The performance of several multiple-sequence alignment programs in relation to secondary-structure features for an rRNA sequence

The performances of five global multiple-sequence alignment programs (CLUSTAL W, Divide and Conquer, Malign, PileUp, and TreeAlign) were evaluated using part of the animal mitochondrial small subunit (12S) rRNA molecule. Conserved sequence motifs derived from an alignment based on secondary structural information were used to score how well each program aligned a data set of five vertebrate and five invertebrate taxa over a range of parameter values. All of the programs could align the motifs with reasonable accuracy for at least one set of parameter conditions, although if the whole sequence was considered, similarity to the structural alignment was only 25%-34%. Use of small gap costs generally gave more accurate results, although Malign and TreeAlign generated longer alignments when gap costs were low. The programs differed in the consistency of the alignments when gap cost was varied; CLUSTAL W, Divide and Conquer, and TreeAlign were the most accurate and robust, while PileUp performed poorly as gap cost values increased, and the accuracy of Malign fluctuated. Default settings for the programs did not give the best results, and attempting to select similar parameter values in different programs did not always result in more similar alignments. Poor alignment of even well-conserved motifs can occur if these are near sites with insertions or deletions. Since there is no a priori way to determine gap costs and because such costs can vary over the gene, alignment of rRNA sequences, particularly the less well conserved regions, should be treated carefully and aided by secondary structure and conserved motifs. Some motifs are single bases and so are often invisible to alignment programs. Our tests involved the most conserved regions of the 12S rRNA gene, and alignment of less well conserved regions will be more problematical. None of the alignments we examined produced a fully resolved phylogeny for the data set, indicating that this portion of 12S rRNA is insufficient for resolution of distant evolutionary relationships.     

Structural and functional diversity of lectin repertoires in invertebrates, protochordates and ectothermic vertebrates

During the past few years, substantial progress has been accomplished in the elucidation of the structural diversity of the lectin repertoires of invertebrates, protochordates and ectothermic vertebrates, providing particularly valuable information on those groups that constitute the invertebrate/vertebrate 'boundary'. Although representatives of lectin families typical of mammals, such as C-type lectins, galectins and pentraxins, have been described in these taxa, the detailed study of selected model species has yielded either novel variants of the structures described for the mammalian lectin representatives or novel lectin families with unique sequence motifs, multidomain arrangements and a new structural fold. Along with the high structural diversity of the lectin repertoires in these taxa, a wide spectrum of biological roles is starting to emerge, underscoring the value of invertebrate and lower vertebrate models for gaining insight into structural, functional and evolutionary aspects of lectins.     

Phylogenetic analysis of molluscan mitochondrial LSU rDNA sequences and secondary structures

Mollusks are an extraordinarily diverse group of animals with an estimated 200,000 species, second only to the phylum Arthropoda. We conducted a comparative analysis of complete mitochondrial ribosomal large subunit sequences (LSU) of a chiton, two bivalves, six gastropods, and a cephalopod. In addition, we determined secondary structure models for each of them. Comparative analyses of nucleotide variation revealed substantial length variation among the taxa, with stylommatophoran gastropods possessing the shortest lengths. Phylogenetic analyses of the nucleotide sequence data supported the monophyly of Albinaria, Euhadra herklotsi + Cepaea nemoralis, Stylommatophora, Cerithioidea, and when only transversions are included, the Bivalvia. The phylogenetic limits of the mitochondrial LSU rRNA gene within mollusks appear to be up to 400 million years, although this estimate will have to be tested further with additional taxa. Our most novel finding was the discovery of phylogenetic signal in the secondary structure of rRNA of mollusks. The absence of entire stem/loop structures in Domains II, III, and V can be viewed as three shared derived characters uniting the stylommatophoran gastropods. The absence of the aforementioned stem/loop structure explains much of the observed length variation of the mitochondrial LSU rRNA found within mollusks. The distribution of these unique secondary structure characters within mollusks should be examined.     

Invertebrate intracellular fatty acid binding proteins

Fatty acid binding proteins are multigenic cytosolic proteins largely distributed along the zoological scale. Their overall identity at primary and tertiary structure is conserved. They are involved in the uptake and transport of hydrophobic ligands to different cellular fates. The precise functions of each FABP type remain imperfectly understood, since sub-specialization of functions is suggested. Evolutionary studies have distinguished major subfamilies that could have been derived from a common ancestor close to vertebrate/invertebrate split. Since the isolation of the first invertebrate FABP from Schistocerca gregaria in 1990, the number of FABPs isolated from invertebrates has been increasing. Differences at the sequence level are appreciable and relationships with vertebrate FABPs are not clear, and lesser among invertebrate proteins, introducing some uncertainty to infer functional relatedness and phylogenetic relationships. The objective of this review is to summarize the information available on invertebrate FABPs to elucidate their mutual relationships, the relationship with their vertebrate counterparts and putative functions. Structure, gene structure, putative functions, expression studies and phylogenetic relationships with vertebrate counterparts are analyzed. Previous suggestions of the ancestral position concerning the heart-type of FABPs are reinforced by evidence from invertebrate models.     

Characterization and Evolution of the Mitochondrial DNA Control Region in Hornbills (Bucerotiformes)

We determined the mitochondrial DNA control region sequences of six Bucerotiformes. Hornbills have the typical avian gene order and their control region is similar to other avian control regions in that it is partitioned into three domains: two variable domains that flank a central conserved domain. Two characteristics of the hornbill control region sequence differ from that of other birds. First, domain I is AT rich as opposed to AC rich, and second, the control region is approximately 500 bp longer than that of other birds. Both these deviations from typical avian control region sequence are explainable on the basis of repeat motifs in domain I of the hornbill control region. The repeat motifs probably originated from a duplication of CSB-1 as has been determined in chicken, quail, and snowgoose. Furthermore, the hornbill repeat motifs probably arose before the divergence of hornbills from each other but after the divergence of hornbills from other avian taxa. The mitochondrial control region of hornbills is suitable for both phylogenetic and population studies, with domains I and II probably more suited to population and phylogenetic analyses, respectively.     

Application of the secondary structure model of rRNA for phylogeny: D2-D3 expansion segments of the LSU gene of plant-parasitic nematodes from the family Hoplolaimidae Filipjev, 1934

Knowledge of rRNA structure is increasingly important to assist phylogenetic analysis through reconstructing optimal alignment, utilizing molecule features as an additional source of data and refining appropriate models of evolution of the molecule. We describe a procedure of optimization for alignment and a new coding method for nucleotide sequence data using secondary structure models of the D2 and D3 expansion fragments of the LSU-rRNA gene reconstructed for fifteen nematode species of the agriculturally important and diverse family Hoplolaimidae, order Tylenchida. Using secondary structure information we converted the original sequence data into twenty-eight symbol codes and submitted the transformed data to maximum parsimony analysis. We also applied the original sequence data set for Bayesian inference. This used the doublet model with sixteen states of nucleotide doublets for the stem region and the standard model of DNA substitution with four nucleotide states for loops and bulges. By this approach, we demonstrate that using structural information for phylogenetic analyses led to trees with lower resolved relationships between clades and likely eliminated some artefactual support for misinterpreted relationships, such as paraphyly of Helicotylenchus or Rotylenchus. This study as well as future phylogenetic analyses is herein supported by the development of an on-line database, NEMrRNA, for rRNA molecules in a structural format for nematodes. We also have developed a new computer program, RNAstat, for calculation of nucleotide statistics designed and proposed for phylogenetic studies.     

Evolution of the cetacean mitochondrial D-loop region.

We sequenced the mitochondrial DNA D-loop regions from two cetacean species and compared these with the published D-loop sequences of several other mammalian species, including one other cetacean. Nucleotide substitution rates, DNA sequence simplicity, possible open reading frames (ORFs), and potential RNA secondary structure were investigated. The substitution rate is an order of magnitude lower than would be expected on the basis of reports on human sequence variation in this region but are consistent with interspecific primate and rodent D-loop sequence variation and with estimates of substitution rates from whole mitochondrial genomes. Deletions/insertions are less common in the cetacean D-loop than in other vertebrate species. Areas of high sequence simplicity (clusters of short repetitive motifs) across the region correspond to areas of high sequence divergence. Three regions predicted to form secondary structures are homologous to such putative structures in other species; however, the presumptive structures most conserved in cetaceans are different from those reported for other taxa. While all three species have possible long ORFs, only a short sequence of seven amino acids is shared with other mammalian species, and those changes that had occurred within it are all nonsynonymous. We conclude that DNA slippage, in addition to point mutation, contributes to the evolution of the D-loop and that regions of conserved secondary structure in cetaceans and an ORF are unlikely to contribute significantly to the conservation of the central region.     

Evaluation of the internal transcribed spacer 2 (ITS2) as a molecular marker for phylogenetic inference using sequence and secondary structure information in blow flies (Diptera: Calliphoridae)

The internal transcribed spacer 2 (ITS2) is a small non-coding region located inside the nuclear ribosomal DNA cluster. ITS2 sequence variability is thought to be appropriate to differentiate species and for phylogenetic reconstructions analyses, which can be further improved if structural information is considered. We evaluated the potential of ITS2 as a molecular marker for phylogenetic inference in Calliphoridae (Diptera: Brachycera) using a broad range of inference methods and different substitution models, accounting or not for structural information. Sequence analyses revealed a hierarchically organized pattern of sequence variation and a small level of nucleotide substitution saturation. Intragenomic variation due to small sequence repeats was found mainly in the most variable domain (IV), but it has no significant impact on the phylogenetic signal at the species level. Inferred secondary structures revealed that GC pairs are more frequently found flanking bulges and loops regions in more conserved domains, thus ensuring structure stability. In the phylogenetic analyses, the use of substitution models accounting for structural information significantly improves phylogenetic inference in both neighbour-joining and Bayesian analyses, although the former provides limited resolution for dealing with highly divergent sequences. For Bayesian analyses, a significant improvement in likelihood was observed when considering structure information, although with small changes in topology and overall support, probably reflecting better evolutionary rates estimates. Based on these findings, ITS2 is a suitable molecular marker for phylogenetic analyses in Calliphoridae, at both species and generic level.     

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.     

The crocodilian mitochondrial control region: general structure,conserved sequences,and evolutionary implications

We present the first comprehensive analysis of the crocodilian control region. We have analyzed sequences from all three families of Crocodylia (Crocodylidae, Gavialidae, Alligatoridae), incorporating all genera except Paleosuchus and Melanosuchus. Within the control region of other vertebrates, several sequence motifs and their order appear to be conserved. Herein, we compare aligned crocodilian D-loop sequences to homologous sequences from other vertebrates ranging from fish to birds. Among other findings, we have discovered that while domain I tends to be shorter than the same region in mammals and birds, it contains sequences similar in structure to both the goose-hairpin and termination associated sequences (TAS). Domain II is highly conservative with regard to size among the taxa examined and contains several of the conserved sequence boxes characterized in other vertebrates. Domain III contains several interesting sequence motifs including tandemly repeated sequences, a long poly-A region in the Crocodylidae, and possible bidirection promoter sequences.     

Monophyly of clade III nematodes is not supported by phylogenetic analysis of complete mitochondrial genome sequences

Background

The orders Ascaridida, Oxyurida, and Spirurida represent major components of zooparasitic nematode diversity, including many species of veterinary and medical importance. Phylum-wide nematode phylogenetic hypotheses have mainly been based on nuclear rDNA sequences, but more recently complete mitochondrial (mtDNA) gene sequences have provided another source of molecular information to evaluate relationships. Although there is much agreement between nuclear rDNA and mtDNA phylogenies, relationships among certain major clades are different. In this study we report that mtDNA sequences do not support the monophyly of Ascaridida, Oxyurida and Spirurida (clade III) in contrast to results for nuclear rDNA. Results from mtDNA genomes show promise as an additional independently evolving genome for developing phylogenetic hypotheses for nematodes, although substantially increased taxon sampling is needed for enhanced comparative value with nuclear rDNA. Ultimately, topological incongruence (and congruence) between nuclear rDNA and mtDNA phylogenetic hypotheses will need to be tested relative to additional independent loci that provide appropriate levels of resolution.

Results

For this comparative phylogenetic study, we determined the complete mitochondrial genome sequences of three nematode species, Cucullanus robustus (13,972 bp) representing Ascaridida, Wellcomia siamensis (14,128 bp) representing Oxyurida, and Heliconema longissimum (13,610 bp) representing Spirurida. These new sequences were used along with 33 published nematode mitochondrial genomes to investigate phylogenetic relationships among chromadorean orders. Phylogenetic analyses of both nucleotide and amino acid sequence datasets support the hypothesis that Ascaridida is nested within Rhabditida. The position of Oxyurida within Chromadorea varies among analyses; in most analyses this order is sister to the Ascaridida plus Rhabditida clade, with representative Spirurida forming a distinct clade, however, in one case Oxyurida is sister to Spirurida. Ascaridida, Oxyurida, and Spirurida (the sampled clade III taxa) do not form a monophyletic group based on complete mitochondrial DNA sequences. Tree topology tests revealed that constraining clade III taxa to be monophyletic, given the mtDNA datasets analyzed, was a significantly worse result.

Conclusion

The phylogenetic hypotheses from comparative analysis of the complete mitochondrial genome data (analysis of nucleotide and amino acid datasets, and nucleotide data excluding 3^rd positions) indicates that nematodes representing Ascaridida, Oxyurida and Spirurida do not share an exclusive most recent common ancestor, in contrast to published results based on nuclear ribosomal DNA. Overall, mtDNA genome data provides reliable support for nematode relationships that often corroborates findings based on nuclear rDNA. It is anticipated that additional taxonomic sampling will provide a wealth of information on mitochondrial genome evolution and sequence data for developing phylogenetic hypotheses for the phylum Nematoda.

     

Complete mitochondrial genome of the miiuy croaker Miichthys miiuy (Perciformes, Sciaenidae) with phylogenetic consideration

The complete sequence of the 16,493 nucleotide mitochondrial genome from the single species of the family Sciaenidae, the miiuy croaker, Miichthys miiuy, was determined. The nucleotide sequences of M. miiuy mitochondrial DNA have been compared with those of three other Sciaenidae fishes. The contents of the M. miiuy mitochondrial genome are 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes, and two non-coding regions (L-strand replication origin and control region), the gene order of which is identical to that observed in most vertebrates. The L-strand replication origin of M. miiuy is not pyrimidine-rich compared to those of most bony fishes. Within the control region, we identified the extended termination associated sequence domain, the central conserved sequence block domain and the conserved sequence block domain, while the typical central conserved blocks CSB-D, -E and -F could not be detected in the three other Sciaenidae species. In the ML phylogenetic analyses, the monophyly of Pseudosciaeniae was not supported, which is against with the morphological results. Collichthys niveatus is most closely related to Larimichthys polyactis, and Collichthys and Larimichthys may be merged into one genus, based on the current datasets.     

Secondary structure models of the nuclear internal transcribed spacer regions and 5.8S rRNA in Calciodinelloideae (Peridiniaceae) and other dinoflagellates

Secondary structure models of the 5.8S rRNA and both internal transcribed spacers (ITS1 and ITS2) are proposed for Calciodinelloideae (Peridiniaceae) and are also plausible for other dinoflagellates. The secondary structure of the 5.8S rRNA corresponds to previously developed models, with two internal paired regions and at least one 5.8S rRNA–28S rRNA interaction. A general secondary structure model of ITS1 for Calciodinelloideae (and other dinoflagellates), consisting of an open multibranch loop with three major helices, is proposed. The homology of these paired regions with those found in other taxa, published in previous studies (e.g. yeast, green algae and Platyhelmithes) remains to be determined. Finally, a general secondary structure model of ITS2 for Calciodinelloideae (and other dinoflagellates) is reconstructed. Based on the 5.8S rRNA–28S rRNA interaction, it consists of a closed multibranch loop, with four major helices. At least helix III and IV have homology with paired regions found in other eukaryotic taxa (e.g. yeast, green algae and vertebrates). Since the secondary structures of both ITS regions are more conserved than the nucleotide sequences, their analysis helps in understanding molecular evolution and increases the number of structural characters. Thus, the structure models developed in this study may be generally useful for future phylogenetic analyses.     

Molecular characterization of PAB2, a member of the multigene family coding for poly(A)-binding proteins in Arabidopsis thaliana.

The poly(A) tail of eukaryotic mRNAs associates with poly(A)-binding (PAB) proteins whose role in mRNA translation and stability is being intensively investigated. Very little is known about the structure and function of the PAB genes in plants. We have cloned multiple PAB-related sequences from Arabidopsis thaliana. Results suggest that PAB proteins are encoded by a multigene family. One member of this family (PAB2) is expressed in root and shoot tissues. The complete nucleotide sequence of PAB2 was determined. Study of the predicted PAB2 protein reveals a similarity in structure among vertebrate, insect, yeast, and plant PAB proteins. All contain two highly conserved domains: an amino-terminal sequence formed by four RNA recognition motifs and an uncharacterized carboxyl-terminal region of 69 to 71 amino acids. Possible roles for the carboxyl-terminal conserved domain are discussed in view of recently published data concerning the structure and function of PAB proteins.     

A common ancestor DNA motif for invertebrate and vertebrate hormone response elements.

The ecdysone-responsive DNA sequence of the Drosophila hsp27 gene promoter contains four direct and inverted repeats reminiscent of those that compose the vertebrate palindromic estrogen response element (ERE) and the thyroid hormone/retinoic acid response element (TRE/RRE). Interestingly, a 3 bp substitution in the wild-type Hsp27 ecdysone response element (EcdRE) increases both its similarity with the vertebrate ERE and TRE/RRE and its capacity to confer ecdysone responsiveness to a heterologous promoter. Remarkably, increasing the spacing between the inverted repeats of this strong EcdRE by two nucleotides converts it into an ERE. Inversely, decreasing the spacing between the two inverted repeats of the vertebrate consensus palindromic ERE, from three to one nucleotide, converts it into a functional EcdRE. Thus, the only difference between an invertebrate EcdRE and a vertebrate palindromic ERE or TRE/RRE is in the spacing between the conserved inverted repeated motifs forming these palindromic HREs. The finding that the sequence motif 5'-GGTCA-3' present in the vertebrate ERE and TRE/RRE is also a functionally important characteristic of an invertebrate HRE, suggests that a common ancestor regulatory DNA sequence gave rise to all HREs known so far. We discuss the possibility that this progenitor motif is the GGTCA sequence.     

Evolutionary origin of peptidoglycan recognition proteins in vertebrate innate immune system

Background  

Innate immunity is the ancient defense system of multicellular organisms against microbial infection. The basis of this first line of defense resides in the recognition of unique motifs conserved in microorganisms, and absent in the host. Peptidoglycans, structural components of bacterial cell walls, are recognized by Peptidoglycan Recognition Proteins (PGRPs). PGRPs are present in both vertebrates and invertebrates. Although some evidence for similarities and differences in function and structure between them has been found, their evolutionary history and phylogenetic relationship have remained unclear. Such studies have been severely hampered by the great extent of sequence divergence among vertebrate and invertebrate PGRPs. Here we investigate the birth and death processes of PGRPs to elucidate their origin and diversity.     

Evolution and phylogenetic information content of mitochondrial genomic structural features illustrated with acrodont lizards

DNA sequences from 195 squamate reptiles indicate that mitochondrial gene order is the most reliable phylogenetic character establishing monophyly of acrodont lizards and of the snake families Boidae, Colubridae, and Viperidae. Gene order shows no evidence of evolutionary parallelisms or reversals in these taxa. Derived secondary structures of mitochondrial tRNAs also prove to be useful phylogenetic characters showing no reversals. Parallelisms for secondary structures of tRNAs are restricted to deep lineages that are separated by at least 200 million years of independent evolution. Presence of a stem-and-loop structure between the genes encoding tRNA(Asn) and tRNA(Cys), where the replication origin for light-strand synthesis is typically located in vertebrate mitochondrial genomes, is found to undergo at least three and possibly as many as seven evolutionary shifts, most likely parallel losses. This character is therefore a less desirable phylogenetic marker than the other structural changes examined. Sequencing regions that contain multiple genes, including tRNA genes, may be preferable to the common practice of obtaining single-gene fragments for phylogenetic inference because it permits observation of major structural changes in the mitochondrial genome. Such characters may occasionally provide phylogenetic information on relatively short internal branches for which base substitutional changes are expected to be relatively uninformative.     

Complete mitochondrial genome of the <Emphasis Type="Italic">Pleuronichthys lighti</Emphasis> (Pleuronectiformes,Pleuronectidae) with phylogenetic consideration

Mitochondrial genome has been used to shed light on many fields of both basic and applied research, including the study of molecular evolution. The complete mitochondrial genome sequence of 17368 bp nucleotides from the Pleuronichthys lighti was determined. It was a circular double-stranded DNA molecule with identical set of 22 transfer RNA genes, 2 ribosomal RNA genes, 13 protein-coding genes as well as a non-coding control region. Stand asymmetry in the nucleotide composition was reflected in the codon usage of genes oriented in opposite directions. In the control region, we identified the extended termination associated sequence domain, the central conserved sequence block domain and the conserved sequence block domain, and two complete repeat region. They were “TTACAATA” and “TGTTGTAA”, respectively. All known 12 mitochondrial genomes of Pleuronectinae fishes were downloaded and analyzed; there were 5570 variable sites in the consensus sequences of 15241 base pairs, calculation of total sites were 35.5%. The highest sequence divergence was 50% (ATP8) and the Kimura-2-parameter genetic distance was 0.235 (ND6), whereas the COIII had the lowest sequence divergence (28.8%) and genetic distance (0.128); the protein coding genes were mainly acted by purifying selection which was detected by selection tests. Analysis of confidence and the information content for per nucleotide revealed ND5, ATP6, COI and ND4 genes were suitable molecular markers for phylogenetic study of Pleuronectinae fishes. Phylogenetic analysis using Bayesian computational algorithms based on COI genes provided support for the taxonomic status of P. lighti, which was consistent with the traditional taxonomy.