoans and many taxa of protists are represented in databases of 18S rRNA gene sequences. The elements of the phylogenetic tree of Metazoa inferred from 18S rRNA genes are characterized by unequal validity: some of them seem to be well grounded; others are not adequately supported, and probably will be revised later. The validity of phylogenetic reconstruction is influenced by two main factors: (1) erroneous grouping of long branches that occur because of abnormally high evolution rate; (2) deficit of phylogenetically informative characters. A method for overcoming these difficulties is suggested in addition to known tools: using phylogenetic markers that are stable within individual taxa and evolve by punctuated equilibrium. These markers are least influenced by the convergence caused by a high evolution rate of the entire gene. The nature of these markers of ancient taxa, paradoxical from the perspective of neutral evolution, is discussed, as well as their importance for establishing monophyly of both new large-scale taxonomic groups of invertebrates (Bilateria + Rhombozoa + Orthonectida + Myxozoa + Cnidaria + Placozoa and Echinodermata + Hemichordata) and some major taxa of Nematoda.     

Conditionally neutral phylogenetic markers of major taxa: a new aspect of the evolution of macromolecules

The current phase of molecular phylogenetics can be named the 18S rRNA gene era, which is now approaching the end. To date, almost all phyla of metazoans and many taxa of protists are represented in databases of 18S rRNA gene sequences. The elements of the phylogenetic tree of Metazoa inferred from 18S rRNA genes are characterized by unequal validity: some of them seem to be well grounded; others are not adequately supported, and probably will be revised later. The validity of phylogenetic reconstruction is influenced by two main factors: (1) erroneous grouping of long branches that occur because of abnormally high evolution rate; (2) deficit of phylogenetically informative characters. A method for overcoming these difficulties is suggested in addition to known tools: using phylogenetic markers that are stable within individual taxa and evolve by punctuated equilibrium. These markers are least influenced by the convergence caused by a high evolution rate of the entire gene. The nature of these markers of ancient taxa, paradoxical from the perspective of neutral evolution, is discussed, as well as their importance for establishing monophyly of both new large-scale taxonomic groups of invertebrates (Bilateria + Rhombozoa + Orthonectida + Myxozoa + Cnidaria + Placozoa and Echinodermata + Hemichordata) and some major taxa of Nematoda.     

Genetic diversity of nuclear ITS1-5.8S-ITS2 rDNA sequence in Clonorchis sinensis Cobbold, 1875 (Trematoda: Opisthorchidae) from the Russian Far East

The present study examined the molecular organisation and sequence variation in the nuclear ribosomal DNA (rDNA) region, including the two internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the Clonorchis sinensis from the Russian Far East. The relevant sequences from other parts of this species' area were downloaded from GenBank. The results showed 100% identity for all investigated 5.8S-ITS2 rDNA sequences. In contrast, two levels of intraspecific variations were revealed in the complete ITS1 sequences. The intra-genomic variation resulted from a C/T polymorphism in a single position. The inter-individual differences between the ITS1 sequences were both due to nucleotide and size polymorphisms resulting from a varying number of five-nucleotide repeats and followed by two ITS1 length variants. These variant frequencies correlate with the clonorchiasis level in some geographical localities. ITS1 differences, both in the mutation profile and mutation localisation, were revealed between northern and southern geographical samples. The presence of GC boxes that are identical to known regulatory motifs in eukaryotes was detected within the ITS1 sub-repeats. The predicted secondary structures for ITS1 consist of two large branches, one of which was invariable, while another depended on ITS1 length. The predicted secondary structure for ITS2 includes four helices around the core. The main differences between C. sinensis and other opisthorchids were localised on the tops of helices 2, 3, and 4. A phylogenetic MST reconstruction subdivided all ITS1 sequences into two well differentiated clusters, each with the major widespread ribotype, and showed that ribotype diversity in both Russia and Korea is much lower than in China. The results obtained demonstrate the feasibility of complete ITS1 sequences in C. sinensis population genetics and can be considered as a basis for further studies of the parasite infection because they may help to elucidate the molecular mechanisms of pathogen evolution and adaptation.     

Predicting genes expressed via -1 and +1 frameshifts

Computational identification of ribosomal frameshift sites in genomic sequences is difficult due to their diverse nature, yet it provides useful information for understanding the underlying mechanisms and discovering new genes. We have developed an algorithm that searches entire genomic or mRNA sequences for frameshifting sites, and implements the algorithm as a web-based program called FSFinder (Frameshift Signal Finder). The current version of FSFinder is capable of finding -1 frameshift sites on heptamer sequences X XXY YYZ, and +1 frameshift sites for two genes: protein chain release factor B (prfB) and ornithine decarboxylase antizyme (oaz). We tested FSFinder on approximately 190 genomic and partial DNA sequences from a number of organisms and found that it predicted frameshift sites efficiently and with greater sensitivity and specificity than existing approaches. It has improved sensitivity because it considers many known components of a frameshifting cassette and searches these components on both + and - strands, and its specificity is increased because it focuses on overlapping regions of open reading frames and prioritizes candidate frameshift sites. FSFinder is useful for discovering unknown genes that utilize alternative decoding, as well as for analyzing frameshift sites. It is freely accessible at http://wilab.inha.ac.kr/FSFinder/.     

The phylogeny of land plants inferred from 18S rDNA sequences: pushing the limits of rDNA signal?

Previous studies of the phylogeny of land plants based on analysis of 18S ribosomal DNA (rDNA) sequences have generally found weak support for the relationships recovered and at least some obviously spurious relationships, resulting in equivocal inferences of land plant phylogeny. We hypothesized that greater sampling of both characters and taxa would improve inferences of land plant phylogeny based on 18S rDNA sequences. We therefore conducted a phylogenetic analysis of complete (or nearly complete) 18S rDNA sequences for 93 species of land plants and 7 green algal relatives. Parsimony analyses with equal weighting of characters and characters state changes and parsimony analyses weighting (1) stem bases half as much as loop bases and (2) transitions half as much as transversions did not produce substantially different topologies. Although the general structure of the shortest trees is consistent with most hypotheses of land plant phylogeny, several relationships, particularly among major groups of land plants, appear spurious. Increased character and taxon sampling did not substantially improve the performance of 18S rDNA in phylogenetic analyses of land plants, nor did analyses designed to accommodate variation in evolutionary rates among sites. The rate and pattern of 18S rDNA evolution across land plants may limit the usefulness of this gene for phylogeny reconstruction at deep levels of plant phylogeny. We conclude that the mosaic structure of 18S rDNA, consisting of highly conserved and highly variable regions, may contain historical signal at two levels. Rapidly evolving regions are informative for relatively recent divergences (e.g., within angiosperms, seed plants, and ferns), but homoplasy at these sites makes it difficult to resolve relationships among these groups. At deeper levels, changes in the highly conserved regions of small-subunit rDNAs provide signal across all of life. Because constraints imposed by the secondary structure of the rRNA may affect the phylogenetic information content of 18S rDNA, we suggest that 18S rDNA sequences be combined with other data and that methods of analysis be employed to accommodate these differences in evolutionary patterns, particularly across deep divergences in the tree of life.