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     

The origin and evolution of variable-region helices in V4 and V7 of the small-subunit ribosomal RNA of branchiopod crustaceans

We sequenced the V4 and V7 regions of the small-subunit ribosomal RNA (SSU rRNA) from 38 species of branchiopod crustaceans (e.g., Artemia, Daphnia, Triops) representing all eight extant orders. Ancestral large-bodied taxa in the orders Anostraca, Notostraca, Laevicaudata, and Spinicaudata (limnadiids and cyzicids) possess the typical secondary structure in these regions, whereas the spinicaudatan Cyclestheria and all of the cladocerans (Anomopoda, Ctenopoda, Onychopoda, and Haplopoda) possess three unique helices. Although the lengths and primary sequences of the distal ends of these helices are extremely variable, their locations, secondary structures, and primary sequences at the proximal end are conserved, indicating that they are homologous. This evidence supports the classical view that Cladocera is a monophyletic group and that the cyclestheriids are transitional between spinicaudatans and cladocerans. The single origin and persistence since the Permian of the unique cladoceran helices suggests that births and deaths of variable region helices have been rare. The broad range of sequence divergences observed among the cladoceran helices permitted us to make inferences about their evolution. Although their proximal ends are very GC-biased, there is a significant negative correlation between length and GC content due to an increasing proportion of U at their distal ends. Slippage-like processes occurring at unpaired nucleotides or bulges, which are very U-biased, are associated with both helix origin and runaway length expansion. The overall GC contents and lengths of V4 and V7 are highly correlated. More surprisingly, the lengths of these SSU rRNA variable regions are also highly correlated with the length of the large-subunit rRNA expansion segment, D2, indicating that mechanisms affecting length variation do so both across single genes and across genes in the rRNA gene family.     

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     

Two Theileria cervi SSU RRNA gene sequence types found in isolates from white-tailed deer and elk in North America.

Two Theileria cervi SSU rRNA gene sequence Types, F and G, from white-tailed deer (Odocoileus virginianus) and elk (Cervus elaphus canadensis) isolates in North America were confirmed. Previously, nucleotide sequencing through a single variable (V4) region showed the presence of SSU rRNA gene Types F and G in T. cervi isolates from white-tailed deer and an elk. In this study, both sequence types were found in four T. cervi isolates (two from deer and two from elk). Microheterogeneity only appeared in the Type G gene, resulting in Subtypes G1, G2 and G3. Subtype G1 was found in two elk and one white-tailed deer T. cervi isolate; Subtypes G2 and G3 were found in a white-tailed deer T. cervi isolate. The Type F SSU rRNA genes were identical in nucleotide sequence in both elk and white-tailed deer T. cervi isolates. The high degree of conservation in the Type F variable regions may be exploited to design specific oligonucleotide primers for parasite detection by the polymerase chain reaction in cervine or tick hosts.     

The secondary structure of the unusually long 18S ribosomal RNA of the myxozoan Sphaerospora truttae and structural evolutionary trends in the Myxozoa

The nearly complete 18S rRNA sequence of the myxozoan parasite Sphaerospora truttae shows an extraordinary length (2,552bp) in comparison with other myxozoans and with metazoans in general (average 1,800-1,900bp). The sequence shows nucleotide insertions in most variable regions of the 18S rRNA (V2, V4, V5 and V7), with especially large expansion segments in V4 and V7. In the myxozoans, nucleotide insertions and specific secondary structures in these regions of the gene were found to be strongly related to large scale phylogenetic clustering and thus with the invertebrate host type. Whereas expansion segments were generally found to be absent in the malacasporeans and the clade of primary marine myxozoan species, they occur in all taxa of the clade containing freshwater species, where they showed a consistent secondary structure throughout. The longest expansion segments occur in S. truttae, Sphaerospora elegans and Leptotheca ranae, which represent a clade that has emerged after the malacosporeans and before the radiation of all other myxozoan genera. These three species demonstrate structural links to the malacosporeans as well as other unique features. A smaller number of nucleotide insertions in different subhelices and specific secondary structures appear to have evolved independently in two marine genera, i.e. Ceratomyxa and Parvicapsula. The secondary structural elements of V4 and V7 of the myxozoan 18S rRNAs were found to be highly informative and revealed evolutionary trends of various regions of the gene hitherto unknown, since previous analyses have been based on primary sequence data excluding these regions. Furthermore, the unique features of the V4 region in S. truttae allowed for the design of a highly specific PCR assay for this species.     

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.     

Sequence Diversity in the 16S–23S Intergenic Spacer Region (ISR) of the rRNA Operons in Representatives of the Escherichia coli ECOR Collection

The ribosomal RNA multigene family in Escherichia coli comprises seven rrn operons of similar, but not identical, sequence. Four operons (rrnC, B, G, and E) contain genes in the 16S–23S intergenic spacer region (ISR) for tRNA^Glu-2 and three (rrnA, D, and H) contain genes for tRNA^Ile-1 and tRNA^Ala-1B. To increase our understanding of their molecular evolution, we have determined the ISR sequence of the seven operons in a set of 12 strains from the ECOR collection. Each operon was specifically amplified using polymerase chain reaction primers designed from genes or open reading frames located upstream of the 16S rRNA genes in E. coli K12. With a single exception (ECOR 40), ISRs containing one or two tRNA genes were found at the same respective loci as those of strain K12. Intercistronic heterogeneity already found in K12 was representative of most variation among the strains studied and the location of polymorphic sites was the same. Dispersed nucleotide substitutions were very few but 21 variable sites were found grouped in a stem-loop, although the secondary structure was conserved. Some regions were found in which a stretch of nucleotides was substituted in block by one alternative, apparently unrelated, sequence (as illustrated by the known putative insertion of rsl in K12). Except for substitutions of different sizes and insertions/deletions found in the ISR, the pattern of nucleotide variation is very similar to that found for the 16S rRNA gene in E. coli. Strains K12 and ECOR 40 showed the highest intercistronic heterogeneity. Most strains showed a strong tendency to homogenization. Concerted evolution could explain the notorious conservation of this region that is supposed to have low functional restrictions. Received: 31 July 1997 / Accepted: 17 October 1997     

Putative secondary structures of unusually long strepsipteran SSU rRNAs and its phylogenetic implications.

We constructed the putative secondary structures of the small subunit rRNAs (SSU rRNA) from three strepsipteran insects. The primary sequences of the strepsipteran SSU rRNAs are unusually long due to unique and long insertions. In spite of these insertions, the basic shapes of their secondary structures are well maintained as shown in those of other eukaryotes, because these insertions appear mainly in the variable regions. The secondary structures for the V1, V3, V5, V8, and V9 regions are well conserved, even though the primary structures of V1, V5, and V8 regions are quite variable. However, the predicted secondary structures for the V2, V4, and V7 regions are quite different from those of other insects. In the V4 and V7 regions, helices specific to the Strepsiptera exist. These helices have not been reported in other organisms so far. Similarly, four eukaryotic specific helices (E8-1, E10-2, E23-4 and E45-1) not reported in insects exist in the V2, V4, and V8 regions. These helices are formed by the inserted sequences. The secondary structures of the expanded segments of the strepsipteran SSU rRNA were applied to infer the phylogenetic position of Strepsiptera, one of the most enigmatic problems in insect phylogeny. Only the secondary structure of the V7 region showed the weak Strepsiptera/Diptera sister-group relationship.     

Primary and Secondary Structure of the Small-Subunit Ribosomal RNA of the Naked, Marine Amoeba Vannella anglica: Phylogenetic Implications

The primary and secondary structure of the small-subunit ribosomal RNA (ssrRNA) gene from the naked, marine amoeba, Vannella anglica (subclass Gymnamoebia), was determined. The ssrRNA is 1962 nucleotides in length, with a low G+C content of 37.1%. The ssrRNA is composed of several uncommon secondary structure features including helix E8-1, which may be a useful target for rRNA probes for the direct identification of isolates in mixed culture. Phylogenetic analysis of sequence data showed that V. anglica branched prior to the rapid diversification of the eukaryotes. It did not associate with the other naked, lobose amoebae represented by Acanthamoeba and Hartmannella, indicating that Vannella represents a separate amoeboid lineage and the subclass Gymnamoebia is polyphyletic. Received: 9 July 1998 / Accepted: 16 November 1998     

Limitations of Metazoan 18S rRNA Sequence Data: Implications for Reconstructing a Phylogeny of the Animal Kingdom and Inferring the Reality of the Cambrian Explosion

We document the phylogenetic behavior of the 18S rRNA molecule in 67 taxa from 28 metazoan phyla and assess the effects of among-site rate variation on reconstructing phylogenies of the animal kingdom. This empirical assessment was undertaken to clarify further the limits of resolution of the 18S rRNA gene as a phylogenetic marker and to address the question of whether 18S rRNA phylogenies can be used as a source of evidence to infer the reality of a Cambrian explosion. A notable degree of among-site rate variation exists between different regions of the 18S rRNA molecule, as well as within all classes of secondary structure. There is a significant negative correlation between inferred number of nucleotide substitutions and phylogenetic information, as well as with the degree of substitutional saturation within the molecule. Base compositional differences both within and between taxa exist and, in certain lineages, may be associated with long branches and phylogenetic position. Importantly, excluding sites with different degrees of nucleotide substitution significantly influences the topology and degree of resolution of maximum-parsimony phylogenies as well as neighbor-joining phylogenies (corrected and uncorrected for among-site rate variation) reconstructed at the metazoan scale. Together, these data indicate that the 18S rRNA molecule is an unsuitable candidate for reconstructing the evolutionary history of all metazoan phyla, and that the polytomies, i.e., unresolved nodes within 18S rRNA phylogenies, cannot be used as a single or reliable source of evidence to support the hypothesis of a Cambrian explosion. Received: 9 December 1997 / Accepted: 23 March 1998     

The Mitochondrial Genome of Chlorogonium elongatum Inferred from the Complete Sequence

The 22,704-bp circular mitochondrial DNA (mtDNA) of the chlamydomonad alga Chlorogonium elongatum was completely cloned and sequenced. The genome encodes seven proteins of the respiratory electron transport chain, subunit 1 of the cytochrome oxidase complex (cox1), apocytochrome b (cob), five subunits of the NADH dehydrogenase complex (nad1, nad2, nad4, nad5, and nad6), a set of three tRNAs (Q, W, M), and the large (LSU)- and small (SSU)-subunit ribosomal RNAs. Six group-I introns were found, two each in the cox1, cob, and nad5 genes. In each intron an open reading frame (ORF) related to maturases or endonucleases was identified. Both the LSU and the SSU rRNA genes are split into fragments intermingled with each other and with other genes. Although the average A + T content is 62.2%, GC-rich clusters were detected in intergenic regions, in variable domains of the rRNA genes, and in introns and intron-encoded ORFs. A comparison of the genome maps reveals that C. elongatum and Chlamydomonas eugametos mtDNAs are more closely related to one another than either is to Chlamydomonas reinhardtii mtDNA. Received: 3 November 1997 / Accepted: 12 January 1998     

Gene Conversion Drives Within Genic Sequences: Concerted Evolution of Ribosomal RNA Genes in Bacteria and Archaea

Multiple copies of a given ribosomal RNA gene family undergo concerted evolution such that sequences of all gene copies are virtually identical within a species although they diverge normally between species. In eukaryotes, gene conversion and unequal crossing over are the proposed mechanisms for concerted evolution of tandemly repeated sequences, whereas dispersed genes are homogenized by gene conversion. However, the homogenization mechanisms for multiple-copy, normally dispersed, prokaryotic rRNA genes are not well understood. Here we compared the sequences of multiple paralogous rRNA genes within a genome in 12 prokaryotic organisms that have multiple copies of the rRNA genes. Within a genome, putative sequence conversion tracts were found throughout the entire length of each individual rRNA genes and their immediate flanks. Individual conversion events convert only a short sequence tract, and the conversion partners can be any paralogous genes within the genome. Interestingly, the genic sequences undergo much slower divergence than their flanking sequences. Moreover, genomic context and operon organization do not affect rRNA gene homogenization. Thus, gene conversion underlies concerted evolution of bacterial rRNA genes, which normally occurs within genic sequences, and homogenization of flanking regions may result from co-conversion with the genic sequence. Received: 31 March 2000 / Accepted: 15 June 2000     

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.     

Algae or Protozoa: Phylogenetic Position of Euglenophytes and Dinoflagellates as Inferred from Mitochondrial Sequences

The chloroplasts of euglenophytes and dinoflagellates have been suggested to be the vestiges of endosymbiotic algae acquired during the process of evolution. However, the evolutionary positions of these organisms are still inconclusive, and they have been tentatively classified as both algae and protozoa. A representative gene of the mitochondrial genome, cytochrome oxidase subunit I (coxI), was chosen and sequenced to clarify the phylogenetic positions of four dinoflagellates, two euglenophytes and one apicomplexan protist. This is the first report of mitochondrial DNA sequences for dinoflagellates and euglenophytes. Our COXI tree shows clearly that dinoflagellates are closely linked to apicomplexan parasites but not with algae. Euglenophytes and algae appear to be only remotely related, with euglenophytes sharing a possible evolutionary link with kinetoplastids. The COXI tree is in general agreement with the tree based on the nuclear encoded small subunit of ribosomal RNA (SSU rRNA) genes, but conflicts with that based on plastid genes. These results support the interpretation that chloroplasts present in euglenophytes and dinoflagellates were captured from algae through endosymbioses, while their mitochondria were inherited from the host cell. We suggest that dinoflagellates and euglenophytes were originally heterotrophic protists and that their chloroplasts are remnants of endosymbiotic algae. Received: 24 March 1997 / Accepted: 21 April 1997     

Characteristics of Nucleotide Substitution in the Hepatitis C Virus Genome: Constraints on Sequence Change in Coding Regions at Both Ends of the Genome

Comparison of complete genome sequences for different variants of hepatitis C virus (HCV) reveals several different constraints on sequence change. Synonymous changes are suppressed in coding regions at both 5′ and 3′ ends of the genome. No evidence was found for the existence of alternative reading frames or for a lower mutation frequency in these regions. Instead, suppression may be due to constraints imposed by RNA secondary structures identified within the core and NS5b genes. Nonsynonymous substitutions are less frequent than synonymous ones except in the hypervariable region of E2 and, to a lesser extent, in E1, NS2, and NS5b. Transitions are more frequent than transversions, particularly at the third position of codons where the bias is 16:1. In addition, nucleotide substitutions may not occur symmetrically since there is a bias toward G or C at the third position of codons, while T ↔ C transitions were twice as frequent as A ↔ G transitions. These different biases do not affect the phylogenetic analysis of HCV variants but need to be taken into account in interpreting sequence change in longitudinal studies. Received: 9 September 1996 / Accepted: 20 April 1997     

Complete Mitochondrial DNA Sequence of Conger myriaster (Teleostei: Anguilliformes): Novel Gene Order for Vertebrate Mitochondrial Genomes and the Phylogenetic Implications for Anguilliform Families

The complete nucleotide sequence of the mitochondrial genome was determined for a conger eel, Conger myriaster (Elopomorpha: Anguilliformes), using a PCR-based approach that employs a long PCR technique and many fish-versatile primers. Although the genome [18,705 base pairs (bp)] contained the same set of 37 mitochondrial genes [two ribosomal RNA (rRNA), 22 transfer RNA (tRNA), and 13 protein-coding genes] as found in other vertebrates, the gene order differed from that recorded for any other vertebrates. In typical vertebrates, the ND6, tRNA^Glu, and tRNA^Pro genes are located between the ND5 gene and the control region, whereas the former three genes, in C. myriaster, have been translocated to a position between the control region and the tRNA^Phe gene that are contiguously located at the 5′ end of the 12S rRNA gene in typical vertebrates. This gene order is similar to the recently reported gene order in four lineages of birds in that the latter lack the ND6, tRNA^Glu, and tRNA^Pro genes between the ND5 gene and the control region; however, the relative position of the tRNA^Pro to the ND6–tRNA^Glu genes in C. myriaster was different from that in the four birds, which presumably resulted from different patterns of tandem duplication of gene regions followed by gene deletions in two distantly related groups of organisms. Sequencing of the ND5–cyt b region in 11 other anguilliform species, representing 11 families, plus one outgroup species, revealed that the same gene order as C. myriaster was shared by another 4 families, belonging to the suborder Congroidei. Although the novel gene orders of four lineages of birds were indicated to have multiple independent origins, phylogenetic analyses using nucleotide sequences from the mitochondrial 12S rRNA and cyt b genes suggested that the novel gene orders of the five anguilliform families had originated in a single ancestral species. Received: 13 July 2000 / Accepted: 30 November 2000     

Structural constraints in expansion segments from a midge 26S rDNA

DNA sequences representing approximately 40% of the large-subunit rRNA gene from the lower dipteran Chironomus thummi were analyzed. Once aligned with their Drosophila counterparts, sequence and base content comparisons were carried out. Sequence identity was found to be high overall, except for six regions that displayed a local bias in nucleotide composition toward AT. These regions were identified as expansion segments D3, D4, D5, D6, D7a, and D12. Besides base sequence divergence, differences in length were observed between the respective variable domains of the two species, particularly for D7a. Prediction of secondary structure showed that the folding of the Chironomus expansion segments analyzed is in agreement with the general patterns proposed for eukaryotic LSU rRNA. The comparison with Drosophila revealed also that the Chironomus secondary structures of the variable domains are supported by multiple compensatory substitutions or even compensatory insertions. Chironomus D7a displayed an unusual structural feature with respect to the insect D7a models that have been inferred up to now. The structural constraint observed in the expansion segments of Diptera so distantly related as midges and Drosophila suggests that these regions contribute to some functional role. Concerning the D7a of insects so far analyzed, there can be, in addition to a conserved secondary structure, a nucleotide composition constraint that might be important for the process giving rise to the alpha and beta halves of the 26S rRNA. Correspondence to: E. Gorab     

The rate of mitochondrial 12S rRNA gene evolution is similar in freshwater turtles and marsupials

Assertions that the ``conventional' rate of mitochondrial DNA (mtDNA) evolution is reduced in poikilotherms in general and turtles in particular were tested for side-necked turtles (Pleurodira: Chelidae). Homologous data sets of mitochondrial 12S rRNA gene sequences were used to compare the average divergence between the Australian and South American species for two Gondwanan groups: the chelid turtles and the marsupials. The mean nucleotide divergences between continental groups for both the turtles and the marsupials are remarkably similar. These data suggest that the rate of evolution of mitochondrial 12S rRNA gene is not substantially slower in turtles than in the homeothermic marsupials. Received: 24 February 1997 / Accepted: 30 June 1997