The mitochondrial genome of the primary screwworm fly Cochliomyia hominivorax (Diptera: Calliphoridae)

The complete sequence of the mitochondrial genome of the screwworm Cochliomyia hominivorax was determined. This genome is 16,022 bp in size and corresponds to a typical Brachycera mtDNA. A Serine start codon for COI and incomplete termination codons for COII, NADH 5 and NADH 4 genes were described. The nucleotide composition of C. hominivorax mtDNA is 77% AT-rich, reflected in the predominance of AT-rich codons in protein-coding genes. Non-optimal codon usage was commonly observed in C. hominivorax mitochondrial genes. Phylogenetic analysis distributed the Acalypterate species as a monophyletic group and assembled the C. hominivorax (Calyptratae) and the Acalyptratae in a typical Brachycera cluster. The identification of diagnostic restriction sites on the sequenced mitochondrial genome and the correlation with previous RFLP analysis are discussed.     

Analysis of mitochondrial DNA and development of PCR-based diagnostic molecular markers for Mediterranean fruit fly (Ceratitis capitata) populations

A 2.99 kb mtDNA fragment containing two variable restriction endonuclease sites ( EcoRV and Xbal ) was subcloned and sequenced from the Mediterranean fruit fly (Ceratitis capitata). This fragment represents approximately one-fifth of the entire mitochondrial sequence. The sequence was aligned with the comparable region from Drosophila yakuba and Anopheles gambiae , resulting in 81.8% and 76.7% identity at the nucleotide level, and 77% and 67.7% identity, respectively, at the amino acid level. The sequenced region includes the complete genes for NADH dehydrogenase 4, NADH dehydrogenase 4L, NADH dehydrogenase 6, and transfer RNAs for proline, threonine and histidine, and part of the genes for NADH dehydrogenase 5 and cytochrome b. Oligonucleotide primers were designed to asymmetrically bracket each of two variable restriction endonuclease sites to allow PCR amplification and subsequent restriction endonuclease analysis of individual fly samples.     

The mitochondrial genome of the mediterranean fruit fly, Ceratitis capitata

The complete sequence of the mitochondrial genome of Ceratitis capitata has been determined. The circular genome is 15 980 bp long and contains a standard gene complement, i.e. the large and small ribosomal RNA subunits, twenty-two transfer RNA (tRNA) genes and thirteen genes encoding mitochondrial proteins. When comparing the sequence to fragments previously sequenced from other isolates it becomes apparent that interstrain polymorphisms are not rare. These differences are potentially useful for the development of diagnostic tools for population analysis applications, such as determining the source of recent introductions. Moreover, they could help obtain a solution to the long-lasting controversy on the possible eradication of the Medfly from certain locations.     

The white gene of the tephritid fruit fly Bactrocera tryoni is characterized by a long untranslated 5' leader and a 12 kb first intron

A 300 bp fragment from exon 6 of the white gene of Bactrocera tryoni was used to screen a B. tryoni genomic library. One positive (∼14 kb) insert contained exons 2–6 of white by nucleotide and amino acid sequence similarity to the white genes of D. melanogaster (O'Hare et al ., 1984; Pepling & Mount, 1990). Lucilia cuprina (Garcia et al ., 1996). Ceratitis capitata (Zwiebel et al ., 1995) and Anopheles gambiae (Besansky et al ., 1995). A white 5' cDNA fragment containing exons 1, 2 and part of exon 3 was amplified, cloned and sequenced. An inverse PCR fragment of genomic DNA was generated, containing the exon 1 coding region plus ∼2.1 kb of upstream sequence, encompassing the putative promoter of the gene. Exon 1 was found to be 728 bp long, encoding the first twenty-five amino acids. The full length of intron 1 was shown to be 12 kb (amplified using long PCR protocols), up to 3 times the length of the longest white intron 1 isolated to date.     

Sequence analysis of the D1 and D2 regions of 28S rDNA in the hornet (Vespa crabro) (Hymenoptera, Vespinae)

The two variable domains D1 and D2 near the 5 end of the 28S ribosomal RNA gene (large subunit rRNA) have been sequenced for Vespa crabro. The sequence was aligned to corresponding rDNA regions of the wasp species Nasonia vitripennis, Melittobia digitata and the fruit fly Drosophila melanogaster. We analysed the nucleotide composition and sequence similarity for the different regions of the investigated sequences and present the inferred secondary structure of Vespa crabro .     

The mitochondrial genome of the mosquito Anopheles gambiae: DNA sequence, genome organization, and comparisons with mitochondrial sequences of other insects

The entire 15,363 bp mitochondrial genome was cloned and sequenced from the mosquito Anopheles gambiae. With respect to the protein-coding genes, rRN A genes and the control region, the gene order was identical to that reported for other insects. There were significant differences, however, in the position and orientation of specific tRNA loci. The overall nucleotide composition was heavily biased towards adenine and thymine, which accounted for 77.6% of all nucleotides. Comparisons were made with the mitochondrial genomes of other insects on the basis genome size and organization, DNA and putative amino acid sequence data, nucleotide substitutions, codon usage and bias, and patterns of AT enrichment.     

Complete base sequence for the mitochondrial large subunit ribosomal RNA of the gypsy moth Lymantha dispar(L.)

A 1355 bp sequence (accession number L32141) isolated from a gypsy moth (Lymantria dispar) cDNA library showed 68–74% sequence identity to mitochondrial large subunit ribosomal RNA (mt IrRNA) sequences of Locusta migratoria, Apis mellifera, Aedes albopictus, Anopheles gambiae and two Dros-ophila species. A comparison of the primary sequences of the mt IrRNAs from the above insects in four orders and from Esherichia coli demonstrated regions of conservation which presumably correspond to regions of functional and/or structural homology. A secondary structure for the gypsy moth mt IrRNA sequence was derived based on the proposed secondary structures of Drosophila yakuba and Aedes albopictus mt IrRNAs (Gutell & Fox, Nucleic Acid Res 16 (Suppl.), r175-r269, 1988). This sequence was found to hybridize to about 10–15% of the clones in several (eleven) gypsy moth cDNA libraries.     

The mitochondrial genome of the Korean hairstreak, Coreana raphaelis (Lepidoptera: Lycaenidae)

We determined the complete nucleotide sequences of the mitochondrial genome (mitogenome) of the Korean hairstreak, Coreana raphaelis (Lepidoptera: Lycaenidae). The entire mitochondrial DNA (mtDNA) molecule was 15,314 bp long. The C. raphaelis genes were in the same order and orientation as the completely sequenced mitogenomes of other lepidopteran species, except for the presence of an extra copy of tRNA(Ser)(AGN). High similarity in primary sequence and secondary structure between the two tandemly located copies of the tRNA(Ser)(AGN) suggest a recent duplication of an original single tRNA(Ser)(AGN). The DHU arm of the two copies of tRNA(Ser)(AGN) formed a simple loop as seen in many other metazoan mt tRNA(Ser)(AGN). The putative initiation codon for the C. raphaelis COI gene appears to be a tetranucleotide, TTAG, found commonly in the sequenced lepidopterans. ATPase8, ATPase6, ND4L and ND6 genes, which are next to another protein-coding gene at their 3' end all had the sequences potential to form a hairpin structure, suggesting the importance of such a structure for precise cleavage of the mature protein-coding genes.     

PCR primers for the amplification of four insect mitochondrial gene fragments

Insect mitochondrial genome (mtDNA) analysis is a powerful tool for the study of population genetics and phylogenetics. In the past few years primer sequences for the PCR amplification of various insect mtDNA genes have been published. The objectives of this study were (1) present new primer sequences for six insect mitochondrial genes and (2) test primers designed in our laboratory and some previously published primers on a wide range of insects to determine if amplification of the target fragment could be obtained. The primers for the amplification of the two ribosomal RNA gene (16S and 12S rRNA) fragments are universal for insects and related groups; the primers for NADH5 and NADH4 dehydrogenase gene fragments and cytochrome c oxidase I gene fragment are applicable broadly.     

The mitochondrial genome of the olive fly Bactrocera oleae: two haplotypes from distant geographical locations

The complete sequence of the olive fly (Bactrocera oleae) mitochondrial genome has been determined. Two independent haplotypes, from flies of distant geographical origin (Italy and Portugal) were completely sequenced. The molecule is 15815 bp long, and shows the gene content and organization typical of insects, namely thirteen protein coding genes (PCGs) encoding proteins involved in oxidative phosphorylation, two rRNAs, twenty-two tRNAs and a long (949 bp) noncoding region. The genomes of the two fly specimens share the same arrangement, differing by a mere thirty-one point mutations. The differences are mostly transitions (26) and synonymous substitutions in PCGs (21). The two new sequences are compared with others already present in the database.     

Linkage map organization of expressed sequence tags and sequence tagged sites in the mosquito,Aedes aegypti

A composite genetic linkage map for the yellow fever mosquito Aedes aegypti was constructed based on restriction fragment length polymorphism (RFLP), single nucleotide polymorphism (SNP) and single strand conformation polymorphism (SSCP) markers. The map consists of 146 marker loci distributed across 205 cM, and includes several morphological mutant marker loci. Most of the genetic markers are derived from random cDNAs or Ae. aegypti genes of known function. A number of markers are derived from random genomic DNAs, including several cloned RAPD-PCR fragments, and also several cDNAs from Drosophila melanogaster. Most of the random cDNAs (80.2%) have high BlastX sequence identities to known genes, with the majority of matches to genes from D. melanogaster. Access to sequence data for all markers will facilitate their continued development for use in high-throughput SNP marker analyses and also provides additional physical anchor points for an anticipated genome sequencing effort.     

Genetic similarity among pheromone and voltinism races of Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae)

The genetic variability of seven European corn borer populations, Ostrinia nubilalis, from North America and Europe was assessed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing. The nuclear ribosomal internal transcribed spacer 1 (ITS-1) region (≈ 500 base pair [bp]) and four mitochondrial (mtDNA) regions (1550 bp total) were examined. The smartweed borer, Ostrinia obumbratalis, and south-Western corn borer, Diatraea grandiosella, were used for comparisons. Of 106 restriction sites identified (80 in mtDNA and 26 in ITS-1), none differentiated geographical populations, pheromone races, or voltine ecotypes of the European corn borer. The lack of variation in the ITS-1 of European corn borer was confirmed by DNA sequence analysis. The genetic similarity of European corn borer populations, despite their wide geographical range and physiological differences, may be explained by a relatively recent origin for the voltinism and pheromone races, gene flow among races, and/or expansion from genetic bottlenecks.     

Sequence and organization of the mitochondrial genome of the Chagas disease vector, Triatoma dimidiata

The 17 019 bp mitochondrial genome of Triatoma dimidiata is composed of thirteen protein coding sequences, twenty‐two tRNAs, small and large ribosomal units, and a control region. The gene order and orientation are identical to that of Drosophila yakuba. The nucleotide composition is biased toward adenine and thymine (69.5% A + T). The 2.1 kb putative control region, known as the A + T rich region in most insects, has an A + T bias of 66%, but contains a 400 bp sequence that is 77.5% A + T and two other distinct regions: (1) one with a lower A + T bias (60.1%) and (2) a region of eight tandem repeat units. The identified 1.4 kb nuclear copy of mitochondrial sequences encompasses the string of Gs and the beginning of the cytochrome c oxidase 1 gene but lacks the 1.8 kb region spanning the eight tandem repeats and the 5′ end of the NADH dehydrogenase subunit II gene.     

The mitochondrial genome of the bristletail Petrobius brevistylis (Archaeognatha: Machilidae)

The complete mitochondrial genome of the bristletail Petrobius brevistylis has been determined. The genome is 15,698 bp long and bears the standard set of genes common to all arthropods as well as a major non-coding A+T-rich region, the putative mitochondrial control region. A unique gene order was revealed as it differs from other hexapod and crustacean mitochondrial genomes in the position of tRNA-Tyr. Genome features like nucleotide composition and codon usage are compared with that of other insect taxa. A+T content is similar in species of Archaeognatha and Zygentoma, but obviously lower than in Collembola and Pterygota. This A+T bias significantly affects also amino acid frequencies and may be a problem for phylogenetic analyses.     

A muscle-specific actin gene from the Mediterranean fruit fly, Ceratitis capitata

A characterization of an actin gene isolated from the genome of the Mediterranean fruit fly, Ceratitis capitata , including the complete sequencing of the coding, 3' and 5' flanking regions of this gene and a partial cDNA was carried out. The partial cDNA was derived from the 3' untranslated region of the actin gene described here, and has been used to identify this gene uniquely. The DNA sequence data presented here, together with the pattern of expression exhibited by this gene during development, strongly support the interpretation that this is a muscle-specific actin gene. Peaks of expression are seen in tissues and during temporal phases of development where muscle differentiation is occurring. The derived protein sequence of the Medfly actin gene shows the highest degrees of similarity, 98.4 and 96.6% respectively, with the two muscle-specific actin genes 79B and 88F from D. melanogaster . The Medfly actin gene also has a single intervening sequence, and an intron is found at the same position in the 79B and 88F actin genes. In the coding region at the DNA level, 17.2 and 16.4% nucleotide differences, respectively, are observed between the Medfly actin gene and these same two D. melanogaster actin genes. The disparity between the amino acid and nucleotide comparisons can be explained, in part, by a high level of synonymous changes in the DNA sequence. In addition, despite the many similarities, codon usage appears to be very different between the actin genes of these species.     

Structure and evolution of the mitochondrial DNA complete control region in the Drosophila subobscura subgroup

The complete A + T-rich region of mitochondrial DNA (mtDNA) has been cloned and sequenced in the species of the Drosophila subobscura subgroup D. subobscura, D. madeirensis and D. guanche. Comparative analysis of these sequences with others already published has identified new sequence motifs that are conserved in Drosophila and other insects. A putative bi-directional promoter and a stop signal are proposed to be involved in the primary mtDNA strand replication of Drosophila. This region strongly resolves relationships of the species included in a phylogenetic analysis, both for closely related species and also at deeper phylogenetic levels when only the left and central domains are taken into account.     

丙型肝炎病毒NS5A区插入序列因子的初步研究

研究丙型肝炎病毒ＮＳ５Ａ区插入序列（ＩＳ） 因子及其来源。以ＲＴ－ＰＣＲ法扩增ＨＣＶ ＮＳ５Ａ区第６８７１至７１２１ 区段， 对扩增产物进行核苷酸测序并与ＨＣＶ－Ｊ株相应区段序列相比较。结果发现ＨＣＶ ＮＳ５Ａ 区插入一个４８ ｂｐ 的ＩＳ因子， 将ＩＳ因子与ＨＣＶ－Ｊ株全核苷酸序列进行同源性对照， 发现该片段来源于ＨＣＶ Ｅ１区。该研究首次报告了ＨＣＶ ＮＳ５Ａ区有来自Ｅ１区的漂移基因片段， 并对其临床和生物学意义进行了初步讨论。     

Comprehensive scanning of the entire mitochondrial genome for mutations

BACKGROUND: Definitive molecular diagnosis of mitochondrial disorders has been greatly hindered by the tremendous clinical and genetic heterogeneity, the heteroplasmic condition of pathogenic mutations, and the presence of numerous homoplasmic mitochondrial DNA (mtDNA) variations with unknown significance. We used temporal temperature gradient gel electrophoresis (TTGE) to detect heteroplasmic mutations from homoplasmic variations in the whole mitochondrial genome. METHODS: We screened 179 unrelated patients by TTGE with use of 32 overlapping primer pairs. Mutations were identified by direct sequencing of the PCR products and confirmed by PCR with allele-specific oligonucleotide or restriction fragment length polymorphism analysis. RESULTS: We detected 71 heteroplasmic and 647 homoplasmic banding patterns. Sequencing of the heteroplasmic fragments identified 68 distinct novel mutations and 132 reported sequence variations and mutations; most of them occurred only once. The deleterious nature of some of the novel mutations was established by analyzing the asymptomatic family members and the biochemical and molecular characteristics of the mutation. When the number of mutations was normalized to the size of the region, the occurrence of mutations was 2.4 times more frequent in the tRNA genes than in the mRNA (protein coding) regions. CONCLUSIONS: Screening by TTGE detects low proportions of mutant mtDNA and distinguishes heteroplasmic from homoplasmic variations. Results from comprehensive molecular analysis should be followed up with clinical correlation to establish a guideline for complete mutational analysis of the entire mitochondrial genome and to facilitate the diagnosis of mitochondrial disorders.     

Mitochondrial DNA sequence divergence in weevils of the Pissodes strobi species complex (Coleoptera: Curculionidae)

Mitochondrial DNA sequence divergence is unusually high between several species of the Pissodes strobi complex, in contrast to low allozyme and morphological divergences, and the ability to hybridize in the laboratory. We sequenced an 810 bp segment in seven individuals, representing four species in the P. strobi complex and one outgroup species, P. affinis Randall. The 810 bp segment covered the 3' half of the cytochrome oxidase subunit I (COI) gene. We also sequenced one specimen of P. strobi (Peck) over a 2301 bp region of mtDNA, extending from the 5' end of COI to the 3' end of COII. Uncorrected sequence divergences were below 1.1% among three specimens of P. schwarzi Hopkins, and between P. terminalis Hopping and P. nemorensis Germar. All other interspecific combinations in the P. strobi complex showed divergences of 6.0–7.5%. The outgroup species, P. affinis , had an average divergence of 12.8% from members of the P. strobi group. As in most other insects, A+T content in Pissodes mtDNA was high; transition:transversion ratio was high among lineages exhibiting low divergences, but declined with increasing sequence divergence; and inferred amino acid sequence divergences were low. The degree of sequence divergence differs markedly across different functional regions of the COI gene. The high mtDNA divergences within the P. strobi complex calculated from direct sequencing support earlier reports based on restriction site surveys; however, cladograms based on mtDNA sequence differ from those based on earlier work.