Secondary structure prediction for complete rDNA sequences (18S, 5.8S,and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.     

Cryptic diversity of the symbiotic cyanobacterium Synechococcus spongiarum among sponge hosts

Cyanobacteria are common members of sponge-associated bacterial communities and are particularly abundant symbionts of coral reef sponges. The unicellular cyanobacterium Synechococcus spongiarum is the most prevalent photosynthetic symbiont in marine sponges and inhabits taxonomically diverse hosts from tropical and temperate reefs worldwide. Despite the global distribution of S. spongiarum , molecular analyses report low levels of genetic divergence among 16S ribosomal RNA (rRNA) gene sequences from diverse sponge hosts, resulting either from the widespread dispersal ability of these symbionts or the low phylogenetic resolution of a conserved molecular marker. Partial 16S rRNA and entire 16S–23S rRNA internal transcribed spacer (ITS) genes were sequenced from cyanobacteria inhabiting 32 sponges (representing 18 species, six families and four orders) from six geographical regions. ITS phylogenies revealed 12 distinct clades of S. spongiarum that displayed 9% mean sequence divergence among clades and less than 1% sequence divergence within clades. Symbiont clades ranged in specificity from generalists to specialists, with most (10 of 12) clades detected in one or several closely related hosts. Although multiple symbiont clades inhabited some host sponges, symbiont communities appear to be structured by both geography and host phylogeny. In contrast, 16S rRNA sequences were highly conserved, exhibiting less than 1% sequence divergence among symbiont clades. ITS gene sequences displayed much higher variability than 16S rRNA sequences, highlighting the utility of ITS sequences in determining the genetic diversity and host specificity of S. spongiarum populations among reef sponges. The genetic diversity of S. spongiarum revealed by ITS sequences may be correlated with different physiological capabilities and environmental preferences that may generate variable host–symbiont interactions.     

Evolutionarily conserved structural features in the ITS2 of mammalian pre-rRNAs and potential interactions with the snoRNA U8 detected by comparative analysis of new mouse sequences.

Mechanisms of ITS2 excision from pre-rRNA remain largely elusive. In mammals, at least two endonucleolytic cleavages are involved, which result in the transient accumulation of precursors to 5.8S rRNA termed 8S and 12S RNAs. We have sequenced ITS2 in four new species of the Mus genus and investigated its secondary structure using thermodynamic prediction and comparative approach. Phylogenetic evidence supports an ITS2 folding organized in four domains of secondary structure extending from a preserved structural core. This folding is also largely conserved for the previously available mammalian ITS2 sequences, rat and human, despite their extensive sequence divergence relative to the Mus species. Conserved structural features include the structural core, containing the 3' end of 8S pre-rRNA within a single-stranded sequence, and a stem containing the 3' end of the 12S pre-rRNA species. A putative, phylogenetically preserved pseudoknot has been detected 1 nt downstream from the 12S 3' end. Two long complementarities have also been identified, in sequences conserved among vertebrates, between the pre-rRNA 32S and the snoRNA (small nucleolar RNA) U8 which is required for the excision of Xenopus ITS2. The first complementarity involves the 5.8S-ITS2 junction and 13 nt at the 5' end of U8, whereas the other one occurs between a mature 28S rRNA segment known to be required for ITS2 excision and positions 15-25 of snoRNA U8. These two potential interactions, in combination with ITS2 folding, could organize a functional pocket containing three cleavage sites and key elements for pre-rRNA processing, suggesting a chaperone role for the snoRNA U8.     

紫芝基因组rDNA序列特征及ITS2二级结构比较

紫芝栽培品种‘紫芝S2’(武芝2号)的ITS序列与NCBI数据库中5个紫芝菌株/分离株相似度高达99.79%-100%,在系统进化树上相聚成一类。本研究预测‘紫芝S2’基因组与参考基因组中的rRNA基因簇,分析rDNA结构及各构件序列间的多态性。从高质量‘紫芝S2’基因组中挖掘得到完整rDNA,序列全长40.377 kb,由4组串联重复的(18S、5.8S、28S、5S) rRNA基因簇组成,并含有完整的基因内间隔区(ITS1、ITS2)和基因间间隔区(IGS1、IGS2)。在紫芝S2的rDNA中,高度保守的28S rRNA基因间出现3个SNP和2个插入(1 bp,10 bp)位点;虽然第4条ITS2中有1个SNP位点,但紫芝S2的4条ITS2在二级结构上的分子形态高度一致,与ITS2数据库中其他紫芝菌株仅存在螺旋区间夹角的微小差异。由‘紫芝S2’基因组rDNA的ITS2生成的DNA条形码与二维码,可以作为该栽培品种鉴定与同源物种其他菌株鉴别的分子标记。     

Molecular phylogeny of Pseudokeronopsis (Protozoa, Ciliophora, Urostylida), with reconsideration of three closely related species at inter- and intra-specific levels inferred from the small subunit ribosomal RNA gene and the ITS1-5.8S-ITS2 region sequences

In order to address the confused taxonomy of morphotypes within the genus Pseudokeronopsis , the small subunit ribosomal RNA (SS rRNA) gene and the internal transcribed spacer (ITS1)-5.8S-ITS2 region for 13 populations/clones of morphologically similar Pseudokeronopsis species, Pseudokeronopsis flava , Pseudokeronopsis rubra and Pseudokeronopsis carnea , were sequenced and compared at inter- and intra-specific levels. Different geographical/time interval isolates belonging to the same species were slightly divergent, while clones from the same strain had identical SS rRNA gene sequences. Compared with the SS rRNA gene sequence, the sequence of the ITS1-5.8S-ITS2 region of Pseudokeronopsis revealed higher diversity, with nucleotide dissimilarities of 3.9–4.9% at the intra-specific level and 8.0–10.5% at the inter-specific level. There were several minor differences among ITS2 secondary structures of three Pseudokeronopsis species, while three Pseudok. carnea populations were identical. Phylogenetic analyses using multiple algorithms and different species selections confirm that Pseudok. flava , Pseudok. rubra and Pseudok. carnea form a monophyletic group within the urostylids. The result also supports the closest relationship of Pseudokeronopsis and Pseudourostyla that forms the so-called Acaudalia Berger (2006) .     

Homology modeling revealed more than 20,000 rRNA internal transcribed spacer 2 (ITS2) secondary structures

Structural genomics meets phylogenetics and vice versa: Knowing rRNA secondary structures is a prerequisite for constructing rRNA alignments for inferring phylogenies, and inferring phylogenies is a precondition to understand the evolution of such rRNA secondary structures. Here, both scientific worlds go together. The rRNA internal transcribed spacer 2 (ITS2) region is a widely used phylogenetic marker. Because of its high variability at the sequence level, correct alignments have to take into account structural information. In this study, we examine the extent of the conservation in structure. We present (1) the homology modeled secondary structure of more than 20,000 ITS2 covering about 14,000 species; (2) a computational approach for homology modeling of rRNA structures, which additionally can be applied to other RNA families; and (3) a database providing about 25,000 ITS2 sequences with their associated secondary structures, a refined ITS2 specific general time reversible (GTR) substitution model, and a scoring matrix, available at http://its2.bioapps.biozentrum.uni-wuerzburg.de.     

松萝属的5.8S-ITS2二级结构比较分析

本研究以松萝属为研究对象,构建了该属核糖体5.8S和第二转录区间（ITS2）的二级结构模型,并对种间的结构差异进行了比较。结果显示,松萝属的结构与先前发表的真核生物ITS2二级结构模型非常相似。属内种间的结构差异主要集中在第二转录区间的臂上。核酸序列分析所产生的系统树与结构特征的比较结果相一致,表明ITS2的二级结构信息可以作为一种辅助的分类手段,适用于松萝属的种的鉴定和亲缘关系研究。     

Identification and typing of miso and soy sauce fermentation yeasts, Candida etchellsii and C. versatilis, based on sequence analyses of the D1D2 domain of the 26S ribosomal RNA gene, and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2 (ITS sequence) of the miso and soy sauce fermentation yeasts, Candida etchellsii and Candida versatilis, in order to evaluate the usefulness of this sequence analysis for identification and typing of these two species. In the 26S rDNA sequence method, the numbers of base substitutions among C. etchellsii strains were up to 2 in 482 bp (99.6% similarity), and they were divided into three types (types A, B, and C). Those of C. versatilis strains were also up to 2 in 521 bp (99.6% similarity) and they were divided into three types (types 1, 2, and 3). In the ITS sequence method, those of C. etchellsii strains were zero in 433 bp (type a, 100% similarity). Those of C. versatilis were 5 in 409 bp (98.8% similarity), divided into 4 types (types I, II, III and IV). It was found that molecular methods based on the sequences of the 26S rDNA D1D2 domain and the ITS region were rapid and precise compared with the physiological method for the identification and typing of these two species.     

Sequencing and analysis of the internal transcribed spacers (ITSs) and coding regions in the EcoR I fragment of the ribosomal DNA of the Japanese pond frog Rana nigromaculata

The rDNA of eukaryotic organisms is transcribed as the 40S-45S rRNA precursor, and this precursor contains the following segments: 5' - ETS - 18S rRNA - ITS 1 - 5.8S rRNA - ITS 2 - 28S rRNA - 3'. In amphibians, the nucleotide sequences of the rRNA precursor have been completely determined in only two species of Xenopus. In the other amphibian species investigated so far, only the short nucleotide sequences of some rDNA fragments have been reported. We obtained a genomic clone containing the rDNA precursor from the Japanese pond frog Rana nigromaculata and analyzed its nucleotide sequence. The cloned genomic fragment was 4,806 bp long and included the 3'-terminus of 18S rRNA, ITS 1, 5.8S rRNA, ITS 2, and a long portion of 28S rRNA. A comparison of nucleotide sequences among Rana, the two species of Xenopus, and human revealed the following: (1) The 3'-terminus of 18S rRNA and the complete 5.8S rRNA were highly conserved among these four taxa. (2) The regions corresponding to the stem and loop of the secondary structure in 28S rRNA were conserved between Xenopus and Rana, but the rate of substitutions in the loop was higher than that in the stem. Many of the human loop regions had large insertions not seen in amphibians. (3) Two ITS regions had highly diverged sequences that made it difficult to compare the sequences not only between human and frogs, but also between Xenopus and Rana. (4) The short tracts in the ITS regions were strictly conserved between the two Xenopus species, and there was a corresponding sequence for Rana. Our data on the nucleotide sequence of the rRNA precursor from the Japanese pond frog Rana nigromaculata were used to examine the potential usefulness of the rRNA genes and ITS regions for evolutionary studies on frogs, because the rRNA precursor contains both highly conserved regions and rapidly evolving regions.     

Isolation of cDNA clones coding for mitochondrial 16S ribosomal RNA from the crustacean Artemia

cDNA clones coding for Artemia mitochondrial 16S ribosomal RNA (rRNA) have been isolated. The clones cover from nucleotide 650 of the RNA molecule to its 3' end. The comparison of Artemia sequence with both vertebrate and invertebrate mitochondrial 16S rRNA sequences has shown the existence of regions of high similarity between them. A model for the secondary structure of the 3' half of Artemia mitochondrial 16S rRNA is proposed. The size of the rRNA molecule has been estimated at 1.35 kb. Despite the similarity of the Artemia gene to insect rRNA in size, sequence and secondary structure, the G + C content of the Artemia gene (42%) is closer to that of mammals than to the insect genes. The number of mitochondria in Artemia has been estimated at 1500 per diploid genome in the cyst and 4000 in the nauplius. In contrast, the amount of mt 16S rRNA is constant at all stages of Artemia development.     

Sequence and secondary structure variation in the Gyrodactylus (Platyhelminthes: Monogenea) ribosomal RNA gene array

Nucleotide sequences were determined for the rRNA internal transcribed spacers 1 and 2 (ITS1 and 2) and the 5' terminus of the large subunit rRNA in selected Gyrodactylus species. Examination of primary sequence variation and secondary structure models in ITS2 and variable region V4 of the small subunit rRNA revealed that structure was largely conserved despite significant variation in sequence. ITS1 sequences were highly variable, and models of structure were unreliable but, despite this, show some resemblance to structures predicted in Digenea. ITS2 models demonstrated binding of the 3' end of 5.8S rRNA to the 5' end of the large subunit rRNA and enabled the termini of these genes to be defined with greater confidence than previously. The structure model shown here may prove useful in future phylogenetic analyses.     

Phylogenetic analysis and predicted secondary structures of the rDNA internal transcribed spacers of the powdery mildew fungi (Erysiphaceae)

The nucleotide sequences of the internal transcribed spacer (ITS) regions of the ribosomal DNA including the 5.8S rRNA gene and the 5′ end of the 28S rRNA gene have been determined for 19 species in 10 genera of the powdery mildew fungi in order to analyze their phylogenetic relationship. These fungi were divided into two large groups based on the nucleotide length of the ITS regions, and this grouping was in line with that based on the morphological characters of the anamorphic stage rather than the teleomorphic stage. Although the variable ITS sequences were often ambiguously aligned, conserved sites were also found. Thus, a neighbor-joining tree was constructed using the nucleotide sequence data of the conserved sites of the ITS regions, the 5.8S rRNA gene, and the 5′ end of the 28S rRNA gene. The phylogenetic tree displayed the presence of four groups in the powdery mildews, which were distinguished by their morphology and/or host ranges. In the ITS2 region, the presence of a common secondary structure having four hairpin domains was suggested, in spite of the highly variable nucleotide sequences of this region. The predicted secondary structure was supported by the compensatory mutations as well as compensatory conserved sequences and high G+C content in the predicted stem regions. Contribution No. 142 from the Laboratory of Plant Pathology, Mie University.     

The structure of a single unit of ribosomal RNA gene (rDNA) including intergenic subrepeats in the Australian bulldog ant Myrmecia croslandi (Hymenoptera: Formicidae)

A complete single unit of a ribosomal RNA gene (rDNA) of M. croslandi was sequenced. The ends of the 18S, 5.8S and 28S rRNA genes were determined by using the sequences of D. melanogaster rDNAs as references. Each of the tandemly repeated rDNA units consists of coding and non-coding regions whose arrangement is the same as that of D. melanogaster rDNA. The intergenic spacer (IGS) contains, as in other species, a region with subrepeats, of which the sequences are different from those previously reported in other insect species. The length of IGSs was estimated to be 7-12 kb by genomic Southern hybridization, showing that an rDNA repeating unit of M. croslandi is 14-19 kb-long. The sequences of the coding regions are highly conserved, whereas IGS and ITS (internal transcribed spacer) sequences are not. We obtained clones with insertions of various sizes of R2 elements, the target sequence of which was found in the 28S rRNA coding region. A short segment in the IGS that follows the 3' end of the 28S rRNA gene was predicted to form a secondary structure with long stems.     

裸子植物5S rRNA基因序列变异及二级结构特征

在高等植物中,5SrRNA基因一级结构是高度保守的,二级结构也相当一致。通过比较18种裸子植物5SrRNA基因序列和二级结构变异,发现55％的核苷酸位点是可变的,这种变异有68％发生在干区（双链区）,其中一些变异,如双链的互补性核苷酸替代,GU配对等能够维系5SrRNA二级结构的稳定性。环区相对保守,这与5SrRNA三级结构折叠或在转录翻译过程中蛋白质、RNA的结合相关。另外,首次报道了松属环E区核苷酸的变异性,这可能与其他区域的变异一样,是假基因造成的结果。5SrRNA基因信息可反映大分类群的系统进化关系,但由于基因长度短,信息量小,其在近缘种系统分类的应用受到限制。     

POLYPHASIC STUDY OF ANTARCTIC CYANOBACTERIAL STRAINS1

We isolated 59 strains of cyanobacteria from the benthic microbial mats of 23 Antarctic lakes, from five locations in two regions, in order to characterize their morphological and genotypic diversity. On the basis of their morphology, the cyanobacteria were assigned to 12 species that included four Antarctic endemic taxa. Sequences of the ribosomal RNA gene were determined for 56 strains. In general, the strains closely related at the 16S rRNA gene level belonged to the same morphospecies. Nevertheless, divergences were observed concerning the diversity in terms of species richness, novelty, and geographical distribution. For the 56 strains, 21 operational taxonomic units (OTUs, defined as groups of partial 16S rRNA gene sequences with more than 97.5% similarity) were found, including nine novel and three exclusively Antarctic OTUs. Sequences of Petalonema cf. involvens and Chondrocystis sp. were determined for the first time. The internally transcribed spacer (ITS) between the 16S and the 23S rRNA genes was sequenced for 33 strains, and similar groupings were observed with the 16S rRNA gene and the ITS, even when the strains were derived from different lakes and regions. In addition, 48 strains were screened for antimicrobial and cytotoxic activities, and 17 strains were bioactive against the gram‐positive Staphylococcus aureus, or the fungi Aspergillus fumigatus and Cryptococcus neoformans. The bioactivities were not in coincidence with the phylogenetic relationships, but rather were specific to certain strains.     

Nucleotide sequence of Halobacterium cutirubrum ribosomal 5 S ribonucleic acid. An altered secondary structure in halophilic organisms

The nucleotide sequence of ribosomal 5 S RNA from a halophilic bacterium, Halobacterium cutirubrum, grown in 4 M sodium chloride is U-U-A-A-G-G-C-G-G-C-C-A-U-A-G-C-G-G-U-G-G-G-G-U-U-A-C-U-C-C-C-G-U-A-C-C-C-A-U-C-C-C-G-A-A-C-A-C-G-G-A-A-G-A-U-A-A-G-C-C-C-G-C-C-U-G-C-G-U-U-C-C-G-G-U-C-A-G-U-A-C-U-G-G-A-G-U-G-C-G-A-G-C-C-U-C-U-G-G-G-A-A-A-U-C-C-G-G-U-U-C-G-C-C-G-C-C-U-A-C-U. This nucleotide sequence is the longest prokaryotic 5 S rRNA to be reported and unlike other 5 S species does not contain a terminal mononucleoside diphosphate residue at its 5'-end. When compared to other 5 S rRNA's, the sequence homology is greatest (about 68%) with Bacillus subtilis; there is a lower but similar degree of homology (about 58%) with either Escherichia coli or human 5 S RNA. The comparisons further indicate that among 5 S RNA's, eleven of the nucleotide residues are unique to H. cutirubrum. Estimates of the secondary structure of the H. cutirubrum 5 S RNA molecule contain one additional stable hairpin loop which is not found in other 5 S rRNA species; this unusual structure is probably an adaptation to the high salt environment within H. cutirubrum cells.     

Molecular characterization of the Trichomonas gallinae morphologic complex in the United States

Forty-two Trichomonas gallinae isolates were molecularly characterized to determine whether isolates differed in genetic sequence of multiple gene targets depending on host species or geographical location. The 5.8S ribosomal RNA (rRNA) and flanking internal transcribed spacer (ITS) gene regions were amplified by polymerase chain reaction, and the sequences were analyzed phylogenetically. The results of the sequence analysis strongly suggest at least 2 species may exist within the T. gallinae morphologic complex. Based on ITS sequences, one group demonstrated high nucleotide identity to the 3 T. gallinae sequences available in GenBank, whereas the second group was more closely related to T. vaginalis (98%) than to T. gallinae (92%). Two common ground-dove (Columbina passerina) isolates shared a 95% identity with T. vaginalis and a 92% identity with T. gallinae and T. tenax. Sequence analysis of both the 18S rRNA and alpha-tubulin genes from a subset of the isolates supports the 5.8S-ITS sequence results. All of the T. vaginalis-like isolates originated from Arizona, California, or Texas, whereas T. gallinae isolates were found in all sampled states. Both T. vaginalis-like and T. gallinae isolates were involved in trichomoniasis outbreaks in California and Arizona.     

Functional analysis of internal transcribed spacer 2 of Saccharomyces cerevisiae ribosomal DNA.

Using the previously described "tagged ribosome" (pORCS) system for in vivo mutational analysis of yeast rDNA, we show that small deletions in the 5'-terminal portion of ITS2 completely block maturation of 26 S rRNA at the level of the 29 SB precursor (5.8 S rRNA-ITS2-26 S rRNA). Various deletions in the 3'-terminal part, although severely reducing the efficiency of processing, still allow some mature 26 S rRNA to be formed. On the other hand, none of the ITS2 deletions affect the production of mature 17 S rRNA. Since all of the deletions severely disturb the recently proposed secondary structure of ITS2, these findings suggest an important role for higher order structure of ITS2 in processing. Analysis of the effect of complete or partial replacement of S. cerevisiae ITS2 with its counterpart sequences from Saccharomyces rosei or Hansenula wingei, points to helix V of the secondary structure model as an important element for correct and efficient processing. Direct mutational analysis shows that disruption of base-pairing in the middle of helix V does not detectably affect 26 S rRNA formation. In contrast, introduction of clustered point mutations at the apical end of helix V that both disrupt base-pairing and change the sequence of the loop, severely reduces processing. Since a mutant containing only point mutations in the sequence of the loop produces normal amounts of mature 26 S rRNA, we conclude that the precise (secondary and/or primary) structure at the lower end of helix V, but excluding the loop, is of crucial importance for efficient removal of ITS2.     

Genetic Diversity and Structure of the Invasive Toxic Cyanobacterium <Emphasis Type="Italic">Cylindrospermopsis raciborskii</Emphasis>

Strains of the invasive toxic cyanobacteria Cylindrospermopsis raciborskii were genetically evaluated with four genetic markers encompassing in total 2.9 kb (16S rRNA, ITS longer spacer, ITS shorter spacer and rpoC1) to assess the phylogenetic relationships, genetic variation and population differentiation of the species across all five continents. The phylogenetic analysis showed that the C. raciborskii strains grouped into three well-supported distinct clusters: (I) European (II) African/American, and (III) Asian/Australian. The European group presented a high genetic similarity with the Asian and the Australian isolates than with the African and American isolates. Several Portuguese isolates were analyzed (n = 7) and revealed a low genetic differentiation with little geographical structure. The genetic distance among groups and phylogenetic relationships obtained in this study suggest that the recent invasion of C. raciborskii in Portuguese and other European temperate environments could have had its origin in the Asian and/or Australian continents.