Complete DNA sequence of the mitochondrial genome ofCepaea nemoralis (Gastropoda: Pulmonata)

The nucleotide sequence of a mitochondrial genome of the pulmonate gastropod molluscCepaea nemoralis has been determined. Contained within the 14,100 basepairs (bp) are the two ribosomal RNA genes and 13 protein coding genes typical of metazoan mitochondrial genomes. TheCepaea mtDNA does contain a gene for ATPase subunit 8, like the clausiliid pulmonate,Albinaria, and the chiton,Katharina, but unlike the bivalve mollusc,Mytilus. The mitochondrial genetic code ofCepaea is proposed to be the same as that ofMytilus, Katharina, andDrosophila. Only 14 putative tRNA genes are presented, although there is sufficient unassigned sequence to encode the remainder of the expected total of 22 tRNA genes. These 14 tRNA genes are a mixture of standard cloverleaf structures and nonstandard structures containing TV replacement loops as seen in nematode and mosquito mitochondrial genomes. If the eight unidentified tRNA genes are indeed present, very little unassigned sequence would remain to serve as a control region. Genes are transcribed from both strands of the molecule. Base composition is the least biased for any reported animal mitochondrial genome and is also very little skewed between strands using measures independent of base composition. TheCepaea mitochondrial gene order is quite unlike that of any other reported metazoan mtDNA, with the exception of the recently reported partial sequences ofAlbinaria. No gene bound-aries are shared among all the reported molluscan taxa, demonstrating a complete lack of conservation of mitochondrial gene order across the phylum Mollusca.     

Novel features of metazoan mtDNA revealed from sequence analysis of three mitochondrial DNA segments of the land snail Albinaria turrita (Gastropoda: Clausiliidae)

The mitochondrial DNA (mtDNA) size of the terrestrial gastropod Albinaria turrita was determined by restriction enzyme mapping and found to be approximately 14.5 kb. Its partial gene content and organization were examined by sequencing three cloned segments representing about one-fourth of the mtDNA molecule. Complete sequences of cytochrome c oxidase subunit II (COII), and ATPase subunit 8 (ATPase8), as well as partial sequences of cytochrome c oxidase subunit I (COI), NADH dehydrogenase subunit 6 (ND6), and the large ribosomal RNA (IrRNA) genes were determined. Nine putative tRNA genes were also identified by their ability to conform to typical mitochondrial tRNA secondary structures. An 82-nt sequence resembles a noncoding region of the bivalve Mytilus edulis, even though it might contain a tenth tRNA gene with an unusual 5-nt overlap with another tRNA gene. The genetic code of Albinaria turrita appears to be the same as that of Drosophila and Mytilus edulis. The structures of COI and COII are conservative, but those of ATPase8 and ND6 are diversified. The sequenced portion of thelrRNA gene (1,079 nt) is characterized by conspicuous deletions in the 5 and 3 ends; this gene represents the smallest coelomate IrRNA gene so far known. Sequence comparisons of the identified genes indicate that there is greater difference between Albinaria and Mytilus than between Albinaria and Drosophila. An evolutionary analysis, based on COII sequences, suggests a possible nonmonophyletic origin of molluskan mtDNA. This is supported also by the absence of the ATPase8 gene in the mtDNA of Mytilus and nematodes, while this gene is present in the mtDNA of Albinaria and Cepaea nemoralis and in all other known coelomate metazoan mtDNAs.     

The Mitochondrial Sequences of Heptathela hangzhouensis and Ornithoctonus huwena Reveal Unique Gene Arrangements and Atypical tRNAs

We have sequenced the complete mitochondrial genomes of the spiders Heptathela hangzhouensis and Ornithoctonus huwena. Both genomes encode 13 protein-coding genes, 22 tRNA genes, and 2 ribosomal RNA genes. H. hangzhouensis, a species of the suborder Mesothelae and a representative of the most basal clade of Araneae, possesses a gene order identical to that of Limulus polyphemus of Xiphosura. On the other hand, O. huwena, a representative of suborder Opisthothelae, infraorder Mygalomorphae, was found to have seven tRNA genes positioned differently from those of Limulus. The rrnL–trnL1–nad1 arrangement shared by the araneomorph families Salticidae, Nesticidae, and Linyphiidae and the mygalomorph family Theraphosidae is a putative synapomorphy joining the mygalomorph with the araneomorph. Between the two species examined, base compositions also differ significantly. The lengths of most protein-coding genes in H. hangzhouensis and O. huwena mtDNA are either identical to or slightly shorter than their Limulus counterparts. Usage of initiation and termination codons in these protein-coding genes seems to follow patterns conserved among most arthropod and some other metazoan mitochondrial genomes. The sequences of the 3 ends of rrnS and rrnL in the two species are similar to those reported for Limulus, and the entire genes are shortened by about 100–250 nucleotides with respect to Limulus. The lengths of most tRNA genes from the two species are distinctly shorter than those of Limulus and the sequences reveal unusual inferred tRNA secondary structures. Our finding provides new molecular evidence supporting that the suborder Mesothelae is basal to opisthothelids.Reviewing Editor Dr. Rafael Zardoya     

Mitochondrial DNA of the blowflyPhormia regina: restriction analysis and gene localization

A study of an invertebrate mitochondrial genome, that of the blowflyPhormia regina, has been initiated to compare its structural and functional relatedness to other metazoan mitochondrial genomes. A restriction map of mitochondrial DNA (mtDNA) isolated from sucrose gradient-purified mitochondria has been established using a combination of single and double restriction endonuclease digestions and hybridizations with isolated mtDNA fragments, revealing a genome size of 17.5 kilobases (kb). A number of mitochondrial genes including those encoding the 12 S and 16 S ribosomal RNA, the cytochromec oxidase I subunit (COI) and an unidentified open reading frame (URF2) have been located on thePhormia mtDNA by Southern blot analysis using as probes both isolated mtDNA fragments and oligonucleotides derived from the sequences of previously characterized genes from rat andDrosophila yakuba mtDNAs. These data indicate that for those regions examined, the mitochondrial genome organization of blowfly mtDNA is the same as that ofDrosophila yakuba, the order being COI-URF2-12 S-16 S. These data also report the presence of an A + T-rich region, located as a 2.5-kb region between the URF2 and the 12 S rRNA genes, and its amplification by the polymerase chain reaction is described.     

The complete mitochondrial genome of the oriental fruit moth <Emphasis Type="Italic">Grapholita molesta</Emphasis> (Busck) (Lepidoptera: Tortricidae)

The oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae) currently is one of the economically most destructive pest species of stone and pome fruits worldwide. Here we sequenced the complete mitochondrial genome of this pest. This genome is 15,776 bp long, with an A + T content of 81.24%, containing 37 typical animal mitochondrial genes and an A + T-rich region. All gene are arranged as hypothesized ancestral gene order of insects except for trnM, which was shuffled from 3′ downstream of trnQ to 5′ upstream of trnI. cox1 gene uses unusual CGA start codon, as that in all other sequenced lepidopteran mitochondrial genome. The secondary structures for the two rRNA genes were predicted. All helices typically present in insect mitochondrial rRNA genes are generated. A microsatellite sequence was inserted into the region of H2347 in rrnL in G. molesta and two other sequenced tortricid mitochondrial genomes, indicating that the insertion event in this helix might occurred anciently in family Tortricidae. All of the 22 typical animal tRNA genes have a typical cloverleaf structure except for trnS2, in which the D-stem pairings in the DHU arm are absent. An intergenic sequence is present between trnQ and nad2 as well as in other sequenced lepidopteran mitochondrial genomes, which was presumed to be a remnant of trnM gene and its boundary sequences after the duplication of trnM to the upstream of trnI in Lepidoptera. The A + T-rich region is 836 bp, containing six repeat sequences of “TTATTATTATTATTAAATA(G)TTT.”     

The complete mitochondrial genome of the Sichuan taimen (Hucho bleekeri): repetitive sequences in the control region and phylogenetic implications for Salmonidae

The complete mitochondrial DNA genome of the Sichuan taimen (Hucho bleekeri) was determined by the long and accurate polymerase chain reaction (LA-PCR) and primer walking sequence method. The entire mitochondrial genome of this species is 16,997 bp in length, making it the longest among the completely sequenced Salmonidae mitochondrial genomes. It consists of two ribosomal RNA (rRNA) genes, 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and one control region (CR). The gene arrangement, nucleotide composition, and codon usage pattern of the mitochondrial genome are similar to those of other teleosts. A T-type mononucleotide microsatellite and an 82 bp tandem repeat were identified in the control region, which were almost identical among the three H. bleekeri individuals examined. Both phylogenetic analyses based on 12 concatenated protein-coding genes of the heavy strand and on just the control region show that H. bleekeri is a basal species in Salmoninae. In addition, Salmo, Salvelinus and Oncorhynchus all represent monophyletic groups, respectively. All freshwater species occupied basal phylogenetic positions, and also possessed various tandem repeats in their mitochondrial control regions. These results support established phylogenetic relationships among genera in Salmonidae based on morphological and molecular analyses, and are consistent with the hypothesis that Salmonidae evolved from freshwater species.     

Genetic structure of the endangered species <Emphasis Type="Italic">Pinna nobilis</Emphasis> (Mollusca: Bivalvia) inferred from mtDNA sequences

This study examines the population genetic structure of the endangered bivalve Pinna nobilis (Mollusca: Bivalvia), based on novel mtDNA sequences (partial COI and 16S rDNA mtDNA genes). The analyzed nucleotide sequences of COI were 729 bp in size, coding for a 243 amino acid peptide, while the analyzed nucleotide sequences of 16S rDNA were 489 bp in size. These sequences of P. nobilis were the first DNA sequences of the species submitted to any Genetic Data Base. Population samples from four geographic regions from Greece, as well as a population sample of Atrina fragilis (as an outgroup) were used. High values of haplotypic diversity were found in the population samples of P. nobilis, based on the COI sequences. A single base in the analyzed 16S rDNA sequences was different in all analyzed individuals from a single population sample (Chios island) differentiating it from the other ones. These mtDNA sequences could be informative for further genetic analyses of the endangered species, contributing in conservation plans for its protection and/or aquaculture investigations.     

Molecular phylogenetic systematics of twelve species of Acipenseriformes based on mtDNAND4L—ND4 gene sequence analysis

Acipenseriformes is an endangered primitive fish group, which occupies a special place in the history of ideas concerning fish evolution, even in vertebrate evolution. However, the classification and evolution of the fishes have been debated. The mitochondrial DNA (mtDNA)ND4L and partialND4 genes were first sequenced in twelve species of the order Acipenseriformes, including endemic Chinese species. The following points were drawn from DNA sequences analysis: (i) the two species ofHuso can be ascribed toAcipenser, (ii)A. dabryanus is the mostly closely related toA. sinensis, and most likely the landlocked form ofA. sinensis; (iii) genusAcipenser in trans-Pacific region might have a common origin; (iv) mtDNAND4L andND4 genes are the ideal genetic markers for phylogenetic analysis of the order Acipenseriformes.     

The complete nucleotide sequence and multipartite organization of the tobacco mitochondrial genome: comparative analysis of mitochondrial genomes in higher plants

Tobacco is a valuable model system for investigating the origin of mitochondrial DNA (mtDNA) in amphidiploid plants and studying the genetic interaction between mitochondria and chloroplasts in the various functions of the plant cell. As a first step, we have determined the complete mtDNA sequence of Nicotiana tabacum. The mtDNA of N. tabacum can be assumed to be a master circle (MC) of 430,597 bp. Sequence comparison of a large number of clones revealed that there are four classes of boundaries derived from homologous recombination, which leads to a multipartite organization with two MCs and six subgenomic circles. The mtDNA of N. tabacum contains 36 protein-coding genes, three ribosomal RNA genes and 21 tRNA genes. Among the first class, we identified the genes rps1 and rps14, which had previously been thought to be absent in tobacco mtDNA on the basis of Southern analysis. Tobacco mtDNA was compared with those of Arabidopsis thaliana, Beta vulgaris, Oryza sativa and Brassica napus. Since repeated sequences show no homology to each other among the five angiosperms, it can be supposed that these were independently acquired by each species during the evolution of angiosperms. The gene order and the sequences of intergenic spacers in mtDNA also differ widely among the five angiosperms, indicating multiple reorganizations of genome structure during the evolution of higher plants. Among the conserved genes, the same potential conserved nonanucleotide-motif-type promoter could only be postulated for rrn18-rrn5 in four of the dicotyledonous plants, suggesting that a coding sequence does not necessarily move with the promoter upon reorganization of the mitochondrial genome.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. Hagemann     

Homology between the ribosomal DNA of Escherichia coli and mitochondrial DNA preparations of maize is principally to sequences other than mitochondrial rRNA genes

E. coli ribosomal DNA has been used to probe maize mitochondrial DNA. It hybridizes primarily with chloroplast ribosomal DNA sequences and with fungal and bacterial sequences which may contaminate the mtDNA preparations. It also hybridizes to the chloroplast 16S ribosomal RNA gene sequence present in the mitochondrial genome (1) as well as to the mitochondrial 18S ribosomal RNA gene sequence. Weak sequence homology was detected between E. coli rDNA and the mitochondrial 26S ribosomal RNA gene.     

Evolution of the noncoding regions inDrosophila mitochondrial DNA: Two intergenic regions

The sequences of the mitochondrial DNA (mtDNA) segment containing the two intergenic regions were determined for six species belonging to theDrosophila immigrans species group and compared to the corresponding segments ofDrosophila species which had been studied previously. We found remarkable differences in the evolutionary rates of the two intergenic regions. The Intergenic I region, which lies between thetRNA ^gln and thetRNA ^ile genes, was found to be highly conserved in terms of both size (30 ntp) and nucleotide sequence among the species studied. In contrast, the sequences of the Intergenic II region, which lies between thetRNA ^f-met and thetRNA ^ile genes, showed considerable variation. The size of the Intergenic II region ranged from 0 to 88 ntp, and accurate alignment was possible only among sequences from geographical strains or very closely related species in thenasuta species subgroup. The observed differences in conservation of the two mtDNA intergenic regions are discussed in light of functional constraints on mtDNA sequences.     

Novel Repetitive Structures, Deviant Protein-Encoding Sequences andUnidentified ORFs in the Mitochondrial Genome of the BrachiopodLingula anatina

Complete sequence determination of the brachiopod Lingula anatina mtDNA (28,818 bp) revealed an organization that is remarkably atypical for an animal mt-genome. In addition to the usual set of 37 animal mitochondrial genes, which make up only 57% (16,555 bp) of the entire sequence, the genome contains lengthy unassigned sequences. All the genes are encoded in the same DNA strand, generally in a compact way, whereas the overall gene order is highly divergent in comparison with known animal mtDNA. Individual genes are generally longer and deviate considerably in sequence from their homologues in other animals. The genome contains two major repeat regions, in which 11 units of unassigned sequences and six genes (atp8, trnM, trnQ, trnV, and part of cox2 and nad2) are found in repetition, in the form of nested direct repeats of unparalleled complexity. One of the repeat regions contains unassigned repeat units dispersed among several unique sequences, novel repetitive structure for animal mtDNAs. Each of those unique sequences contains an open reading frame for a polypeptide between 80 and 357 amino acids long, potentially encoding a functional molecule, but none of them has been identified with known proteins. In both repeat regions, tRNA genes or tRNA gene-like sequences flank major repeated units, supporting the view that those structures play a role in the mitochondrial gene rearrangements. Although the intricate repeated organization of this genome can be explained by recurrent tandem duplications and subsequent deletions mediated by replication errors, other mechanisms, such as nonhomologous recombinations, appear to explain certain structures more easily.     

Drosophila mitochondrial DNA: Conserved sequences in the A+T-rich region and supporting evidence for a secondary structure model of the small ribosomal RNA

Summary The sequence of a segment of theDrosophila virilis mitochondrial DNA (mtDNA) molecule that contains the A+T-rich region, the small rRNA gene, the tRNA^f-met, tRNA^gln, and tRNA^ile genes, and portions of the ND2 and tRNA^val genes is presented and compared with the corresponding segment of theD. yakuba mtDNA molecule. The A+T-rich regions ofD. virilis andD. yakuba contain two correspondingly located sequences of 49 and 276/274 nucleotides that appear to have been conserved during evolution. In each species the replication origin of the mtDNA molecule is calculated to lie within a region that overlaps the larger conserved sequence, and within this overlap is found a potential hairpin structure. Substitutions between the larger conserved sequences of the A+T-rich regions, the small mt-rRNA genes, and the ND2 genes are biased in favor of transversions, 71–97% of which are AT changes. There is a 13.8 times higher frequency of nucleotide differences between the 5 halves than between the 3 halves of theD. virilis andD. yakuba small mt-rRNA genes. Considerations of the effects of observed substitutions and deletion/insertions on possible nucleotide pairing within the small mt-rRNA genes ofD. virilis andD. yakuba strongly support the secondary structure model for theDrosophila small mt-rRNA that we previously proposed.     

Platyhelminth mitochondrial DNA: Evidence for early evolutionary origin of a tRNAserAGN that contains a dihydrouridine arm replacement loop,and of serine-specifying AGA and AGG codons

Summary The nucleotide sequence of a segment of the mitochondrial DNA (mtDNA) molecule of the liver flukeFasciola hepatica (phylum Platyhelminthes, class Trematoda) has been determined, within which have been identified the genes for tRNA^ala, tRNA^asp, respiratory chain NADH dehydrogenase subunit I (ND1), tRNA^asn, tRNA^pro, tRNA^ile, tRNA^lys, ND3, tRNA^serAGN, tRNA^trp, and cytochromec oxidase subunit I (COI). The 11 genes are arranged in the order given and are all transcribed from the same strand of the molecule. The overall order of theF. hepatica mitochondrial genes differs from what is found in other metazoan mtDNAs. All of the sequenced tRNA genes except the one for tRNA^serAGN can be folded into a secondary structure with four arms resembling most other metazoan mitochondrial tRNAs, rather than the tRNAs that contain a TψC arm replacement loop, found in nematode mtDNAs. TheF. hepatica mitochondrial tRNA^serAGN gene contains a dihydrouridine arm replacement loop, as is the case in all other metazoan mtDNAs examined to date. AGA and AGG are found in theF. hepatica mitochondrial protein genes and both codons appear to specify serine. These findings concerningF. hepatica mtDNA indicate that both a dihydrouridine arm replacement loop-containing tRNA^serAGN gene and the use of AGA and AGG codons to specify serine must first have occurred very early in, or before, the evolution of metazoa.     

The complete nucleotide sequence of the mitochondrial genome of <Emphasis Type="Italic">Phthonandria atrilineata</Emphasis> (Lepidoptera: Geometridae)

Using long-polymerase chain reaction (Long-PCR) method, we determined the complete nucleotide sequence of the mitochondrial genome (mitogenome) of Phthonandria atrilineata. The complete mtDNA from P. atrilineata was 15,499 base pairs in length and contained 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a control region. The P. atrilineata genes were in the same order and orientation as the completely sequenced mitogenomes of other lepidopteran species. The nucleotide composition of P. atrilineata mitogenome was biased toward A + T nucleotides (81.02%), and the 13 PCGs show different A + T contents that range from 73.25% (cox1) to 92.12% (atp8). Phthonandria had the canonical set of 22 tRNA genes, that fold in the typical cloverleaf structure described for metazoan mt tRNAs, with the unique exception of trnS(AGN). The phylogenetic relationships were reconstructed with the concatenated sequences of the 13 PCGs of the mitochondrial genome, which confirmed that P. atrilineata is most closely related to the superfamily Bombycoidea.     

Phylogenetic analysis of six species of pacific abalone (Haliotidae) based on DNA sequences of 16s rRNA and cytochrome c oxidase subunit I mitochondrial genes

Six species of abalones (Haliotidae) are found on the Korean coasts. Identification and characterization of these abalones are usually based on morphologic characters. In this research we compared the partial sequences of the mitochondrial 16S ribosomal RNA and cytochrome c oxidase subunit I genes to identify species using molecular data and to determine their phylogenetic relationships. Sequence alignments and phylogenetic analysis revealed that the 6 species fell into 2 distinct groups which were genetically distant from each other and exhibited little internal phylogenetic resolution. One group included Haliotis discus hannai, H. discus discus, H. madaka, and H. gigantea, while the other group contained H. diversicolor supertexta and H. diversicolor diversicolor. The 16S rRNA sequences were relatively more conserved than to the COI sequences, but both gene sequences provided sufficient phylogenetic information to distinguish among the 6 species of Pacific abalone, and thus could be valuable molecular characters for species identification.