ATP6 Gene Research Articles

New features of Asian Crassostrea oyster mitochondrial genomes: A novel alloacceptor tRNA gene recruitment and two novel ORFs

A feasible way to perform evolutionary analyses is to compare characters divergent enough to observe significant differences, but sufficiently similar to exclude saturation of the differences that occurred. Thus, comparisons of invertebrate mitochondrial (mt) genomes at low taxonomic levels can be extremely helpful in investigating patterns of variation and evolutionary dynamics of genomes, as intermediate stages of the process may be identified. Fortunately, in this study, we newly sequenced the mt genome of the eighth member of Asian Crassostrea oysters which can provide necessary intermediate characters for us to believe that the variation of Crassostrea mt genomes is considerably greater than previously acknowledged. Several new features of Asian Crassostrea oyster mitochondrial genomes were revealed, and our results are particularly significant as they 1) suggest a novel model of alloacceptor tRNA gene recruitment, namely “vertical” tRNA gene recruitment, which can be successfully used to explain the origination of the unusually additional trnK and trnQ genes (annotated as trnK2 and trnQ2 respectively) in the mt genomes of the five Asian oysters, and we speculate that this recruitment progress may be a common phenomenon in the evolution of the tRNA multigene family; 2) reveal the existence of two additional, lineage-specific, mtDNA-encoded genes that may originate from duplication of nad2 followed by rapid evolutionary change. Each of these two genes encodes a unique amino terminal signal peptide, thus each might possess an unknown function; and 3) identify for the first time the atp8 gene in oysters. The present study thus gives further credence to the comparison of congeneric bivalves as a meaningful strategy to investigate mt genomic evolutionary trends in genome organization, tRNA multigene family, and gene loss and/or duplication that are difficult to undertake at higher taxonomic levels. In particular, our study provides new evidence for the identification and characterization of ORFs in the “non-coding region” of animal mt genomes.

Marine turtle mitogenome phylogenetics and evolution

The sea turtles are a group of cretaceous origin containing seven recognized living species: leatherback, hawksbill, Kemp’s ridley, olive ridley, loggerhead, green, and flatback. The leatherback is the single member of the Dermochelidae family, whereas all other sea turtles belong in Cheloniidae. Analyses of partial mitochondrial sequences and some nuclear markers have revealed phylogenetic inconsistencies within Cheloniidae, especially regarding the placement of the flatback. Population genetic studies based on D-Loop sequences have shown considerable structuring in species with broad geographic distributions, shedding light on complex migration patterns and possible geographic or climatic events as driving forces of sea-turtle distribution. We have sequenced complete mitogenomes for all sea-turtle species, including samples from their geographic range extremes, and performed phylogenetic analyses to assess sea-turtle evolution with a large molecular dataset. We found variation in the length of the ATP8 gene and a highly variable site in ND4 near a proton translocation channel in the resulting protein. Complete mitogenomes show strong support and resolution for phylogenetic relationships among all sea turtles, and reveal phylogeographic patterns within globally-distributed species. Although there was clear concordance between phylogenies and geographic origin of samples in most taxa, we found evidence of more recent dispersal events in the loggerhead and olive ridley turtles, suggesting more recent migrations (<1Myr) in these species. Overall, our results demonstrate the complexity of sea-turtle diversity, and indicate the need for further research in phylogeography and molecular evolution.

Complete mitochondrial genome of the Asian green mussel Perna viridis (Bivalvia, Mytilidae)

The complete mitochondrial (mt) genome of the Asian green mussel Perna viridis (16,627 bp), an economically important bivalve, was newly sequenced and annotated. P. viridis is the shortest and has a comparatively highest overall A+T content (68%) among six available genomes of marine mussels to date. The atp8 gene's length (49 a.a.) of the green mussel is unexpectedly greatly shorter than that of other marine mussels (87 a.a.). Comparison of the gene order demonstrated that the six marine mussels share no identical gene blocks although they belong to the same family, which indicates that this group should be a good model to study mtDNA evolution and mitochondria inheritance.

Mitochondrial SCAR and SSR Markers for distinguishing cytoplasmic male sterile lines from their isogenic maintainer lines in cotton

With 8 figures and 3 tablesAbstractCytoplasmic male sterility (CMS) line P30A of Upland cotton has been used in commercial production of F1 seeds. However, cytoplasm‐specific molecular markers in this ‘three‐line’ (CMS line P30A, maintainer line P30B and restorer line Y18R) system are mostly unknown. Twenty mitochondrial gene probes were used to identify the differences between P30B fertile (N‐) and P30A sterile (S‐) cytoplasm. Among six genes that revealed restriction fragment length polymorphisms (RFLPs), the gene for α‐subunit of F1ATPase (atpA) revealed significant differences between N‐ and S‐cytoplasm. All of the EcoRI restriction bands of atpA were amplified by inverse PCR (iPCR) technique, indicating that both N‐ and S‐cytoplasm contained an intact and a 3′ truncated atpA copy, but the truncating site (breakpoint) was different. According to the chimerical sequences following the breakpoints, three cytoplasm‐specific sequence characterized amplified region (SCAR) markers were developed. In addition, a simple sequence repeat (SSR) locus in the 3′ flanking sequences of the intact atpA gene was found between N‐ and S‐cytoplasm. The four PCR markers were used to distinguish CMS lines from maintainer lines.

Supplementation of culture medium with L-carnitine improves development and cryotolerance of bovine embryos produced in vitro

High lipid content in embryos is associated with low freezing tolerance. This study assessed the effects of exogenous L-carnitine, an enhancer of lipid metabolism, on the in vitro development and freezing survival of bovine embryos. Also, effects on metabolic activity, reactive oxygen species (ROS) and apoptosis were investigated. Supplementation of embryo culture medium with 1.518 mM or 3.030 mM L-carnitine significantly increased the rates of zygote development to the blastocyst stage and blastocyst cell numbers whereas 6.072 mM of this compound did not improve embryo development. Survival rates after slow freezing of blastocysts were significantly higher when embryos were cultured in the presence of 1.518 mM or 3.030 mM L-carnitine compared with the control. A lower density of lipid droplets was detected in L-carnitine-treated blastocysts compared with the control. L-carnitine significantly reduced ROS levels in 2-cell embryos but did not reduce ROS levels at later stages. The apoptotic cell rate was not different between control and L-carnitine-treated blastocysts. L-carnitine significantly increased ATP levels in 2-cell embryos but not at the 8-cell or blastocyst stages. L-carnitine increased the expression of metabolism-related ATP6 and COX1 genes in blastocysts. In conclusion, L-carnitine supplementation enhanced lipid metabolism in embryos resulting in improved development and cryotolerance of bovine blastocysts produced in vitro.

The complete mitochondrial genome of an ectoparasitic monopisthocotylean fluke Benedenia hoshinai (Monogenea: Platyhelminthes)

An exponential growth of mitochondrial genome information has brought significant progress in understanding the organismal phylogeny and mitochondrial genome evolution for many metazoans including platyhelminth groups. In this study, we determined the complete mitochondrial genome sequence for Benedenia hoshinai, an ectoparasitic monogenean species, and compared it with its congener Benedenia seriolae. The complete mitochondrial genome is 13,554 bp in length and contains 12 protein-coding genes (lacking the atp8 gene), 2 rRNA genes, and 22 tRNA genes, all encoded in the same direction as found in all other platyhelminth species sequenced to date. The gene arrangement of B. hoshinai mtDNA is almost identical to B. seriolae, differing only by the translocation of trnT between cox1 and rrnL. It is unclear whether the shared position of trnT between B. hoshinai and Gyrodactylus represents evidence for their phylogenetic affinity; testing this hypothesis requires further mitogenomic evidence.

Transcriptional organization of the large and the small ATP synthase operons, atpI/H/F/A and atpB/E, in Arabidopsis thaliana chloroplasts

The ATP synthase is a ubiquitous enzyme which is found in bacteria and eukaryotic organelles. It is essential in the photosynthetic and respiratory processes, by transforming the electrochemical proton gradient into ATP energy via proton transport across the membranes. In Escherichia coli, the atp genes coding for the subunits of the ATP synthase enzyme are grouped in the same transcriptional unit, while in higher plants the plastid atp genes are organized into a large (atpI/H/F/A) and a small (atpB/E) atp operon. By using the model plant Arabidopsis thaliana, we have investigated the strategy evolved in chloroplasts to overcome the physical separation of the atp gene clusters and to coordinate their transcription. We show that all the identified promoters in the two atp operons are PEP dependent and require sigma factors for specific recognition. Our results indicate that transcription of the two atp operons is initiated by at least one common factor, the essential SIG2 factor. Our data show that SIG3 and SIG6 also participate in transcription initiation of the large and the small atp operon, respectively. We propose that SIG2 might be the factor responsible for coordinating the basal transcription of the plastid atp genes and that SIG3 and SIG6 might serve to modulate plastid atp expression with respect to physiological and environmental conditions. However, we observe that in the sigma mutants (sig2, sig3 and sig6) the deficiency in the recognition of specific atp promoters is largely balanced by mRNA stabilization and/or by activation of otherwise silent promoters, indicating that the rate-limiting step for expression of the atp operons is mostly post-transcriptional.

Heteroplasmy levels of mtDNA1555A>G mutation is positively associated with diverse phenotypes and mutation transmission in a Chinese family

The mtDNA 1555A>G mutation was considered to be one of the most common causes of aminoglycoside-induced and non-syndromic hearing loss. However, this mutation was always found in homoplasmy with high phenotypic heterogeneity. Recently this mutation in heteroplasmy has been reported in several studies. In the present study, we have collected a large Chinese family harboring heteroplasmic mtDNA 1555A>G mutation with diverse clinical phenotypes. To investigate the relationship between the mutation load and the severity of hearing loss under Eastern Asian background, we performed clinical, molecular, genetic and phylogenic analysis. This pedigree was characterized by coexistence of eight subjects with homoplasmic mutation and ten subjects with various degrees of heteroplasmy, and the results suggested that there was a strong correlation between the mutation load and the severity/age-onset of hearing loss (r=0.758, p<0.001). We noticed that the mutation level of offspring was associated with their mothers’ in this pedigree, which indicated that maybe exist a regular pattern during the process of the heteroplasmic transmission. In addition, analysis of the complete mtDNA genome of this family revealed that it belonged to Eastern Asian haplogroup B4C1. In addition, a rare homoplasmic mtDNA 9128T>C variant was identified, it located at a strictly conserved site of mtDNA ATP6 gene.

Population structure and conservation of a high-altitude specialist, the Andean cat Leopardus jacobita

The Andean cat Leopardus jacobita is considered one of the rarest felids in the world, although it has a large latitudinal distribution. Due to its preference for upland habitats, it has been suggested that this cat could have naturally fragmented populations. Despite great concern regarding the conservation status of this species, very little is known about its population struc- ture, which is crucial information for appropriate management plans. In this study, we investi- gated its genetic diversity, population structure and evolutionary history by analysing 459 base pairs of the mitochondrial DNA control region, 789 base pairs of the NADH-5, ATP-8 and 16S mitochondrial genes, and 11 nuclear microsatellites, with the aim of identifying conservation units. The analyses were made on 30 skins and 65 recently collected faecal samples from through- out the known range of the species. These analyses revealed a total of 56 individuals. Our results confirm that Andean cat populations harbour extremely low mitochondrial and nuclear genetic diversity. The population structure of this species suggests the existence of 2 evolutionarily signif- icant units (ESUs), with a latitudinal separation between 26 and 35° S. In addition, 2 genetically distinct groups within the northern ESU could be considered separate management units.

Molecular characterization of lactic acid bacteria and in situ amylase expression during traditional fermentation of cereal foods

Lactic acid bacteria play an important role in traditional fermented foods consumed in different countries. Study of their taxonomic structure and diversity is necessary for starter culture selection, improved safety and nutritional enhancement. To achieve these objectives, microbial genomic typing methods were used to study genetic differences of autochthonous bacteria and their distribution in two traditional African fermented cereal foods. A total of 85 predominant bacterial species were isolated from ogi and kunu-zaki obtained from Northern and Southern geographical region of Nigeria. They were identified using combination of 16S rRNA gene sequencing, multilocus sequence analysis (MLSA) based on rpoA, pheS and atpA genes as well as M13-PCR gel fingerprints. The results showed that Lactobacillus fermentum was the most frequently isolated species in ogi (71.4%) and kunu-zaki (84.5%). Other species of lactic acid bacteria (LAB) identified were Lactobacillus plantarum, Streptococcus gallolyticus subsp. macedonicus and Pediococcus pentosaceus. Non lactic acid bacteria isolated from these foods were species belonging to the Bacillus and Staphylococcus. Non-metric multidimensional scaling (nMDS) analysis of the M13-PCR fingerprints for LAB strains showed clonal diversity among strains of the same species. In vitro and in situ expression of amylase gene during fermentation by amylolytic L. plantarum ULAG11 was detected, indicating the potential usefulness of such species for development of starter cultures and for controlled fermentation processes.

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

An evolutionarily conserved mitochondrial orf108 is associated with cytoplasmic male sterility in different alloplasmic lines of Brassica juncea and induces male sterility in transgenic Arabidopsis thaliana

Nuclear-mitochondrial gene interactions governing cytoplasmic male sterility (CMS) in angiosperms have been found to be unique to each system. Fertility restoration of three diverse alloplasmic CMS lines of Brassica juncea by a line carrying the fertility-restorer gene introgressed from Moricandia arvensis prompted this investigation to examine the molecular basis of CMS in these lines. Since previous studies had found altered atpA transcription associated with CMS in these lines, the atpA genes and transcripts of CMS, fertility-restored, and euplasmic lines were cloned and compared. atpA coding and downstream sequences were conserved among CMS and euplasmic lines but major differences were found in the 5' flanking sequences of atpA. A unique open reading frame (ORF), orf108, co-transcribed with atpA, was found in male sterile flowers of CMS lines carrying mitochondrial genomes of Diplotaxis berthautii, D. catholica, or D. erucoides. In presence of the restorer gene, the bicistronic orf108-atpA transcript was cleaved within orf108 to yield a monocistronic atpA transcript. Transgenic expression of orf108 with anther-specific Atprx18 promoter in Arabidopsis thaliana gave 50% pollen sterility, indicating that Orf108 is lethal at the gametophytic stage. Further, lack of transmission of orf108 to the progeny showed for the first time that mitochondrial ORFs could also cause female sterility. orf108 was found to be widely distributed among wild relatives of Brassica, indicating its ancient origin. This is the first report that shows that CMS lines of different origin and morphology could share common molecular basis. The gametic lethality of Orf108 offers a novel opportunity for transgene containment.

Identification of lactic acid bacteria in traditional fermented yak milk and evaluation of their application in fermented milk products

In this study, 53 strains of lactic acid bacteria (LAB) isolated from Xueo, a traditional fermented yak milk in the western Sichuan Plateau of China, were identified and their use in fermented milk was evaluated. All gram-positive and catalase-negative strains were divided into 6 groups at the level of 87% similarity using amplified ribosomal DNA restriction analysis. These groups were identified as 6 species using 16S rDNA sequence analysis and atpA gene analysis. The dominant LAB strains in Xueo were Enterococcus durans, Lactobacillus fermentum, and Lactobacillus paracasei, accounting for 45.3, 22.6, and 17.0% of isolates, respectively. Milk fermented with most of the representative strains was high in quality, exhibiting relatively high viscosity, moderate acidity, good sensory quality, and high counts of viable LAB. Fermented milk of E. durans SCA16 and L. fermentum SCA52 achieved the highest scores for overall sensory quality. Most strains displayed antimicrobial activity against at least 1 of 9 spoilage microorganisms. Lactic acid was the main factor inhibiting the growth of spoilage bacteria, and H2O2 was also inhibitory to some extent. Excluding the influence of acid and H2O2, strains SCA52 (L. fermentum) and SCA7 (Lactobacillus plantarum) were antagonistic against some of the indicators, suggesting that the 2 strains may produce a bacteriocin-like substance. Therefore, the development of superior strains isolated from Xueo to ferment milk with similar flavor and texture to Xueo is expected.

Phylogenetic placement of Geastrum melanocephalum and polyphyly of Geastrum triplex

Geastrum melanocephalum, originally described as Trichaster melanocephalus, is characterized by large basidiomata and an evanescent endoperidium. Although Trichaster was recently treated as a synonym of Geastrum and the specific name G. melanocephalum has often been used, it is still controversial whether Trichaster is an independent genus. Although a close affinity of G. melanocephalum and G. triplex has been suggested based on some morphological similarities, it is highly likely that G. triplex is polyphyletic because of its high morphological variability. To clarify the phylogenetic position of G. melanocephalum, it is therefore critical to evaluate the monophyly of G. triplex. This study sampled ITS, LSU, and atp6 genes from 144 specimens of Geastrales including G. melanocephalum and G. triplex from several continents. Results of phylogenetic analyses demonstrated G. melanocephalum is nested within Geastrum and is most closely related to the European and North American group of G. triplex. Morphological similarities of G. melanocephalum and European and North American G. triplex are also suggested. Based on phylogenetic and morphological evidence, we confirm Trichaster is a synonym of Geastrum, and the scientific name Geastrum melanocephalum should be accepted. Moreover, the present study revealed that taxa tentatively identified as “G. triplex” are highly polyphyletic, and a taxonomic revision of “G. triplex” is therefore needed.

Mitochondrial Gene Diversity Associated with the atp9 Stop Codon in Natural Populations of Wild Carrot (Daucus carota ssp. carota)

Mitochondrial genomes extracted from the wild populations of Daucus carota have been used as a genetic resource by breeders of cultivated carrot, yet little is known concerning the extent of their diversity in nature. Of special interest is an SNP in the putative stop codon of the mitochondrial gene atp9 that has been associated previously with male-sterile and male-fertile phenotypic variants. In this study, either the sequence or PCR/RFLP genotypes were obtained from the mitochondrial genes atp1, atp9, and cox1 found in D. carota individuals collected from 24 populations in the eastern United States. More than half of the 128 individuals surveyed had a CAA or AAA, rather than TAA, genotype at the position usually thought to function as an atp9 stop codon in this species. We also found no evidence for mitochondrial RNA editing (Cytosine to Uridine) of the CAA stop codon in either floral or leaf tissue. Evidence for intragenic recombination, as opposed to the more common intergenic recombination in plant mitochondrial genomes, in our data set is presented. Indel and SNP variants elsewhere in atp9, and in the other 2 genes surveyed, were nonrandomly associated with the 3 atp9 stop codon variants, though further analysis suggested that multilocus genotypic diversity had been enhanced by recombination. Overall the mitochondrial genetic diversity was only modestly structured among populations with an F(ST) of 0.34.

Complete mitochondrial genome sequence from an endangered Indian snake, Python molurus molurus (Serpentes, Pythonidae)

This paper reports the complete mitochondrial genome sequence of an endangered Indian snake, Python molurus molurus (Indian Rock Python). A typical snake mitochondrial (mt) genome of 17258 bp length comprising of 37 genes including the 13 protein coding genes, 22 tRNA genes, and 2 ribosomal RNA genes along with duplicate control regions is described herein. The P. molurus molurus mt. genome is relatively similar to other snake mt. genomes with respect to gene arrangement, composition, tRNA structures and skews of AT/GC bases. The nucleotide composition of the genome shows that there are more A-C % than T-G% on the positive strand as revealed by positive AT and CG skews. Comparison of individual protein coding genes, with other snake genomes suggests that ATP8 and NADH3 genes have high divergence rates. Codon usage analysis reveals a preference of NNC codons over NNG codons in the mt. genome of P. molurus. Also, the synonymous and non-synonymous substitution rates (ka/ks) suggest that most of the protein coding genes are under purifying selection pressure. The phylogenetic analyses involving the concatenated 13 protein coding genes of P. molurus molurus conformed to the previously established snake phylogeny.

Phylogenetic placement of &lt;i&gt;Geastrum melanocephalum&lt;/i&gt; and polyphyly of &lt;i&gt;Geastrum triplex&lt;/i&gt;

Geastrum melanocephalum, originally described as Trichaster melanocephalus, is characterized by large basidiomata and an evanescent endoperidium. Although Trichaster was recently treated as a synonym of Geastrum and the specific name G. melanocephalum has often been used, it is still controversial whether Trichaster is an independent genus. Although a close affinity of G. melanocephalum and G. triplex has been suggested based on some morphological similarities, it is highly likely that G. triplex is polyphyletic because of its high morphological variability. To clarify the phylogenetic position of G. melanocephalum, it is therefore critical to evaluate the monophyly of G. triplex. This study sampled ITS, LSU, and atp6 genes from 144 specimens of Geastrales including G. melanocephalum and G. triplex from several continents. Results of phylogenetic analyses demonstrated G. melanocephalum is nested within Geastrum and is most closely related to the European and North American group of G. triplex. Morphological similarities of G. melanocephalum and European and North American G. triplex are also suggested. Based on phylogenetic and morphological evidence, we confirm Trichaster is a synonym of Geastrum, and the scientific name Geastrum nocephalum should be accepted. Moreover, the present study revealed that taxa tentatively identified as “G. triplex” are highly polyphyletic, and a taxonomic revision of “G. triplex” is therefore needed.

Loss of Mitochondrial Membrane Potential Triggers the Retrograde Response Extending Yeast Replicative Lifespan

In the budding yeast Saccharomyces cerevisiae, loss of mitochondrial DNA (rho0) can induce the retrograde response under appropriate conditions, resulting in increased replicative lifespan (RLS). Although the retrograde pathway has been extensively elaborated, the nature of the mitochondrial signal triggering this response has not been clear. Mitochondrial membrane potential (MMP) was severely reduced in rho0 compared to rho+ cells, and RLS was concomitantly extended. To examine the role of MMP in the retrograde response, MMP was increased in the rho0 strain by introducing a mutation in the ATP1 gene, and it was decreased in rho+ cells by deletion of COX4. The ATP1-111 mutation in rho0 cells partially restored the MMP and reduced mean RLS to that of rho+ cells. COX4 deletion decreased MMP in rho+ cells to a value intermediate between rho+ and rho0 cells and similarly increased RLS. The increase in expression of CIT2, the diagnostic gene for the retrograde response, seen in rho0 cells, was substantially suppressed in the presence of the ATP1-111 mutation. In contrast, CIT2 expression increased in rho+ cells on deletion of COX4. Activation of the retrograde response results in the translocation of the transcription factor Rtg3 from the cytoplasm to the nucleus. Rtg3–GFP translocation to the nucleus was directly observed in rho0 and rho+ cox4Δ cells, but it was blunted in rho0 cells with the ATP1-111 mutation. We conclude that a decrease in MMP is the signal that initiates the retrograde response and leads to increased RLS.

The complete mitochondrial genome sequence of the black-capped capuchin (Cebus apella)

The phylogenetic relationships of primates have been extensively investigated, but key issues remain unresolved. Complete mitochondrial genome (mitogenome) data have many advantages in phylogenetic analyses, but such data are available for only 46 primate species. In this work, we determined the complete mitogenome sequence of the black-capped capuchin (Cebus apella). The genome was 16,538 bp in size and consisted of 13 protein-coding genes, 22 tRNAs, two rRNAs and a control region. The genome organization, nucleotide composition and codon usage did not differ significantly from those of other primates. The control region contained several distinct repeat motifs, including a putative termination-associated sequence (TAS) and several conserved sequence blocks (CSB-F, E, D, C, B and 1). Among the protein-coding genes, the COII gene had lower nonsynonymous and synonymous substitutions rates while the ATP8 and ND4 genes had higher rates. A phylogenetic analysis using Maximum likelihood and Bayesian methods and the complete mitogenome data for platyrrhine species confirmed the basal position of the Callicebinae and the sister relationship between Atelinae and Cebidae, as well as the sister relationship between Aotinae (Aotus) and Cebinae (Cebus/Saimiri) in Cebidae. These conclusions agreed with the most recent molecular phylogenetic investigations on primates. This work provides a framework for the use of complete mitogenome information in phylogenetic analyses of the Platyrrhini and primates in general.

Complete sequence of the mitochondrial genome of the Japanese buff-tip moth, Phalera flavescens (Lepidoptera: Notodontidae)

We sequenced the complete mitochondrial genome of Phalera flavescens. The mitogenome is 15,659 bp in length, including 13 protein-coding genes (atp6, atp8, cox1-3, nad1-6, nad4L, cob), two ribosomal RNAs (rrnS and rrnL), 22 transfer RNAs and an AT-rich region, a putative control region (D-loop). Gene order and orientation were found to be identical to those of other completely sequenced lepidopteran mitogenomes. All 13 protein-coding genes start with the common codon ATN, except for the cox1 gene, which uses CGA as the initial codon. Nine of the 13 protein-coding genes stop with codon TAA, while the cox1, cox2, nad5, and nad4 genes stop with the single nucleotide T. All tRNA genes can be folded into canonical cloverleaf secondary structure, except for trnS1, which loses the ''DHU'' arm. Six overlapping sequences totaling 20 bp (1-8 bp for each sequence) and 16 intergenic spacer sequences, totaling 276 bp (1-58 bp for each sequence) are scattered throughout the genome; the largest intergenic spacer is located between the trnQ and nad2 genes. A microsatellite-like structure (AT)(6)ACC(AT)(6) and 16-bp poly-T elements preceded by the ATTTA motif are present in the D-loop region. Additionally, unexpectedly, an extra 190-bp insertion, with unknown function, was found in the small subunit rRNA gene (rrnS); this gene is the longest known (1020 bp) among all of the Lepidoptera.