直翅目昆虫线粒体基因组研究进展

本文总结了本实验室对40余种直翅目昆虫的线粒体基因组序列的研究方法和主要结果.直翅目线粒体基因组研究中最重要的发现包括：（1）在直翅目昆虫线粒体基因组中发现了3种基因排列次序.蝗亚目除蜢总科外都具有DK排列.蜢总科的变色乌蜢为KD 排列,与蝗亚目其他总科不同,而与螽亚目昆虫的排序方式相同.已测出的螽亚目大多数昆虫的KD 排列顺序与典型节肢动物的完全相同,但在黄脸油葫芦Teleogryllus emma发生了tRNA^Glu,tRNA^Ser和tRNA^Asn的倒置;（2）在疑钩额螽Ruspolia dubia中发现了一种到目前为止具有最短控制区（70 bp）的线粒体基因组;（3）采用多种方法分析了昆虫A+T富集区存在的调控序列和二级结构特征,获得了昆虫A+T富集区保守序列的一致结构.采用Z曲线分析蝗虫的A+T富集区,表明也存在与原核生物复制起点类似的信号;（4）构建了30种蝗虫12S rRNA和16S rRNA的二级结构.在昆虫线粒体基因组非编码链中发现了一些类tRNA结构和tRNA异构体;（5）构建了基于线粒体基因组数据的直翅目昆虫主要亚科以上分类单元之间的系统发育关系.     

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.     

Structure and evolution of teleost mitochondrial control regions

We amplified and sequenced the mitochondrial control region from 23 species representing six families of teleost fish. The length of this segment is highly variable among even closely related species due to the presence of tandemly repeated sequences and large insertions. The position of the repetitive sequences suggests that they arise during replication both near the origin of replication and at the site of termination of the D-loop strand. Many of the conserved sequence blocks (CSBs) observed in mammals are also found among fish. In particular, the mammalian CSB-D is present in all of the fish species studied. Study of potential secondary structures of RNAs from the conserved regions provides little insight into the functional constraints on these regions. The variable structure of these control regions suggests that particular care should be taken to identify the most appropriate segment for studies of intraspecific variation. Correspondence to: T.D. Kocher     

Origin and direction of replication in mitochondrial DNA molecules from the genus Drosophila.

Mitochondrial DNA (mtDNA) obtained from ovaries of Drosophila simulans, D. mauritiana, D. takahashii, D. yakuba and D. virilis was examined by electron microscopy. From a consideration of the structural properties of replicative intermediates, it was concluded that in mtDNA molecules of each species, synthesis on one strand can be up to 97% complete before synthesis on the complementary strand is initiated. MtDNA molecules of each species contain a single A+T-rich region which shows species-specific size variation from 1.0 kb (D. virilis) to 4.8 kb (D. simulans), and maps at the same position in all molecules relative to three common EcoRI sites. The structural properties of complex forms, interpreted as having originated from replicative intermediates, and produced by either partial denaturation or EcoRI digestion, are consistent with the hypothesis that replication is initiated within the A+T-rich region and proceeds unidirectionally around the molecule towards the nearest common EcoRI site. The replication origin is located near the center of the A+T-rich region in D. simulans and D. mauritiana, but lies closer to that end of the A+T-rich region which is distal to the nearest common EcoRI site in D. takahashii, D. yakuba and D. virilis.     

Morphometrical evolution in a Drosophila clade: the Drosophila obscura group

Five morphometrical traits (wing and thorax length, ovariole number, and thoracic and female abdomen pigmentation) were investigated in laboratory stocks of 20 species belonging to the Drosophila obscura group (subgenus Sophophora). These species originated from four biogeographical regions and represent all five of the presently recognized, taxonomic subgroups. Size‐related traits (wing and thorax length) were highly variable across species, and interspecific variation explained more than 90% of total variability. In both traditional and phylogenetic analyses, wing size was positively correlated with latitude of origin. These interspecific correlations were however notably weaker than those for intraspecific correlations. Wing/thorax ratio, which may be related to flight capacity, showed little variation. Ovariole number was highly variable (range 27–53) both within and between species, and was positively correlated with the wing/thorax ratio, suggesting that species with relatively large ovaries have relatively low wing loading. Although many species are completely dark, 11 had some regions of light coloration. A light thorax with a median darkening was observed in six species. A variable pigmentation of abdominal tergites, in females only, was found in nine species, belonging to three subgroups only. With respect to both molecular phylogeny and morphometrical evolution, the D. obscura subgroup is probably now the best investigated clade in Drosophila.     

Molecular Evolution of P Transposable Elements in the Genus Drosophila. II. The obscura Species Group

A phylogenetic analysis of P transposable elements in the Drosophila obscura species group is described. Multiple P sequences from each of 10 species were obtained using PCR primers that flank a conserved region of exon 2 of the transposase gene. In general, the P element phylogeny is congruent with the species phylogeny, indicating that the dominant mode of transmission has been vertical, from generation to generation. One manifestation of this is the distinction of P elements from the Old World obscura and subobscura subgroups from those of the New World affinis subgroup. However, the overall distribution of elements within the obscura species group is not congruent with the phylogenetic relationships of the species themselves. There are at least four distinct subfamilies of P elements, which differ in sequence from each other by as much as 34%, and some individual species carry sequences belonging to different subfamilies. P sequences from D. bifasciata are particularly interesting. These sequences belong to two subfamilies and both are distinct from all other P elements identified in this survey. Several mechanisms are postulated to be involved in determining phylogenetic relationships among P elements in the obscura group. In addition to vertical transmission, these include retention of ancestral polymorphisms and horizontal transfer by an unknown mating-independent mechanism.     

Phylogeny of the Drosophila obscura species group deduced from mitochondrial DNA sequences

Approximately 2 kb corresponding to different regions of the mtDNA of 14 different species of the obscura group of Drosophila have been sequenced. In spite of the uncertainties arising in the phylogenetic reconstruction due to a restrictive selection toward a high mtDNA A+T content, all the phylogenetic analysis carried out clearly indicate that the obscura group is formed by, at least, four well-defined lineages that would have appeared as the consequence of a rapid phyletic radiation. Two of the lineages correspond to monophyletic subgroups (i.e., afftnis and pseudoobscura), whereas the obscura subgroup remains heterogeneous assemblage that could be reasonably subdivided into at least two complexes (i.e., subobscura and obscura).     

Mutational analysis of the bacteriophage phi X174 replication origin

Bacteriophage phi X174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis. A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained. The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type phi X174. Two phi X174 ori mutants with a reduced growth ability spontaneously acquired additional mutations that enhanced the growth rate. The additional mutation was located at the same site as the original mutation or was located in the N-terminal part of the gene A protein. This latter secondary mutation is responsible for a better binding and/or recognition of the gene A protein to the mutated origin. In a Darwinian experiment wild-type phi X174 outgrows all phi X174 ori mutants, indicating the superiority of the wild-type ori sequence for the reproduction of bacteriophage phi 174. Insertions and deletions were constructed at different positions within the phi X174 replication origin cloned in a plasmid. Small insertions and deletions in the A + T-rich spacer region do not inhibit phi X174 gene A protein cleavage in vitro, but severely impair packaging of single-stranded plasmid DNA in viral coats.     

Initiation of DNA replication at cloned origins of bacteriophage T7

Bacteriophage T7 DNA replication is initiated at a site 15% of the distance from the genetic left end of the chromosome. This primary origin contains two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by an A + T-rich region. When the primary origin region is deleted replication initiates at secondary origins. We have analyzed the ability of plasmids containing cloned fragments of T7 to replicate after infection of Escherichia coli with bacteriophage T7. All cloned T7 fragments that support plasmid replication contain a T7 promoter but a T7 promoter alone is not sufficient for replication. Replication of plasmids containing the primary origin is dependent on T7 DNA polymerase and gene 4 protein (helicase/primase) and a portion of the A + T-rich region. The other T7 fragments that support plasmid replication after T7 infection are promoter regions phi OR, phi 13 and phi 6.5 (secondary origins). When both the primary and secondary origins are present simultaneously on compatible plasmids, replication of each is temporally regulated. Such regulation may play a role during T7 DNA replication.     

Concerted evolution of sequence repeats inDrosophila mitochondrial DNA

Summary In the eightDrosophila species of themelanogaster subgroup, the mitochondrial DNA (mtDNA) contains an A+T-rich region in which replication originates. The length of this region, in contrast with that of the coding part of the genome, varies extensively among these species. The A+T-rich region ranges from about 1kbp inD. yakuba, D. teissieri, D. erecta, andD. orena to 5 kbp inD. melanogaster, D. simulans, D. mauritiana, andD. sechellia. The difference in size is due in part to the amplification, in the species with long genomes, of a 470-bp sequence that is present only once in each of the four species with short genomes.Usually three to six repeats of this sequence occur in direct tandem repetition in the species with long genomes. The sequence is characterized by the relative positions of the Hpa I and Acc I cleavage sites. Comparative study of the genomes found in the species with long mtDNA molecules reveals relative homogeneity of the repeat units within a given genome, which contrasts with the variability found among the repeats of different genomes. This result is suggestive of a process of a concerted evolution.The examination of heteroplasmic flies of three species (D. simulans, D. mauritiana, andD. sechellia) has shed light on this process. In most cases the molecular types of mtDNA present in a heteroplasmic individual differ by one repeat unit. Addition or deletion of this sequence appears to be the original mutational event generating transient heteroplasmy. Cycles of addition or deletion may consequently maintain the intragenomic homogeneity of the repeats.Finally, we have analyzed an exceptional isofemale line in which three molecular lengths of mtDNA are found (molecules with four, five, and six repeats, respectively). Individual offspring of this line carry from one to three of the molecular types, in all combinations. This indicates that the remodeling of the mitochondrial genome occurs through a mechanism that is at present unknown, but that is site specific and rather frequent.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

Evolution of the A+T-rich region of mitochondrial DNA in the melanogaster species subgroup of Drosophila

We determined the nucleotide sequences of two regions in the A+T-rich region of mitochondrial DNA (mtDNA) in the siI and siII types of D. simulans, the maII type of D. mauritiana, and D. sechellia. The sequences were aligned with those of the corresponding regions of siIII of D. simulans and maI of D. mauritiana, D. melanogaster, and D. yakuba. The type I and type II elements and the T-stretches were detected in all eight of the mtDNA types compared, indicating that the three elements are essential in the A+T-rich region of this species subgroup. The alignment revealed several short repetitive sequences and relatively large deletions in the central portions of the region. In the highly conserved sequence elements in the type II elements, the substitution rates were not uniform among lineages and acceleration in the substitution rate might have been due to loss of functional constraint in the stem–loop-forming sequences predicted in the type II elements. Patterns of nucleotide substitutions observed in the A+T-rich region were further compared with those in the coding regions and in the intergenic regions of mtDNA. Substitutions between A and T were particularly repressed in the highly conserved sequence elements and in the intergenic regions compared with those in the A+T-rich region excluding the highly conserved sequence elements and in the fourfold degenerate sites in the coding regions. The functional and structural characteristics of the A+T-rich region that might be involved in this substitutional bias are discussed.     

云斑车蝗线粒体基因组全序列测定与分析

采用长距 PCR 扩增及保守引物步移法并结合克隆测序测定并注释了云斑车蝗 Gastrimargus marmoratus （Thunberg）的线粒体基因组全序列。结果表明：云斑车蝗线粒体基因组全序列为15 904 bp（GenBank登录号为EU527334）,A+T含量略高于非洲飞蝗Locusta migratoria,为76.04%,包括13个蛋白质编码基因,22个tRNA 基因,2个rRNA基因和一段1 057 bp的A+T富集区。蛋白质基因的起始密码子中,除COⅠ和ND5为TTG以外,均为昆虫典型的起始密码子ATN。ND5基因使用了不完全终止密码子T,其余基因均为典型的TAA或TAG。预测了22个tRNA基因的二级结构,发现tRNA^Ser（AGN）缺少DHU臂, tRNA^Ser（UGY）的反密码子环上有9个碱基。预测了云斑车蝗12S和16S rRNA二级结构,分别包括3个结构域30个茎环和6个结构域44个茎环。A+T富集区含有3个串联重复序列。     

The complete nucleotide sequence of the mitochondrial DNA genome of the rainbow trout,Oncorhynchus mykiss

The complete nucleotide sequence of the mitochondrial DNA of the rainbow trout, Onchorynchus mykiss, has been determined. The total length of the molecule is 16,660 bp. The rainbow trout mitochondrial DNA has the same organization described in eutherian mammals, the clawed frog (Xenopus laevis), and the two fish species, Oriental stream loach (Crossotoma lacustre) and carp (Cyprinus carpio). Alignment and comparison of the deduced amino acid sequences of the 13 proteins encoded by rainbow trout and other vertebrate mitochondrial genomes allowed us to estimate that COI is the most conserved mitochondrial subunit (amino acid identity ranging from 85.6% to 94.8%) whereas ATPase 8 is the most variable one (amino acid identity ranging from 30.8% to 70.4%). Putative secondary structures for the 22 tRNAs found in the molecule are given along with an extensive comparison of tRNA sequences among representative species of each major group of vertebrates. In this sense, an unusual cloverleaf structure for the tRNA^Ser(AGY) is proposed. A stem-loop structure inferred for the origin of the L-strand replication (O_L) and the presence of a large polycytidine tract in the O_L loop is described. The existence of this stretch instead of the usual T-rich sequence reported so far in mammal mtDNAs is explained in terms of a less-strict template dependence of the RNA primase involved in the initiation of L-strand replication. Correspondence to: J.M. Bautista     

Intraspecific diversity of nucleotide sequences within the adenine + thymine-rich region of mitochondrial DNA molecules of Drosophila mauritiana, Drosophila melanogaster and Drosophila simulans.

Mitochondrial DNA (mtDNA) molecules from Drosophila mauritiana, D. melanogaster, and D. simulans contain a single adenine + thymine (A+T)-rich region, which is similarly located in all molecules, but varies in size among these species. Using agarose gel electrophoresis and electron microscopy, a difference in occurrence of one EcoRI site, and a difference in size (approximately 0.7 kb) of the A+T-rich regions was found between mtDNA molecules of flies of two female lines of D. mauritiana. In heteroduplexes constructed between these two kinds of mtDNA molecules, two or three regions of strand separation, each comprising single strands of unequal length, were apparent near the center of the A+T-rich region. Using the structural differences between D. mauritiana mtDNA molecules it was demonstrated the mtDNA of this species is maternally inherited. Differences in length of A+T-rich regions were also found between mtDNA molecules of two geographically separated strains of D. melanogaster, and between mtDNA molecules of two geographically separated strains of D. simulans. However, in both cases, in heteroduplexes constructed between mtDNA molecules of different strains of one species, the A+T-rich regions appeared completely paired.     

Discrepancy in divergence of the mitochondrial and nuclear genomes ofDrosophila teissieri andDrosophila yakuba

Summary Restriction sites were compared in the mitochondrial DNA (mtDNA) molecules from representatives of two closely related species of fruit flies: nine strains ofDrosophila teissieri and eight strains ofDrosophila yakuba. Nucleotide diversities amongD. teissieri strains and amongD. yakuba strains were 0.07% and 0.03%, respectively, and the nucleotide distance between the species was 0.22%. Also determined was the nucleotide sequence of a 2305-nucleotide pari (ntp) segment of the mtDNA molecule ofD. teissieri that contains the noncoding adenine+thymine (A+T)-rich region (1091 ntp) as well as the genes for the mitochondrial small-subunit rRNA, tRNA^f-met, tRNA^gln, and tRNA^ile, and portions of the ND2 and tRNA^val genes. This sequence differs from the corresponding segment of theD. yakuba mtDNA by base substitutions at 0.1% and 0.8% of the positions in the coding and noncoding regions, respectively. The higher divergence due to base substitutions in the A+T-rich region is accompanied by a greater number of insertions/deletions than in the coding regions. From alignment of theD. teissieri A+T-rich sequence with those ofD. yakuba andDrosophila virilis, it appears that the 40% of this sequence that lies adjacent to the tRNA^ile gene has been highly conserved. Divergence between the entireD. teissieri andD. yakuba mtDNA molecules, estimated from the sequences, was 0.3%; this value is close to the value (0.22%) obtained from the restriction analysis, but 10 times lower than the value estimated from published DNA hybridization results. From consideration of the relationships of mitochondrial nucleotide distance and allozyme genetic distance found among seven species of theDrosophila melanogaster subgroup, the mitochondrial nucleotide distance observed forD. teissieri andD. yakuba is anomalously low in relation to the nuclear genetic distance.     

Nucleotide sequence divergence in the A+T-rich region of mitochondrial DNA in Drosophila simulans and Drosophila mauritiana

We have determined the nucleotide sequences of two regions within the A+T-rich region of mitochondrial DNA (mtDNA) in the siIII type of Drosophila simulans and the maI type of D. mauritiana. The sequences of the two regions in siIII and maI are almost identical. The sequences include elements corresponding to the type I and type II repeats elements and the T-stretches as reported in D. melanogaster; an approximately 340-bp region (A region) adjacent to the tRNA(Ile) gene includes a part of the type II repeat element, and an approximately 440- bp region (B region) includes a central portion of the A+T-rich region between the type I and type II repeat arrays. Each sequence of the two species was compared with those of D. melanogaster and D. yakuba. The sequences of the A region are relatively well conserved among the four species. The alignment of the two sequences of the B region with those of D. melanogaster and D. yakuba requires numerous insertions/deletions. For both regions, nucleotide differences between D. simulans or D. mauritiana and D. melanogaster are similar to those between the two and D. yakuba. The tendency is obvious in a subregion within the type II repeat element in the A region. These findings suggest that the rate of nucleotide substitution in the subregion is accelerated in the lineage leading to D. melanogaster. Loss of functional constraint in the stem-loop-forming sequence is proposed for this acceleration.      

Electron microscope heteroduplex study of Drosophila mitochondrial DNAs: evolution of A+T-rich region.

Mitochondrial DNAs (mtDNA) from three species of the genus Drosophila (D. melanogaster, D. simulons, and D. virilis) were compared by electron microscope heteroduplex mapping. Analysis of heteroduplex molecules revealed that the A + T-rich region of these mtDNAs has undergone quite extensive base sequence divergence, whereas the remainder of the molecule was found to share apparently complete base sequence homology in all three species. The differences in the sizes of the A + T-rich regions, as determined from the heteroduplex measurements, completely account for the differences in the total sizes of these mtDNAs. A segment of approximately 0.1 kb is conserved within the A + T-rich regions of D. simulans and D. virilis mtDNAs, but not within the A + T-rich region of D. melanogaster mtDNA. HaeIII restriction endonuclease analysis of the heteroduplex molecules has further shown that the unique HaeIII site of D. virilis mtDNA molecule is apparently conserved in both D. melanogaster and D. simulans mtDNA molecules. Finally, electrophoretic patterns of HaeIII-digested mtDNAs from all three species were found to be different and distinguishable from each other suggesting that single base substitutions have probably taken place throughout the entire mitochondrial genome.     

Comparison of the two major ARS elements of the ura4 replication origin region with other ARS elements in the fission yeast,Schizosaccharomyces pombe

We have previously peported that the replication orgin region located near the ura4 gene on chromosome III of the fission yeast, Schizosaccharomyces pombe, contains three closely spaced origins, each associated with an autonomously replicating sequence (ARS) element. Here we report the nucleotide sequences of two of these ARS elements, ars3002 and ars3003. The two ARS elements are located on either side of a transcribed 1.5 kb open reading frame. Like 11 other S. pombe ARS elements whose sequences have previously been determined in other laboratories, the 2 new ARS elements are unusually A+T-rich. All 13 ARS elements contain easily unwound stretches of DNA. Each of the ARS elements contains numerous copies, at a higher than expected frequency, of short stretches of A+T-rich DNA in which most of the Ts are on one strand and most of the As are on the complementary strand. We discuss the potential significance for ARS function of these multiple asymmetric A+T-rich sequences.