Proposed secondary structure of eukaryote specific expansion segment 15 in 28S rRNA from mice, rats, and rabbits

The expansion segments in eukaryotic ribosomal RNAs are additional RNA sequences not found in the RNA core common to both prokaryotes and eukaryotes. These regions show large species-dependent variations in sequence and size. This makes it difficult to create secondary structure models for the expansion segments exclusively based on phylogenetic sequence comparison. Here we have used a combination of experimental data and computational methods to generate secondary structure models for expansion segment 15 in 28S rRNA in mice, rats, and rabbits. The experimental data were collected using the structure sensitive reagents DMS, CMCT, kethoxal, micrococcal nuclease, RNase T(1), RNase CL3, RNase V(1), and lead(II) acetate. ES15 was folded with the computer program RNAStructure 3.5 using modification data and phylogenetic similarities between different ES15 sequences. This program uses energy minimization to find the most stable secondary structure of an RNA sequence. The presented secondary structure models include several common structural motifs, but they also have characteristics unique to each organism. Overall, the secondary structure models showed indications of an energetically stable but dynamic structure, easily accessible from the solution by the modification reagents, suggesting that the expansion segment is located on the ribosomal surface.     

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.     

Application of the hypervariable region of the 16S rDNA sequence as an index for the rapid identification of species in the genus Alicyclobacillus

A comparison of the 16S rRNA gene (rDNA) sequences of seven type strains belonging to different Alicyclobacillus species (i.e., five validated species, one proposed species and one genomic species) suggested that the 5' end hypervariable region (259-273 bases in length) of 16S rDNA was specific for the respective type strains. Further phylogenetic analysis based on DNA sequences of the hypervariable region using 24 Alicyclobacillus strains revealed that the strains could be categorized into five species and the A. acidocaldarius-Alicyclobacillus genomic species 1 group. The hypervariable region was highly conserved among the five species: A. acidiphilus, A. acidoterrestris, A. cycloheptanicus, A. herbarius, and A. hesperidum. The strains in the A. acidocaldarius-Alicyclobacillus genomic species 1 group were subdivided into two clusters (Clusters I and II) based on DNA sequences in the hypervariable region. On the basis of phenotypic characteristics, chemotaxonomic and phylogenetic analyses, and DNA-DNA hybridization data, strains in Cluster I were grouped as Alicyclobacillus genomic species 1 and strains in Cluster II were re-identified as A. acidocaldarius, thereby demonstrating that the hypervariable regions were also highly conserved within these two species. These results suggest that as is the case with Bacillus, the hypervariable region is significantly species-specific in the genus Alicyclobacillus to distinguish Alicyclobacillus species by DNA sequence comparison of the hypervariable region.     

Molecular phylogenetics of the lizard genus Microlophus (squamata:tropiduridae): aligning and retrieving indel signal from nuclear introns

We use a multigene data set (the mitochondrial locus and nine nuclear gene regions) to test phylogenetic relationships in the South American "lava lizards" (genus Microlophus) and describe a strategy for aligning noncoding sequences that accounts for differences in tempo and class of mutational events. We focus on seven nuclear introns that vary in size and frequency of multibase length mutations (i.e., indels) and present a manual alignment strategy that incorporates insertions and deletions (indels) for each intron. Our method is based on mechanistic explanations of intron evolution that does not require a guide tree. We also use a progressive alignment algorithm (Probabilistic Alignment Kit; PRANK) and distinguishes insertions from deletions and avoids the "gapcost" conundrum. We describe an approach to selecting a guide tree purged of ambiguously aligned regions and use this to refine PRANK performance. We show that although manual alignment is successful in finding repeat motifs and the most obvious indels, some regions can only be subjectively aligned, and there are limits to the size and complexity of a data matrix for which this approach can be taken. PRANK alignments identified more parsimony-informative indels while simultaneously increasing nucleotide identity in conserved sequence blocks flanking the indel regions. When comparing manual and PRANK with two widely used methods (CLUSTAL, MUSCLE) for the alignment of the most length-variable intron, only PRANK recovered a tree congruent at deeper nodes with the combined data tree inferred from all nuclear gene regions. We take this concordance as an objective function of alignment quality and present a strongly supported phylogenetic hypothesis for Microlophus relationships. From this hypothesis we show that (1) a coded indel data partition derived from the PRANK alignment contributed significantly to nodal support and (2) the indel data set permitted detection of significant conflict between mitochondrial and nuclear data partitions, which we hypothesize arose from secondary contact of distantly related taxa, followed by hybridization and mtDNA introgression.     

Trematode and Monogenean rRNA ITS2 Secondary Structures Support a Four-Domain Model

The secondary structure of rRNA internal transcribed spacer 2 is important in the process of ribosomal biogenesis. Trematode ITS sequences are poorly conserved and difficult to align for phylogenetic comparisons above a family level. If a conserved secondary structure can be identified, it can be used to guide primary sequence alignments. ITS2 sequences from 39 species were compared. These species span four orders of trematodes (Echinostomiformes, Plagiorchiformes, Strigeiformes, and Paramphistomiformes) and one monogenean (Gyrodactyliformes). The sequences vary in length from 251 to 431 bases, with an average GC content of 48%. The monogenean sequence could not be aligned with confidence to the trematodes. Above the family level trematode sequences were alignable from the 5′ end for 139 bases. Secondary structure foldings predicted a four-domain model. Three folding patterns were required for the apex of domain B. The folding pattern of domains C and D varies for each family. The structures display a high GC content within stems. Bases A and U are favored in unpaired regions and variable sites cluster. This produces a mosaic of conserved and variable regions with a structural conformation resistant to change. Two conserved strings were identified, one in domain B and the other in domain C. The first site can be aligned to a processing site identified in yeast and rat. The second site has been found in plants, and structural location appears to be important. A phylogenetic tree of the trematode sequences, aligned with the aid of secondary structures, distinguishes the four recognized orders. Received: 21 November 1997 / Accepted: 9 February 1998     

Analysis of the Primary Sequence and Secondary Structure of the Unusually Long SSU rRNA of the Soil Bug, Armadillidium vulgare

The complete nucleotide sequence of the SSU rRNA gene from the soil bug, Armadillidium vulgare (Crustacea, Isopoda), was determined. It is 3214 bp long, with a GC content of 56.3%. It is not only the longest SSU rRNA gene among Crustacea but also longer than any other SSU rRNA gene except that of the strepsipteran insect, Xenos vesparum (3316 bp). The unusually long sequence of this species is explained by the long sequences of variable regions V4 and V7, which make up more than half of the total length. RT-PCR analysis of these two regions showed that the long sequences also exist in the mature rRNA and sequence simplicity analysis revealed the presence of slippage motifs in these two regions. The putative secondary structure of the rRNA is typical for eukaryotes except for the length and shape variations of the V2, V4, V7, and V9 regions. Each of the V2, V4, and V7 regions was elongated, while the V9 region was shortened. In V2, two bulges, located between helix 8 and helix 9 and between helix 9 and helix 10, were elongated. In V4, stem E23-3 was dramatically expanded, with several small branched stems. In V7, stem 43 was branched and expanded. Comparisons with the unusually long SSU rRNAs of other organisms imply that the increase in total length of SSU rRNA is due mainly to expansion in the V4 and V7 regions. Received: 2 March 1999 / Accepted: 22 July 1999     

Sequence and structural conservation in RNA ribose zippers

The "ribose zipper", an important element of RNA tertiary structure, is characterized by consecutive hydrogen-bonding interactions between ribose 2'-hydroxyls from different regions of an RNA chain or between RNA chains. These tertiary contacts have previously been observed to also involve base-backbone and base-base interactions (A-minor type). We searched for ribose zipper tertiary interactions in the crystal structures of the large ribosomal subunit RNAs of Haloarcula marismortui and Deinococcus radiodurans, and the small ribosomal subunit RNA of Thermus thermophilus and identified a total of 97 ribose zippers. Of these, 20 were found in T. thermophilus 16 S rRNA, 44 in H. marismortui 23 S rRNA (plus 2 bridging 5 S and 23 S rRNAs) and 30 in D. radiodurans 23 S rRNA (plus 1 bridging 5 S and 23 S rRNAs). These were analyzed in terms of sequence conservation, structural conservation and stability, location in secondary structure, and phylogenetic conservation. Eleven types of ribose zippers were defined based on ribose-base interactions. Of these 11, seven were observed in the ribosomal RNAs. The most common of these is the canonical ribose zipper, originally observed in the P4-P6 group I intron fragment. All ribose zippers were formed by antiparallel chain interactions and only a single example extended beyond two residues, forming an overlapping ribose zipper of three consecutive residues near the small subunit A-site. Almost all ribose zippers link stem (Watson-Crick duplex) or stem-like (base-paired), with loop (external, internal, or junction) chain segments. About two-thirds of the observed ribose zippers interact with ribosomal proteins. Most of these ribosomal proteins bridge the ribose zipper chain segments with basic amino acid residues hydrogen bonding to the RNA backbone. Proteins involved in crucial ribosome function and in early stages of ribosomal assembly also stabilize ribose zipper interactions. All ribose zippers show strong sequence conservation both within these three ribosomal RNA structures and in a large database of aligned prokaryotic sequences. The physical basis of the sequence conservation is stacked base triples formed between consecutive base-pairs on the stem or stem-like segment with bases (often adenines) from the loop-side segment. These triples have previously been characterized as Type I and Type II A-minor motifs and are stabilized by base-base and base-ribose hydrogen bonds. The sequence and structure conservation of ribose zippers can be directly used in tertiary structure prediction and may have applications in molecular modeling and design.     

The unusually long small subunit ribosomal RNA gene found in amitochondriate amoeboflagellate Pelomyxa palustris: its rRNA predicted secondary structure and phylogenetic implication

In order to ascertain a phylogenetic position of the freshwater amitochondriate amoeboflagellate Pelomyxa palustris its small subunit (SSU) rRNA gene was amplified and sequenced. It was shown to be 3502 bp long. The predicted secondary structure of its rRNA includes at least 16 separate expansion zones located in all the variable regions (V1-V9), as well as in some conservative gene regions. Most insertions are represented by sequences of low complexity that have presumably arisen by a slippage mechanism. Relatively conservative, uniformly positioned motifs contained in regions V4 and V7, as well as in some others, made it possible to perform folding. In maximum likelihood, maximum parsimony, and neighbor-joining trees, P. palustris tends to cluster with amitochondriate and secondary lost mitochondria amoebae and amoeboflagellates Entamoeba, Endolimax nana, and Phreatamoeba balamuthi, comprising together with them and aerobic lobose amoebae Vannella, Acanthamoeba, Balamuthia, and Hartmannella a monophyletic cluster. Another pelobiont, Mastigamoeba invertens, does not belong to this cluster. No specific similarity was discovered between the SSU rRNA of P. palustris and amitochondriate taxa of 'Archezoa': Diplomonada, Parabasalia, Microsporidia. Pelomyxa palustris SSU rRNA does not occupy a basal position in the phylogenetic trees and could be ascribed to the so-called eukaryotic 'crown' group if the composition of the latter were not so sensitive to the methods of tree building. Thus, molecular and morphological data suggest that P. palustris represents a secondarily modified eukaryotic lineage.     

The role of polymerase eta in somatic hypermutation determined by analysis of mutations in a patient with xeroderma pigmentosum variant

To determine the possible role of polymerase eta (pol eta) in somatic hypermutation of B cells, a mutational analysis of 24 nonproductive rearrangements from a patient with xeroderma pigmentosum variant with a defect in pol eta was conducted. Although the mutational frequency of A and T bases decreased in WA (A/T, A) motifs, regardless of their RGYW (purine, G; pyrimidine, A/T) context, the overall mutational frequency of A or T bases was not affected. Moreover, the overall mutational frequency of the sequences examined was not decreased. There was an apparent increase in the number of insertions and deletions. The results are consistent with the conclusion that pol eta specifically targets WA motifs. However, its overall contribution to the somatic hypermutational process does not appear to be indispensable and in its absence other mechanisms maintain mutational activity.     

A story: unpaired adenosine bases in ribosomal RNAs

In 1985 an analysis of the Escherichia coli 16 S rRNA covariation-based structure model revealed a strong bias for unpaired adenosines. The same analysis revealed that the majority of the G, C, and U bases were paired. These biases are (now) consistent with the high percentage of unpaired adenosine nucleotides in several structure motifs.An analysis of a larger set of bacterial comparative 16 S and 23 S rRNA structure models has substantiated this initial finding and revealed new biases in the distribution of adenosine nucleotides in loop regions. The majority of the adenosine nucleotides are unpaired, while the majority of the G, C, and U bases are paired in the covariation-based structure model. The unpaired adenosine nucleotides predominate in the middle and at the 3' end of loops, and are the second most frequent nucleotide type at the 5' end of loops (G is the most common nucleotide). There are additional biases for unpaired adenosine nucleotides at the 3' end of loops and adjacent to a G at the 5' end of the helix. The most prevalent consecutive nucleotides are GG, GA, AG, and AA. A total of 70 % of the GG sequences are within helices, while more than 70 % of the AA sequences are unpaired. Nearly 50 % of the GA sequences are unpaired, and approximately one-third of the AG sequences are within helices while another third are at the 3' loop.5' helix junction. Unpaired positions with an adenosine nucleotide in more than 50 % of the sequences at the 3' end of 16 S and 23 S rRNA loops were identified and arranged into the A-motif categories XAZ, AAZ, XAG, AAG, and AAG:U, where G or Z is paired, G:U is a base-pair, and X is not an A and Z is not a G in more than 50 % of the sequences. These sequence motifs were associated with several structural motifs, such as adenosine platforms, E and E-like loops, A:A and A:G pairings at the end of helices, G:A tandem base-pairs, GNRA tetraloop hairpins, and U-turns.     

Large expansion segments in 18S rDNA support a new sponge clade (Class Demospongiae, Order Haplosclerida)

Newly emerging molecular phylogenetic hypotheses involving the sponge Order Haplosclerida (Class Demospongiae) are far removed from traditional views on their classification using morphology. In the new grouping of marine haplosclerid taxa by molecular data all members of one highly supported clade were found to have three large indels in the 18S rRNA gene. These indels were not found in this gene in other marine haplosclerids or in any other demosponges analysed. These indels were found in the variable V4 and V7 region of the gene, had high GC contents and formed stable double stranded helices in the 18S rRNA secondary structure. These indels are very important synapomorphies, provide high support for an alternative taxonomic scheme and could help resolve the phylogeny of this order in conjunction with other phylogenetically informative characters.     

Ribosomal RNA secondary structure: compensatory mutations and implications for phylogenetic analysis

Using sequence data from the 28S ribosomal RNA (rRNA) genes of selected vertebrates, we investigated the effects that constraints imposed by secondary structure have on the phylogenetic analysis of rRNA sequence data. Our analysis indicates that characters from both base-pairing regions (stems) and non-base-pairing regions (loops) contain phylogenetic information, as judged by the level of support of the phylogenetic results compared with a well-established tree based on both morphological and molecular data. The best results (the greatest level of support of well-accepted nodes) were obtained when the complete data set was used. However, some previously supported nodes were resolved using either the stem or loop bases alone. Stem bases sustain a greater number of compensatory mutations than would be expected at random, but the number is < 40% of that expected under a hypothesis of perfect compensation to maintain secondary structure. Therefore, we suggest that in phylogenetic analyses, the weighting of stem characters be reduced by no more than 20%, relative to that of loop characters. In contrast to previous suggestions, we do not recommend weighting of stem positions by one-half, compared with that of loop positions, because this overcompensates for the constraints that selection imposes on the secondary structure of rRNA.      

16S～23S RDNA间区在链球菌和流感嗜血杆菌分类中的应用

利用16S~23S rDNA间区（intergenic spacer regions,ISR）在不同细菌中拷贝数、碱基排列、序列长度及所含tRNA基因种类和数目的差异,对15株链球菌和流感嗜血杆菌进行属、种、型和株系的分类鉴定。在16S rDNA的3′端和23S rDNA的5′端的保守区中合成引物,PCR扩增16S~23S rDNA ISR序列,对多态片段切胶纯化直接测序。在GenBank上查找对应细菌的ISR序列。用DNAMAN软件进行系统进化分析。链球菌属为单拷贝16S~23Sr RNA ISR、有一个tRNAAla基因编码区、分子大小在269~446bp之间,序列分成4个保守区和4个可变区,可变区碱基排列方式和数目的不同是种分类的依据。7株链球菌的同源率在78%~88%。同种异株的差异反映在碱基的插入和缺失上。流感嗜血杆菌各生物型均为2个拷贝的ISR,小片段为514~519bp,编码1个tRNAGlu基因,有3个狭窄可变区。大片段富含A T碱基,在I、II和IV型中分别是868、848和856bp,编码一个tRNAIle基因和一个tRNAAla基因。不同生物型小分子ISR与标准菌株比较,同源性在97.3%~99.6 %之间。 ISR作为细菌分类的目的基因具有属、种、型和株特异性与灵敏性。简单的基因分离分析技术为认识病原微生物提供了更多的机会。 Abstract:To facilitate species level identification of bacteria without the requirement of presumptive identification,the paper describes a rapid identification method of bacteria by amplification and direct sequencing 16S~23S rDNA intergenic spacer regions (ISR) of the pathogens which cause the upper respiratory tract infective disease by Streptococcus and Haemophilus.Three pairs of primer targeting conserved sequences flanking the 3′ end of 16S and the 5′end of 23S rRNA were used to amplify 16S~23S rRNA ISR of 7 streptococcus strains and 8 Haemophilus strains.The PCR products were separated by 1% agarose gel electrophoresis and the polymorphisms fragments were purified with the Wizard PCR Min-Prep Kit (Promega) and Protocol-SK131(Sangon).The nucleotide sequences of ISR inserts were determined by using the XEQTM DTCS Kit——Terminator Cycle Sequencing and a CEQTM 2000XL DNA Analysis system (Backman Coulter) automatic DAN sequencer.Then those sequences were compared with known seqnences on the GenBank.The alignment of nucleotide sequence,evolutionary distances and phylogenetic tress were analyzed by software DANMAN version 4.0.The PCR products were showed polymorphism patterns with agarose gel.One band was contained in streptococcus genus.The significant variation was found among the spacer sequences of different species in Streptococcus with the lengths of the spacer varying from 269 to 446bp.All the ISR of the streptococcal species had a tRNA Ala gene in the spacer and the sequence identities varied from 78 to 88% within genera.It was found that some spacer sequence blocks were highly conserved between operons of a genome,whereas the presence of others was variable,three regions showed significant spatial variation.Most of the differences between the sequences came from several bases insertions/deletions and substitutions.There are two major bands in the Haemophilus biotypes(515 and 884bp),the small ISR amplicon contained one tDNA coding for tRNAGlu.In contrast to the large one contained two tRNA genes coding for tRANAla and tRNAIle.Two regions of repeating motifs with only A or T were present in higher copy numbers between tRANAla and tRNAIle.The phylogenetic trees varied from 97.5 to 98.8%.The PCR and direct sequencing of 16S~23S rRAN ISR were successful in the pathogen species identification.     

A case study to estimate the applicability of secondary structures of SSU‐rRNA gene in taxonomy and phylogenetic analyses of ciliates

Phylogenetic studies of ciliates are mainly based on the primary structure information of the nuclear genes. Some regions of the small subunit ribosomal RNA (SSU‐rRNA) gene have distinctive secondary structures, which have demonstrated value as phylogenetic/taxonomic characters. In the current work, we predict the secondary structures of four variable regions (V2, V4, V7 and V9) in the SSU‐rRNA gene of 45 urostylids. Structure comparisons indicate that the V4 region is the most effective in revealing interspecific relationships, while the V9 region appears suitable at the family level or higher. The V2 region also offers some taxonomic information, but is too conserved to reflect phylogenetic relationships at the family or lower level, at least for urostylids. The V7 region is the least informative. We constructed several phylogenetic trees, based on the primary sequence alignment and based on an improved alignment according to the secondary structures. The results suggest that including secondary structure information in phylogenetic analyses provides additional insights into phylogenetic relationships. Using urostylid ciliates as an example, we show that secondary structure information results in a better understanding of their relationships, for example generic relationships within the family Pseudokeronopsidae.     

Characterization of Mitochondrial Small-Subunit Ribosomal RNAs from Holoparasitic Plants

Mitochondrial small-subunit (19S) rDNA sequences were obtained from 10 angiosperms to further characterize sequence divergence levels and structural variation in this molecule. These sequences were derived from seven holoparasitic (nonphotosynthetic) angiosperms as well as three photosynthetic plants. 19S rRNA is composed of a conservative core region (ca. 1450 nucleotides) as well as two variable regions (V1 and V7). In pairwise comparisons of photosynthetic angiosperms to Glycine, the core 19S rDNA sequences differed by less than 1.4%, thus supporting the observation that variation in mitochondrial rDNA is 3–4 times lower than seen in protein coding and rDNA genes of other subcellular organelles. Sequences representing four distinct lineages of nonasterid holoparasites showed significantly increased numbers of substitutions in their core 19S rDNA sequences (2.3–7.6%), thus paralleling previous findings that showed accelerated rates in nuclear (18S) and plastid (16S) rDNA from the same plants. Relative rate tests confirmed the accelerated nucleotide substitution rates in the holoparasites whereas rates in nonparasitic plants were not significantly increased. Among comparisons of both parasitic and nonparasitic plants, transversions outnumbered transitions, in many cases more than two to one. The core 19S rRNA is conserved in sequence and structure among all nonparasitic angiosperms whereas 19S rRNA from members of holoparasitic Balanophoraceae have unique extensions to the V5 and V6 variable domains. Substitution and insertion/deletion mutations characterized the V1 and V7 regions of the nonasterid holoparasites. The V7 sequence of one holoparasite (Scybalium) contained repeat motifs. The cause of substitution rate increases in the holoparasites does not appear to be a result of RNA editing, hence the underlying molecular mechanism remains to be fully documented. Received: 18 May 1997 / Accepted: 11 July 1997