Sequence characterization of the 5S ribosomal DNA and the internal transcribed spacer (ITS) region in four European Donax species (Bivalvia: Donacidae).

BACKGROUND: The whole repeat unit of 5S rDNA and the internal transcribed spacer (ITS) of four European Donax species were analysed. After amplifying, cloning and sequencing several 5S and ITS units, their basic features and their variation were described. The phylogenetic usefulness of 5S and ITS sequences in the inference of evolutionary relationships among these wedge clams was also investigated. RESULTS: The length of the 5S repeat presented little variation among species, except D. trunculus that differed from the rest of the Donax species in 170-210 bp. The deduced coding region covered 120 bp, and showed recognizable internal control regions (ICRs) involved in the transcription. The length of non-transcribed spacer region (NTS) ranged from 157 bp to 165 bp in Donax trunculus and from 335 bp to 367 bp in the other three species. The conservation degree of transcriptional regulatory regions was analysed revealing a conserved TATA-like box in the upstream region. Regarding ITS sequences, the four Donax species showed slight size differences among clones due to the variation occurring in the ITS1 and ITS2, except Donax variegatus did not display size differences in the ITS2. The total length of the ITS sequence ranged between 814 and 1014 bp. Resulting phylogenetic trees display that the two ribosomal DNA regions provide well-resolved phylogenies where the four European Donax species form a single clade receiving high support in nodes. The topology obtained with 5S sequences was in agreement with Donax evolutionary relationships inferred from several sequences of different nature in previous studies. CONCLUSIONS: This is not only a basic research work, where new data and new knowledge is provided about Donax species, but also have allowed the authentication of these wedge clams and offers future applications to provide other genetic resources.

A Tandemly Arranged Pattern of Two 5S rDNA Arrays in Amolops mantzorum (Anura, Ranidae).

In an attempt to extend the knowledge of the 5S rDNA organization in anurans, the 5S rDNA sequences of Amolops mantzorum were isolated, characterized, and mapped by FISH. Two forms of 5S rDNA, type I (209 bp) and type II (about 870 bp), were found in specimens investigated from various populations. Both of them contained a 118-bp coding sequence, readily differentiated by their non-transcribed spacer (NTS) sizes and compositions. Four probes (the 5S rDNA coding sequences, the type I NTS, the type II NTS, and the entire type II 5S rDNA sequences) were respectively labeled with TAMRA or digoxigenin to hybridize with mitotic chromosomes for samples of all localities. It turned out that all probes showed the same signals that appeared in every centromeric region and in the telomeric regions of chromosome 5, without differences within or between populations. Obviously, both type I and type II of the 5S rDNA arrays arranged in tandem, which was contrasting with other frogs or fishes recorded to date. More interestingly, all the probes detected centromeric regions in all karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA.

Linking maternal and somatic 5S rRNA types with different sequence-specific non-LTR retrotransposons.

5S rRNA is a ribosomal core component, transcribed from many gene copies organized in genomic repeats. Some eukaryotic species have two 5S rRNA types defined by their predominant expression in oogenesis or adult tissue. Our next-generation sequencing study on zebrafish egg, embryo, and adult tissue identified maternal-type 5S rRNA that is exclusively accumulated during oogenesis, replaced throughout the embryogenesis by a somatic-type, and thus virtually absent in adult somatic tissue. The maternal-type 5S rDNA contains several thousands of gene copies on chromosome 4 in tandem repeats with small intergenic regions, whereas the somatic-type is present in only 12 gene copies on chromosome 18 with large intergenic regions. The nine-nucleotide variation between the two 5S rRNA types likely affects TFIII binding and riboprotein L5 binding, probably leading to storage of maternal-type rRNA. Remarkably, these sequence differences are located exactly at the sequence-specific target site for genome integration by the 5S rRNA-specific Mutsu retrotransposon family. Thus, we could define maternal- and somatic-type MutsuDr subfamilies. Furthermore, we identified four additional maternal-type and two new somatic-type MutsuDr subfamilies, each with their own target sequence. This target-site specificity, frequently intact maternal-type retrotransposon elements, plus specific presence of Mutsu retrotransposon RNA and piRNA in egg and adult tissue, suggest an involvement of retrotransposons in achieving the differential copy number of the two types of 5S rDNA loci.

Phylogeny of Helieae (Gentianaceae): Resolving taxonomic chaos in a Neotropical clade.

The monophyletic and Neotropical tribe Helieae of the worldwide family Gentianaceae (Gentianales, Asterids, Angiospermae) is well known for its problematic generic classifications. An initial phylogenetic analysis of Helieae shed light onto the relationships between genera, and indicated that traditional generic limits did not correspond to monophyletic groups. In order to obtain a more thorough understanding of generic relationships within the group, we enhanced sampling within the so-called Symbolanthus clade and performed phylogenetic analyses from DNA sequences from one plastid region (matK) and two nuclear regions (ITS and 5S-NTS), plus 112 morphological characters, which were analyzed separately and in combination, using parsimony and Bayesian approaches. A total of 83 individuals representing 20 genera and 51 species of Helieae were sampled; 13 species were included in this study solely based on their morphological characters. Ancestral character reconstructions were performed to identify potential synapomorphies of clades and patterns of homoplasy in the morphological dataset. Our results demonstrate that Prepusa is sister to the remainder of Helieae. Furthermore, the Macrocarpaea clade, the Irlbachia clade and the Symbolanthus clade were also recovered. Within the Symbolanthus clade, our results confirm that Calolisianthus and Chelonanthus are not monophyletic, and also contest the monophyly of Irlbachia as currently circumscribed. Specifically, two species of Calolisianthus group with the type species of Chelonanthus, while the other Calolisianthus species are more closely related to Tetrapollinia and Symbolanthus. Moreover, the green-white-flowered Chelonanthus species and Adenolisianthus are undoubtedly related to Helia and several analyses support Irlbachia pratensis as more closely related to the lineage including the type species of Chelonanthus described above The addition of new characters and taxa led to higher confidence in the relative position of some clades, as well as provided further support for a new generic circumscription of Calolisianthus, Chelonanthus, and Helia. Even though several morphological characters traditionally used in the taxonomy of the group were shown to be homoplasious, most clades can be diagnosed by a combination of morphological character states.

Widespread occurrence of organelle genome-encoded 5S rRNAs including permuted molecules.

5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator.

A phylogenetic model for the detection of epistatic interactions.

Paired epistatic interactions, such as those in the stem regions of RNA, play an important role in many biological processes. However, unlike protein-coding regions, paired epistatic interactions have lacked the appropriate statistical tools for the detection of departures from selective neutrality. Here, a model is presented for the analysis of paired epistatic regions that draws upon the population genetics of the compensatory substitution process to detect the relative strength of natural selection acting against deleterious combinations of alleles. The method is based upon the relative rates of double and single substitution, and can differentiate between nonindependent interactions and negatively epistatic ones. The model is implemented in a fully Bayesian framework for parameter estimation and is demonstrated using a 5S rRNA data set. In addition to the detection of selection, modeling the double and single substitution processes in this manner inherently accounts for a substantial proportion of rate variation among stem positions.

Contrasting patterns of evolution of 45S and 5S rDNA families uncover new aspects in the genome constitution of the agronomically important grass Thinopyrum intermedium (Triticeae).

We employed sequencing of clones and in situ hybridization (genomic and fluorescent in situ hybridization [GISH and rDNA-FISH]) to characterize both the sequence variation and genomic organization of 45S (herein ITS1-5.8S-ITS2 region) and 5S (5S gene + nontranscribed spacer) ribosomal DNA (rDNA) families in the allohexaploid grass Thinopyrum intermedium. Both rDNA families are organized within several rDNA loci within all three subgenomes of the allohexaploid species. Both families have undergone different patterns of evolution. The 45S rDNA family has evolved in a concerted manner: internal transcribed spacer (ITS) sequences residing within the arrays of two subgenomes out of three got homogenized toward one major ribotype, whereas the third subgenome contained a minor proportion of distinct unhomogenized copies. Homogenization mechanisms such as unequal crossover and/or gene conversion were coupled with the loss of certain 45S rDNA loci. Unlike in the 45S family, the data suggest that neither interlocus homogenization among homeologous chromosomes nor locus loss occurred in 5S rDNA. Consistently with other Triticeae, the 5S rDNA family in intermediate wheatgrass comprised two distinct array types-the long- and short-spacer unit classes. Within the long and short units, we distinguished five and three different types, respectively, likely representing homeologous unit classes donated by putative parental species. Although the major ITS ribotype corresponds in our phylogenetic analysis to the E-genome species, the minor ribotype corresponds to Dasypyrum. 5S sequences suggested the contributions from Pseudoroegneria, Dasypyrum, and Aegilops. The contribution from Aegilops to the intermediate wheatgrass' genome is a new finding with implications in wheat improvement. We discuss rDNA evolution and potential origin of intermediate wheatgrass.

The origin of modern 5S rRNA: a case of relating models of structural history to phylogenetic data.

Evolutionary models of molecular structures must incorporate molecular information at different levels of structural complexity and must be phrased within a phylogenetic perspective. In this regard, phylogenetic trees of substructures that are reconstructed from molecular features that contribute to order and thermodynamic stability show that a gradual model of evolution of 5S rRNA structure is more parsimonious than models that invoke large segmental duplications of the molecule. The search for trees of substructures that are most parsimonious, by their very nature, defines an objective strategy to select models of molecular change that best fit structural data. When combined with additional data, such as the age of protein domains that interact with RNA substructures, these trees can be used to falsify unlikely hypotheses.

Codependence of repetitive sequence classes in genomes: phylogenetic analysis of 5S rDNA families in Hordeum (Triticeae: Poaceae).

To complete our study of the genus Hordeum and to elaborate a phylogeny of species based upon 5S rDNA sequences, we have cloned and sequenced PCR amplicons from seven American polyploid species to generate 164 new 5S rRNA gene sequences. These sequences were analysed along with the more than 2000 5S rDNA sequences previously generated from the majority of species in Hordeum to provide a comprehensive picture of the distribution (presence or absence) of 5S rDNA unit classes (orthologous groups) in this genus as well as insights into the phylogeny of Hordeum. Testing of substitution models for each unit class based upon the consensus sequences of all the taxa as well as for each unit class within the genus found that the general best fit was TPM3uf+G, from which a maximum-likelihood tree was calculated. A novel application of cophylogenetic analysis, where relationships among unit classes were treated as host-parasite interactions, depicted some significant pair links under tests of randomness indicative of nonrandom codivergence among several unit classes within the same taxon. The previous classification of four genomic groups is reflected in combinations of unit classes, and it is proposed that current taxa developed from ancient diploidized paleopolyploids and that some were subjected to gene loss, i.e., unit class loss. Finally, separate phylogenetic analyses performed for the tetraploid and hexaploid species were used to derive a working model describing the phylogeny of the polyploid taxa from their putative diploid ancestry.

Analysis and classification of RNA tertiary structures.

There is a fast growing interest in noncoding RNA transcripts. These transcripts are not translated into proteins, but play essential roles in many cellular and pathological processes. Recent efforts toward comprehension of their function has led to a substantial increase in both the number and the size of solved RNA structures. With the aim of addressing questions relating to RNA structural diversity, we examined RNA conservation at three structural levels: primary, secondary, and tertiary structure. Additionally, we developed an automated method for classifying RNA structures based on spatial (three-dimensional [3D]) similarity. Applying the method to all solved RNA structures resulted in a classified database of RNA tertiary structures (DARTS). DARTS embodies 1333 solved RNA structures classified into 94 clusters. The classification is hierarchical, reflecting the structural relationship between and within clusters. We also developed an application for searching DARTS with a new structure. The search is fast and its performance was successfully tested on all solved RNA structures since the creation of DARTS. A user-friendly interface for both the database and the search application is available online. We show intracluster and intercluster similarities in DARTS and demonstrate the usefulness of the search application. The analysis reveals the current structural repertoire of RNA and exposes common global folds and local tertiary motifs. Further study of these conserved substructures may suggest possible RNA domains and building blocks. This should be beneficial for structure prediction and for gaining insights into structure-function relationships.

Molecular confirmation of the genomic constitution of Douglasdeweya (Triticeae: Poaceae): demonstration of the utility of the 5S rDNA sequence as a tool for haplome identification.

A new genus Douglasdeweya containing the two species, Douglasdeweya deweyi and D. wangii was published in 2005 by Yen et al. based upon the results of cytogenetical and morphological findings. The genome constitution of Douglasdeweya-PPStSt-allowed its segregation from the genus Pseudoroegneria which contains the StSt or StStStSt genomes. Our previous work had demonstrated the utility of using 5S rDNA units, especially the non-transcribed spacer sequence variation, for the resolution of genomes (haplomes) previously established by cytology. Here, we show that sequence analysis of the 5S DNA units from these species strongly supports the proposed species relationships of Yen et al. (Can J Bot 83:413-419, 2005), i.e., the PP genome from Agropyron and the StSt genome from Pseudoroegneria. Analysis of the 5S rDNA units constitutes a powerful tool for genomic research especially in the Triticeae.

DNA profiling of Acorus calamus chemotypes differing in essential oil composition.

The phylogenetic relationship of Acorus gramineus and three types of Acorus calamus was analyzed by comparing the 700 bp sequence of a 5S-rRNA gene spacer region. Although there was no intra-specific variation in the essential oil profile of A. gramineus which contained a phenylpropanoid (Z-asarone) as a predominant constituent, A. calamus was classified into two chemotypes: chemotype A in which Z-asarone is a major essential oil constituent and chemotype B which contained sesquiterpenoids predominantly. An intermediate type (M) of these two chemotypes in various ratios was also observable. The NJ tree constructed based on the sequences revealed that A. gramineus was clearly distinguished from any of the chemotypes of A. calamus and that the phylogenetic relationship predicted by the spacer region data correlated well with the essential oil chemotype pattern of A. calamus.

Taxonomy of 5 S ribosomal RNA by the linguistic technique: probing with mitochondrial and mammalian sequences.

Linguistic similarities and dissimilarities between 5 S rRNA sequences allowed taxonomical separation of species and classes. Comparisons with the molecule from mammals distinguished fungi and plants from protists and animals. Similarities to mammalians progressively increased from protists to invertebrates and to somatic-type molecules of the vertebrates lineage. In this, deviations were detected in avian, oocyte type, and pseudogene sequences. Among bacteria, actinobacteria were most similar to the mammalians, which could be related to the high frequency of associations among members of these groups. Some archaebacterial species most similar to the mammalians belonged to the Thermoproteales and Halobacteria groups. Comparisons with the soybean mitochondrial molecule revealed high internal homogeneity among plant mitochondria. The eubacterial groups most similar to it were Thermus and Rhodobacteria gamma-1 and alpha-2. Other procedures have already indicated similarities of Rhodobacteria alpha to mitochondria but the linguistic similarities were on the average higher with the first two groups.

Herbidospora gen. nov., a new genus of the family Streptosporangiaceae Goodfellow et al. 1990.

Eight actinomycete strains originally isolated from soil and plant samples were studied to determine their taxonomic status. All isolates produced branching substrate mycelia, but no distinct aerial hyphae. Relatively short chains of nonmotile spores (10 to 30 spores per chain) were borne on the tips of sporophores arising directly from the agar surface. The chemotaxonomic characteristics of the isolates, with the exception of the menaquinone profile, coincided with those of members of the family Streptosporangiaceae Goodfellow, Stanton, Simpson, and Minnikin 1990. Furthermore, the results of a phylogenetic analysis performed with 5S rRNA support the conclusion that the isolates should be classified in this family. The isolates differed from members of the constituent genera of the Streptosporangiaceae in morphological characteristics and menaquinone composition. Therefore, we propose a new genus for the strains, Herbidospora. The type species and type strain are Herbidospora cretacea sp. nov. and strain K-319 (= JCM 8553), respectively.

Origins of the plant chloroplasts and mitochondria based on comparisons of 5S ribosomal RNAs.

In this paper, we provide macromolecular comparisons utilizing the 5S ribosomal RNA structure to suggest extant bacteria that are the likely descendants of chloroplast and mitochondria endosymbionts. The genetic stability and near universality of the 5S ribosomal gene allows for a useful means to study ancient evolutionary changes by macromolecular comparisons. The value in current and future ribosomal RNA comparisons is in fine tuning the assignment of ancestors to the organelles and in establishing extant species likely to be descendants of bacteria involved in presumed multiple endosymbiotic events.