Comparison of complete mitochondrial genomes of pine wilt nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus (Nematoda: Aphelenchoidea) and development of a molecular tool for species identification

We determined the complete mitochondrial genome sequences for Bursaphelenchus mucronatus, one species of pinewood nematode. The genome is a circular-DNA molecule of 14,583 bp (195 bp smaller than its congener Bursaphelenchus xylophilus) and contains 12 protein-coding genes (lacking atp8), 22 tRNA genes, and 2 rRNA genes encoded in the same direction, consistent with most other nematodes. Based on sequence comparison of mtDNA genomes, we developed a PCR-based molecular assay to differentiate B. xylophilus (highly pathogenic) and B. mucronatus (relatively less virulent) using species-specific primers. The molecular identification system employs multiplex-PCR and is very effective and reliable for discriminating these Bursaphelenchus species, which are economically important, but difficult to distinguish based on morphology. The comparison of the mitochondrial genomes and molecular identification system of the two species of Bursaphelenchus spp. should provide a rich source of genetic information to support the effective control and management (quarantine) of the pine wilt disease caused by pinewood nematodes.     

A unique tRNA gene family and a novel,highly expressed ORF in the mitochondrial genome of the silver-lip pearl oyster, Pinctada maxima (Bivalvia: Pteriidae)

Characteristics of mitochondrial (mt) DNA such as gene content and arrangement, as well as mt tRNA secondary structure, are frequently used in comparative genomic analyses because they provide valuable phylogenetic information. However, most analyses do not characterize the relationship of tRNA genes from the same mt genome and, in some cases, analyses overlook possible novel open reading frames (ORFs) when the 13 expected protein-coding genes are already annotated. In this study, we describe the sequence and characterization of the complete mt genome of the silver-lip pearl oyster, Pinctada maxima. The 16,994-bp mt genome contains the same 13 protein-coding genes (PCGs) and two ribosomal RNA genes typical of metazoans. The gene arrangement, however, is completely distinct from that of all other available bivalve mt genomes, and a unique tRNA gene family is observed in this genome. The unique tRNA gene family includes two trnS^− AGY and trnQ genes, a trnM isomerism, but it lacks trnS^− CUN. We also report the first clear evidence of alloacceptor tRNA gene recruitment (trnP → trnS^− AGY) in mollusks. In addition, a novel ORF (orfUR1) expressed at high levels is present in the mt genome of this pearl oyster. This gene contains a conserved domain, “Oxidored_q1_N”, which is a member of Complex I and thus may play an important role in key biological functions. Because orfUR1 has a very similar nucleotide composition and codon bias to that of other genes in this genome, we hypothesize that this gene may have been moved to the mt genome via gene transfer from the nuclear genome at an early stage of speciation of P. maxima, or it may have evolved as a result of gene duplication, followed by rapid sequence divergence. Lastly, a 319-bp region was identified as the possible control region (CR) even though it does not correspond to the longest non-coding region in the genome. Unlike other studies of mt genomes, this study compares the evolutionary patterns of all available bivalve mt tRNA and atp8 genes.     

The complete mitochondrial genome of Taeniogonalos taihorina (Bischoff) (Hymenoptera: Trigonalyidae) reveals a novel gene rearrangement pattern in the Hymenoptera

The family Trigonalyidae is considered to be one of the most basal lineages in the suborder Apocrita of Hymenoptera. Here, we determine the first complete mitochondrial genome of the Trigonalyidae, from the species Taeniogonalos taihorina (Bischoff, 1914). This mitochondrial genome is 15,927 bp long, with a high A + T-content of 84.60%. It contains all of the 37 typical animal mitochondrial genes and an A + T-rich region. The orders and directions of all genes are different from those of previously reported hymenopteran mitochondrial genomes. Eight tRNA genes, three protein-coding genes and the A + T-rich region were rearranged, with the dominant gene rearrangement events being translocation and local inversion. The arrangements of three tRNA clusters, trnY–trnM–trnI–trnQ, trnW–trnL2–trnC, and trnH–trnA–trnR–trnN–trnS–trnE–trnF, and the position of the cox1 gene, are novel to the Hymenoptera, even the insects. Six long intergenic spacers are present in the genome. The secondary structures of the RNA genes are normal, except for trnS2, in which the D-stem pairing is absent.     

Evolution of the tRNA gene family in mitochondrial genomes of five Meretrix clams (Bivalvia,Veneridae)

In contrast to the extreme conservation of nuclear-encoded tRNAs, organization of the mitochondrial (mt) tRNA gene family in invertebrates is highly dynamic and rapidly evolving. While gene duplication and loss, gene isomerism, recruitment, and rearrangements have occurred sporadically in several invertebrate lineages, little is known regarding the pattern of their evolution. Comparisons of invertebrate mt genomes at a generic level can be extremely helpful in investigating evolutionary patterns of variation, as intermediate stages of the process may be identified. Variation of mitochondrial tRNA organization among Meretrix clams provides good materials to investigate mt tRNA evolution. We characterized the complete mt genome of the lyrate Asiatic hard clam Meretrix lyrata, re-annotated tRNAs of four previously sequenced Meretrix clams, and undertook an intensive comparison of tRNA gene families in these clams. Our results 1) provide evidence that the commonly observed duplication of trnM may have occurred independently in different bivalve lineages and, based on the higher degree of trnM gene similarity, may have occurred more recently than expected; 2) suggest that “horizontal” evolution may have played an important role in tRNA gene family evolution based on frequent gene duplications and gene recruitment events; and 3) reveal the first case of isoacceptor “vertical” tRNA gene recruitment (VTGR) and present the first clear evidence that VTGR allows rapid evolution of tRNAs. We identify the trnS^− UCR gene in Meretrix clams, previously considered missing in this lineage, and speculate that trnS^− UCR lacking the D-arm in both M. lyrata and Meretrix lamarckii may represent the ancestral status. Phylogenetic analysis based on 13 concatenate protein-coding genes provided opportunities to detect rapidly evolved tRNA genes via VTGR and gene isomerism processes. This study suggests that evolution of the mt tRNA gene family in bivalves is more complex than previously thought and that comparison of several congeneric species is a useful strategy in investigating evolutionary patterns and dynamics of mt tRNA genes.     

Characterization of the complete mitochondrial genome of Chilo auricilius and comparison with three other rice stem borers

The mitogenome of Chilo auricilius (Lepidoptera: Pyraloidea: Crambidae) was a circular molecule made up of 15,367 bp. Sesamia inferens, Chilo suppressalis, Tryporyza incertulas, and C. auricilius, are closely related, well known rice stem borers that are widely distributed in the main rice-growing regions of China. The gene order and orientation of all four stem borers were similar to that of other insect mitogenomes. Among the four stem borers, all AT contents were below 83%, while all AT contents of tRNA genes were above 80%. The genomes were compact, with only 121–257 bp of non-coding intergenic spacer. There are 56 or 62-bp overlapping nucleotides in Crambidae moths, but were only 25-bp overlapping nucleotides in the noctuid moth S. inferens. There was a conserved motif ‘ATACTAAA’ between trnS2 (UCN) and nad1 in Crambidae moths, but this same region was ‘ATCATA’ in the noctuid S. inferens. And there was a 6-bp motif ‘ATGATAA’ of overlapping nucleotides, which was conserved in Lepidoptera, and a 14-bp motif ‘TAAGCTATTTAAAT’ conserved in the three Crambidae moths (C. suppressalis, C. auricilius and T. incertulas), but not in the noctuid. Finally, there were no stem-and-loop structures in the two Chilo moths.     

The complete mitogenome of Apocheima cinerarius (Lepidoptera: Geometridae: Ennominae) and comparison with that of other lepidopteran insects

The complete mitochondrial genome (mitogenome) of a female flightless geometrid moth Apocheima cinerarius was found to be 15,722 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a control region. The A + T content of the complete mitogenome is 80.83%. The AT skew value ([A − T] / [A + T]) is 0.027. The 13 PCGs of the mitogenome start with typical ATN codons, except for cox1 with the start codon CGA. All the tRNA genes have typical cloverleaf secondary structures, except for trnSer(AGN). The secondary structures of rrnL and rrnS were predicted. Six structural domains including conserved regions (IV, V) and variable regions (I, II, III, VI) were identified in the secondary structure of rrnL. The secondary structure of rrnS consists of 3 structural domains. The control region of A. cinerarius begins with conserved motifs of “ATAGA” + 19-bp poly T. It also contains a microsatellite-like (TA)₂₆, a stem-and-loop structure, and a poly-A stretch. Phylogenetic analysis showed that Geometroidea is more closely related to Bombycoidea than to Noctuoidea. A. cinerarius is more closely related to Biston panterinaria than to Phthonandria atrilineata, which is in accordance with the conventional morphology-based classification.     

Crystal structure of the E. coli tRNA aminoacyl stem isoacceptor RR-1660 at 2.0 Å resolution

Due to the redundancy of the genetic code there exist six mRNA codons for arginine and several tRNA^Arg isoacceptors which translate these triplets to protein within the context of the mRNA. The tRNA identity elements assure the correct aminoacylation of the tRNA with the cognate amino acid by the aminoacyl-tRNA-synthetases. In tRNA^Arg, the identity elements consist of the anticodon, parts of the D-loop and the discriminator base. The minor groove of the acceptor stem interacts with the arginyl-tRNA-synthetase. We crystallized different Escherichia coli tRNA^Arg acceptor stem helices and solved the structure of the tRNA^Arg isoacceptor RR-1660 microhelix by X-ray structure analysis. The acceptor stem helix crystallizes in the space group P1 with the cell constants a = 26.28, b = 28.92, c = 29.00 Å, α = 105.74, β = 99.01, γ = 97.44° and two molecules per asymmetric unit. The RNA hydration pattern consists of 88 bound water molecules. Additionally, one glycerol molecule is bound within the interface of the two RNA molecules.     

Plastid trnF pseudogenes are present in Jaltomata, the sister genus of Solanum (Solanaceae): Molecular evolution of tandemly repeated structural mutations

Extensive gene duplication arranged in a tandem array is rare in the plastome of embryophytes. Interestingly, we found pseudogene copies of the trnF gene in the genus Jaltomata, the sister genus of Solanum where such gene duplication has been previously reported. In each Jaltomata sequence available we found two pseudogene copies in close 5′-proximity to the original functional gene. The size of each pseudogene copy ranged between 17 and 48 bp and the anticodon domain was identified as the most conserved element. A common ATT(G)_n motif is particularly interesting and its modifications were found to border the 3′ of the duplicated regions. Other motifs were partial residues, or entire parts of the T- and D-domains, and both domains proved to be variable in length among the pseudogenes identified. The residues of the 3′ and 5′ acceptor stem were not found among the copies. We further compared the newly discovered copies of Jaltomata with those ones previously described from Solanum and inferred phylogenetic relationships of the copies aligned. The evolution of Solanum copies, in contrast to Jaltomata, is hard to explain as resulting only in parsimonious changes since reticulate evolutionary patterns were detected among the copies. The dynamic evolutionary patterns of Solanum might be explained by possible inter- or intrachromosomal recombination.     

Complete mitochondrial genome of the atlas moth, Attacus atlas (Lepidoptera: Saturniidae) and the phylogenetic relationship of Saturniidae species

Mitochondrial genome (mitogenome) can provide information for genomic structure as well as for phylogenetic analysis and evolutionary biology. In this study, we present the complete mitogenome of the atlas moth, Attacus atlas (Lepidoptera: Saturniidae), a well-known silk-producing and ornamental insect with the largest wing surface area of all moths. The mitogenome of A. atlas is a circular molecule of 15,282 bp long, and its nucleotide composition shows heavily biased towards As and Ts, accounting for 79.30%. This genome comprises 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and an A + T-rich region. It is of note that this genome exhibits a slightly positive AT skew, which is different from the other known Saturniidae species. All PCGs are initiated by ATN codons, except for COI with CGA instead. Only six PCGs use a common stop codon of TAA or TAG, whereas the remaining seven use an incomplete termination codon T or TA. All tRNAs have the typical clover-leaf structure, with an exception for tRNA^Ser(AGN). The A. atlas A + T-rich region contains non-repetitive sequences, but harbors several features common to the Bombycoidea insects. The phylogenetic relationships based on Maximum Likelihood method provide a well-supported outline of Saturniidae, which is in accordance with the traditional morphological classification and recent molecular works.     

The complete mitochondrial genome of the Ailanthus silkmoth, Samia cynthia cynthia (Lepidoptera: Saturniidae)

The complete mitochondrial genome (mitogenome) of the Ailanthus silkmoth, Samia cynthia cynthia (Lepidoptera: Saturniidae) was determined. The circular genome is 15,345 bp long, and presents a typical gene organization and order for sequenced mitogenomes of Bombycidea species. The nucleotide composition of the genome is highly A+T biased, accounting for 79.86%. The AT skew of the genome is slightly negative, indicating the occurrence of more Ts than As, as found in other Saturniidae species. All protein-coding genes (PCGs) are initiated by ATN codons, except for COI and COII, which are tentatively designated by CGA and GTG, respectively, as observed in other insects. Four of 13 PCGs, including COI, COII, ATP6, and ND3, harbor the incomplete termination codons, T or TA. With an exception for tRNA^Ser(AGN), all other tRNAs can form a typical clover-leaf structure of mitochondrial tRNA. The 359 bp A+T-rich region of S. c. cynthia contains non-repetitive sequences, but harbors several features common to the Bombycidea insects, including the motif ATAGA followed by a poly-T stretch of 19 bp, a microsatellite-like (AT)₇ element preceded by the ATTTA motif, and a poly-A element upstream tRNA^Met. The phylogenetic analyses support the morphology-based current hypothesis that Bombycidae and Saturniidae are monophyletic. Our result confirms that Saturniini and Attacini form a reciprocal monophyletic group within Saturniidae.     

The 1.2 Å crystal structure of an E. coli tRNA acceptor stem microhelix reveals two magnesium binding sites

tRNA identity elements assure the correct aminoacylation of tRNAs by the cognate aminoacyl-tRNA synthetases. tRNA^Ser belongs to the so-called class II system, in which the identity elements are rather simple and are mostly located in the acceptor stem region, in contrast to ‘class I’, where tRNA determinants are more complex and are located within different regions of the tRNA.The structure of an Escherichia coli tRNA^Ser acceptor stem microhelix was solved by high resolution X-ray structure analysis. The RNA crystallizes in the space group C2, with one molecule per asymmetric unit and with the cell constants a = 35.79, b = 39.13, c = 31.37 Å, and β = 111.1°. A defined hydration pattern of 97 water molecules surrounds the tRNA^Ser acceptor stem microhelix. Additionally, two magnesium binding sites were detected in the tRNA^Ser aminoacyl stem.     

RNase J is involved in the 5′-end maturation of 16S rRNA and 23S rRNA in Sinorhizobium meliloti

Sinorhizobium meliloti harbours genes encoding orthologs of ribonuclease (RNase) E and RNase J, the principle endoribonucleases in Escherichia coli and Bacillus subtilis, respectively. To analyse the role of RNase J in S. meliloti, RNA from a mutant with miniTn5-insertion in the RNase J-encoding gene was compared to the wild-type and a difference in the length of the 5.8S-like ribosomal RNA (rRNA) was observed. Complementation of the mutant, Northern blotting and primer extension revealed that RNase J is necessary for the 5′-end maturation of 16S rRNA and of the two 23S rRNA fragments, but not of 5S rRNA.     

The mitochondrial genomes of Amphiascoides atopus and Schizopera knabeni (Harpacticoida: Miraciidae) reveal similarities between the copepod orders Harpacticoida and Poecilostomatoida

Members of subclass Copepoda are abundant, diverse, and—as a result of their variety of ecological roles in marine and freshwater environments—important, but their phylogenetic interrelationships are unclear. Recent studies of arthropods have used gene arrangements in the mitochondrial (mt) genome to infer phylogenies, but for copepods, only seven complete mt genomes have been published. These data revealed several within-order and few among-order similarities. To increase the data available for comparisons, we sequenced the complete mt genome (13,831 base pairs) of Amphiascoides atopus and 10,649 base pairs of the mt genome of Schizopera knabeni (both in the family Miraciidae of the order Harpacticoida). Comparison of our data to those for Tigriopus japonicus (family Harpacticidae, order Harpacticoida) revealed similarities in gene arrangement among these three species that were consistent with those found within and among families of other copepod orders. Comparison of the mt genomes of our species with those known from other copepod orders revealed the arrangement of mt genes of our Harpacticoida species to be more similar to that of Sinergasilus polycolpus (order Poecilostomatoida) than to that of T. japonicus. The similarities between S. polycolpus and our species are the first to be noted across the boundaries of copepod orders and support the possibility that mt-gene arrangement might be used to infer copepod phylogenies. We also found that our two species had extremely truncated transfer RNAs and that gene overlaps occurred much more frequently than has been reported for other copepod mt genomes.     

Solution Structure of an Archaeal RNase P Binary Protein Complex: Formation of the 30-kDa Complex between Pyrococcus furiosus RPP21 and RPP29 Is Accompanied by Coupled Protein Folding and Highlights Critical Features for Protein-Protein and Protein-RNA Interactions

Ribonuclease P (RNase P) is a ribonucleoprotein (RNP) enzyme that catalyzes the Mg²⁺-dependent 5′ maturation of precursor tRNAs. In all domains of life, it is a ribozyme: the RNase P RNA (RPR) component has been demonstrated to be responsible for catalysis. However, the number of RNase P protein subunits (RPPs) varies from 1 in bacteria to 9 or 10 in eukarya. The archaeal RPR is associated with at least 4 RPPs, which function in pairs (RPP21-RPP29 and RPP30-POP5). We used solution NMR spectroscopy to determine the three-dimensional structure of the protein-protein complex comprising Pyrococcus furiosus RPP21 and RPP29. We found that the protein-protein interaction is characterized by coupled folding of secondary structural elements that participate in interface formation. In addition to detailing the intermolecular contacts that stabilize this 30-kDa binary complex, the structure identifies surfaces rich in conserved basic residues likely vital for recognition of the RPR and/or precursor tRNA. Furthermore, enzymatic footprinting experiments allowed us to localize the RPP21-RPP29 complex to the specificity domain of the RPR. These findings provide valuable new insights into mechanisms of RNP assembly and serve as important steps towards a three-dimensional model of this ancient RNP enzyme.     

The potato mitochondrial initiator methionine tRNA gene and its flanking regions: an illustration of the diversity of mitochondrial genome rearrangements among plant species

The initiator methionine transfer RNA (tRNAf ^Met) gene was identified on a 347 bpEco RI-Hind III DNA fragment of the potato mitochondrial (mt) genome. The sequence of this gene shows 1 to 7 nucleotide differences with the other plant mt tRNAsf ^Met or tRNAf ^Met genes studied so far. Whereas the tRNAf ^Met gene is present as a single copy in the potato mt genome, a tRNA pseudogene corresponding to 60% of a complete tRNA (from the 5 end to the variable region) and located at 105 nucleotides upstream of the tRNAf ^Met gene on the opposite strand was shown to be repeated at least three times. Furthermore, the physical environment of the tRNAf ^Met gene in the mt genome is very different among plants, which suggests that the tRNAf ^Met gene region has often been implicated in recombination events of plant mt genomes leading to important rearrangements in gene order.