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     

Evolution of the cetacean mitochondrial D-loop region.

We sequenced the mitochondrial DNA D-loop regions from two cetacean species and compared these with the published D-loop sequences of several other mammalian species, including one other cetacean. Nucleotide substitution rates, DNA sequence simplicity, possible open reading frames (ORFs), and potential RNA secondary structure were investigated. The substitution rate is an order of magnitude lower than would be expected on the basis of reports on human sequence variation in this region but are consistent with interspecific primate and rodent D-loop sequence variation and with estimates of substitution rates from whole mitochondrial genomes. Deletions/insertions are less common in the cetacean D-loop than in other vertebrate species. Areas of high sequence simplicity (clusters of short repetitive motifs) across the region correspond to areas of high sequence divergence. Three regions predicted to form secondary structures are homologous to such putative structures in other species; however, the presumptive structures most conserved in cetaceans are different from those reported for other taxa. While all three species have possible long ORFs, only a short sequence of seven amino acids is shared with other mammalian species, and those changes that had occurred within it are all nonsynonymous. We conclude that DNA slippage, in addition to point mutation, contributes to the evolution of the D-loop and that regions of conserved secondary structure in cetaceans and an ORF are unlikely to contribute significantly to the conservation of the central region.     

Characterization of the red knot (Calidris canutus) mitochondrial control region.

We sequenced the complete mitochondrial control regions of 11 red knots (Calidris canutus). The control region is 1168 bp in length and is flanked by tRNA glutamate (glu) and the gene ND6 at its 5' end and tRNA phenylalanine (phe) and the gene 12S on its 3' end. The sequence possesses conserved sequence blocks F, E, D, C, CSB-1, and the bird similarity box (BSB), as expected for a mitochondrial copy. Flanking tRNA regions show correct secondary structure, and a relative rate test indicated no significant difference between substitution rates in the sequence we obtained versus the known mitochondrial sequence of turnstones (Charadriiformes: Scolopacidae). These characteristics indicate that the sequence is mitochondrial in origin. To confirm this, we sequenced the control region of a single individual using both purified mitochondrial DNA and genomic DNA. The sequences were identical using both methods. The sequence and methods presented in this paper may now serve as a reference for future studies using knot and other avian control regions. Furthermore, the discovery of five variable sites in 11 knots towards the 3' end of the control region, and the variability of this region in contrast to the more conserved central domain in the alignment between knots and other Charadriiformes, highlights the importance of this area as a source of variation for future studies in knots and other birds.     

Structural organization of the mitochondrial DNA control region in Aedes aegypti.

The complete A+T - rich region of Aedes aegypti mitochondrial DNA has been cloned and sequenced. In Argentinean populations of the species, a polymorphism in the length of the amplified fragment was observed. Nucleotide sequence comparison of the shortest and longest A+T - rich amplified fragments detected revealed the presence of 2 types of tandemly repeated blocks. The size variation observed in natural populations is mainly due to the presence of a variable number of a 181 bp tandem repeat unit, located toward the 12S rRNA gene end. The size of the longest A+T - rich region was of 2070 bp, representing the largest control sequence reported for any mosquito species. Few relevant short blocks of primary-sequence similarity conserved in the control region of mosquitoes and other insects were detected scattered throughout the whole region. Five putative stem-loop secondary structures were found, one of them flanked by conserved sequences described in other insects. Our results suggest that there are no universal models of structure-function relations in the control region of insect mtDNA. In addition, we identified a short A+T - rich variable segment in the Ae. aegyti control region that would be suitable for population genetic studies.     

Sequence length and variation in the mitochondrial control region of two freshwater gobiid fishes belonging to Rhinogobius (Teleostei: Gobioidei)

The control region (D-loop) of mitochondrial DNA (mtDNA) was amplified and sequenced for eight samples of the rhinogobies Rhinogobius maculafasciatus and R. giurinus from Taiwan and southern China. The control regions of both species are of 841–842 bp; the length of these sequences being the most compact among all known sequences in teleost fishes. Three conserved sequence blocks (CSB) were observed. The full D-loop and tRNA Phe gene sequences were determined and compared with other fishes. The interspecific sequence divergence between the two species is 11.3–11.7%; and the intraspecific variation in R. guirinus 0.8–1.8%. Results suggest that the control region of Rhinogobius is informative for phylogenetic reconstruction at both intraspecific and interspecific levels in this gobiid genus.     

An unusual source of apparent mitochondrial heteroplasmy: duplicate mitochondrial control regions in Thalassarche albatrosses

Molecular ecologists, in search of suitable molecular markers, frequently PCR-amplify regions of mitochondrial DNA from total DNA extracts. This approach, although common, is prone to the co-amplification of nuclear copies of transposed DNA sequences (numts), which can then generate apparent mitochondrial sequence heteroplasmy. In this study we describe the discovery of apparent mitochondrial sequence heteroplasmy in Thalassarche albatrosses but eliminate the possibility of true sequence heteroplasmy and numts and instead reveal the source of the apparent heteroplasmy to be a duplicated control region. The two control regions align easily but are not identical in sequence or in length. Comparisons of functionally significant conserved sequence blocks do not provide evidence of degeneration in either duplicate. Phylogenetic analyses of domain I of both control region copies in five Thalassarche species indicate that they are largely evolving in concert; however, a short section within them is clearly evolving independently. To our knowledge this is the first time contrasting evolutionary patterns have been reported for duplicate control regions. Available evidence suggests that this duplication may be taxonomically widespread, so the results presented here should be considered in future evolutionary studies targeting the control region of all Procellariiformes and potentially other closely related avian groups.     

Sequence variation in the guillemot (Alcidae: Cepphus) mitochondrial control region and its nuclear homolog

We describe sequence variation in the mitochondrial control region and its nuclear homolog in three species and seven subspecies of guillemots (Cepphus spp.). Nuclear homologs of the 5' end of the control region were found in all individuals. Nuclear sequences were approximately 50% divergent from their mitochondrial counterparts and formed a distinct phylogenetic clade; the mitochondrial-nuclear introgression event must have predated the radiation of Cepphus. As in other vertebrates, the guillemot control region has a relatively conserved central block flanked by hypervariable 5' and 3' ends. Mean pairwise interspecific divergence values among control regions were lower than those in other birds. All individuals were heteroplasmic for the number of simple tandem nucleotide repeats (A(n)C) at the 3' end of the control region. Phylogenetic analyses suggest that black guillemots are basal to pigeon and spectacled guillemots, but evolutionary relationships among subspecies remain unresolved, possibly due to incomplete lineage sorting. Describing molecular variation in nuclear homologs of mitochondrial genes is of general interest in phylogenetics because, if undetected, the homologs may confound interpretations of mitochondrial phylogenies.      

Slow rate of evolution in the mitochondrial control region of gulls (Aves: Laridae)

We sequenced part of the mitochondrial control region and the cytochrome b gene in 72 specimens from 32 gull species (Laridae, Larini) and 2 outgroup representatives (terns: Laridae, Sternini). Our control region segment spanned the conserved central domain II and the usually hypervariable 3' domain III. Apart from some heteroplasmy at the 3' end of the control region, domain III was not more variable than domain II or the cytochrome b gene. Furthermore, variation in the tempo of evolution of domain III was apparent between phyletic species groups. The lack of variation of the gull control region could not be explained by an increase in the proportion of conserved sequences in these birds, and the gull control region showed an organization similar to those of other avian control regions studied to date. A novel invariant direct repeat was identified in domain II of gulls, and in domain III, two to three inverted, sometimes imperfect, repeats are able to form a significantly stable stem-and-loop structure. These putative secondary structures have not been reported before, and a comparison between species groups showed that they are more stable in the group with the more conserved control region. The unusually slow rate of evolution of control region part III of the gulls could thus be partly explained by the existence of secondary structures in domain III of these species.     

DNA sequence variation in the mitochondrial control region of red-backed voles (Clethrionomys)

The complete mitochondrial DNA (mtDNA) control region was sequenced for 71 individuals from five species of the rodent genus Clethrionomys both to understand patterns of variation and to explore the existence of previously described domains and other elements. Among species, the control region ranged from 942 to 971 bp in length. Our data were compatible with the proposal of three domains (extended terminal associated sequences [ETAS], central, conserved sequence blocks [CSB]) within the control region. The most conserved region in the control region was the central domain (12% of nucleotide positions variable), whereas in the ETAS and CSB domains, 22% and 40% of nucleotide positions were variable, respectively. Tandem repeats were encountered only in the ETAS domain of Clethrionomys rufocanus. This tandem repeat found in C. rufocanus was 24 bp in length and was located at the 5' end of the control region. Only two of the proposed CSB and ETAS elements appeared to be supported by our data; however, a "CSB1-like" element was also documented in the ETAS domain.     

DMRT gene cluster analysis in the platypus: new insights into genomic organization and regulatory regions

We isolated and characterized a cluster of platypus DMRT genes and compared their arrangement, location, and sequence across vertebrates. The DMRT gene cluster on human 9p24.3 harbors, in order, DMRT1, DMRT3, and DMRT2, which share a DM domain. DMRT1 is highly conserved and involved in sexual development in vertebrates, and deletions in this region cause sex reversal in humans. Sequence comparisons of DMRT genes between species have been valuable in identifying exons, control regions, and conserved nongenic regions (CNGs). The addition of platypus sequences is expected to be particularly valuable, since monotremes fill a gap in the vertebrate genome coverage. We therefore isolated and fully sequenced platypus BAC clones containing DMRT3 and DMRT2 as well as DMRT1 and then generated multispecies alignments and ran prediction programs followed by experimental verification to annotate this gene cluster. We found that the three genes have 58-66% identity to their human orthologues, lie in the same order as in other vertebrates, and colocate on 1 of the 10 platypus sex chromosomes, X5. We also predict that optimal annotation of the newly sequenced platypus genome will be challenging. The analysis of platypus sequence revealed differences in structure and sequence of the DMRT gene cluster. Multispecies comparison was particularly effective for detecting CNGs, revealing several novel potential regulatory regions within DMRT3 and DMRT2 as well as DMRT1. RT-PCR indicated that platypus DMRT1 and DMRT3 are expressed specifically in the adult testis (and not ovary), but DMRT2 has a wider expression profile, as it does for other mammals. The platypus DMRT1 expression pattern, and its location on an X chromosome, suggests an involvement in monotreme sexual development.     

The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.      

两种毛蚜线粒体基因组比较分析

【目的】线粒体基因组分析已被应用于昆虫系统发育研究。本研究以蚜科Aphididae重要类群毛蚜亚科物种为代表,测定并比较分析了该类蚜虫的线粒体基因组特征,探讨了基于线粒体基因组信息的蚜虫系统发育关系重建。【方法】以毛蚜亚科三角枫多态毛蚜Periphyllus acerihabitans Zhang和针茅小毛蚜Chaetosiphella stipae Hille Ris Lambers,1947为研究对象,利用长短PCR相结合的方法测定线粒体基因组的序列,分析了基因组的基本特征;基于在线t RNAscan-SE Search Server搜索方法预测了t RNA的二级结构;基于12个物种(本研究获得的2个物种和10个Gen Bank上下载的物种数据)的蛋白编码基因(PCGs)序列,利用最大似然法和贝叶斯法重建了蚜科的系统发育关系。【结果】两种毛蚜均获得了约94%的线粒体基因组数据,P.acerihabitans获得了14 908 bp,控制区为1 205 bp;C.stipae获得了13 893 bp,控制区为609 bp。两种毛蚜同时获得33个基因,包含接近完整的13个蛋白编码基因(PCGs)(nad5不完整),18个tRNA,2个rRNA基因;ka/ks值表明,C.stipae的进化速率更快。从基因组组成、基因排列顺序、核苷酸组成分析、密码子使用情况、t RNA二级结构等特征来分析,两种蚜虫线粒体基因组基本特征相似。系统发育重建结果表明毛蚜亚科、蚜亚科的单系性得到了支持,毛蚜亚科位于蚜科的基部位置。【结论】两种毛蚜线粒体基因组的基本特征相似,符合蚜虫线粒体基因组的一般特征,两种线粒体基因组的长度差异主要来自控制区长度的不同;系统发育重建支持毛蚜亚科与蚜亚科的单系性,毛蚜亚科位于蚜科较为基部的位置。研究结果为蚜虫类系统发育重建提供了参考。     

Origin and evolution of homologous repeated sequences in the mitochondrial DNA control region of shrews

The complete mitochondrial DNA (mtDNA) control region was amplified and directly sequenced in two species of shrew, Crocidura russula and Sorex araneus (Insectivora, Mammalia). The general organization is similar to that found in other mammals: a central conserved region surrounded by two more variable domains. However, we have found in shrews the simultaneous presence of arrays of tandem repeats in potential locations where repeats tend to occur separately in other mammalian species. These locations correspond to regions which are associated with a possible interruption of the replication processes, either at the end of the three-stranded D-loop structure or toward the end of the heavy-strand replication. In the left domain the repeated sequences (R1 repeats) are 78 bp long, whereas in the right domain the repeats are 12 bp long in C. russula and 14 bp long in S. araneus (R2 repeats). Variation in the copy number of these repeated sequences results in mtDNA control region length differences. Southern blot analysis indicates that level of heteroplasmy (more than one mtDNA form within an individual) differs between species. A comparative study of the R2 repeats in 12 additional species representing three shrew subfamilies provides useful indications for the understanding of the origin and the evolution of these homologous tandemly repeated sequences. An asymmetry in the distribution of variants within the arrays, as well as the constant occurrence of shorter repeated sequences flanking only one side of the R2 arrays, could be related to asymmetry in the replication of each strand of the mtDNA molecule. The pattern of sequence and length variation within and between species, together with the capability of the arrays to form stable secondary structures, suggests that the dominant mechanism involved in the evolution of these arrays in unidirectional replication slippage.      

The complete DNA sequence of the mitochondrial genome of the self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the first description of duplication of a control region in fish

We isolated Rivulus marmoratus mitochondrial DNA by long-polymerase chain reaction with conserved primers, and sequenced it with 36 sets of internal conserved primers, which were designed from the extensive sequence similarities of mitochondrial DNA from several fish species. The R. marmoratus mitochondrial DNA has 17,329 bp with a conserved structural organization compared to those of other fish. Rivulus marmoratus mitochondrial DNA also has two nearly identical control regions. The basic characteristics of the R. marmoratus mitochondrial genome are discussed.     

Sequence and organization of the complete mitochondrial genomes of spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri).

In this work, the mitochondrial genomes for spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri) were completely sequenced. The entire mitochondrial genome sequences of the spotted halibut and barfin flounder were 17,273 and 17,588 bp in length, respectively. The organization of the two mitochondrial genomes was similar to those reported from other fish mitochondrial genomes containing 37 genes (2 rRNAs, 22 tRNAs and 13 protein-coding genes) and two non-coding regions (control region (CR) and WANCY region). In the CR, the termination associated sequence (ETAS), six central conserved block (CSB-A,B,C,D,E,F), three conserved sequence blocks (CSB1-3) and a region of 61-bp tandem repeat cluster at the end of CSB-3 were identified by similarity comparison with fishes and other vertebrates. The tandem repeat sequences show polymorphism among the different individuals of the two species. The complete mitochondrial genomes of spotted halibut and barfin flounder should be useful for evolutionary studies of flatfishes and other vertebrate species.     

圆斑星鲽及相关种类线粒体DNA控制区结构分析

采用PCR产物直接测序法测定了圆斑星鲽(Verasper variegatus)的24个个体的线粒体控制区(Control region)核苷酸全序列, 并进行了结构分析。结果表明, 圆斑星鲽线粒体控制区核苷酸全序列具有长度多态性, 得到4种长度单元型, 主要表现为控制区中的串联重复序列的长度不同。对鲽形目鱼类如鲽科的条斑星鲽(Verasper moseri)、黄盖鲽(Limanda feruginea)、马舌鲽 (Reinhardtius hippoglossoides), 美洲拟庸鲽(Heppoglossoides platessoides )和鲆科的牙鲆(Paralichthys olivaceus)以及鳎科的欧洲鳎(Solea solea)、塞内加尔鳎(S. senegalensis)和沙鳎(S. lascari)的控制区的比较研究发现, 鲽形目鱼类的线粒体控制区均存在相似的结构, 即线粒体控制区可分为终止相关序列区(ETAS)、中央保守区(包括CSB-A、CSB-B、CSB-C、CSB-D、CSB-E、CSB-F)以及保守序列区(CSB1、CSB2、CSB3)和重复序列区(Repeat region)4个区域。通过与脊椎动物各个纲线粒体控制区序列的比较分析, 发现只有鲽形目(包括鲆、鲽类和鳎类)鱼类和两栖纲的无尾类在CSB-3之后存在相似的串联重复序列。     

Structure of mitochondrial DNA control region of Fenneropenaeus chinensis and phylogenetic relationship among different populations

This paper deals with the structure of mitochondrial DNA control region of Fenneropenaeus chinensis. The termination-associated sequence (TAS), cTAS, CSB-D-CSB-F, and CSB-1 are detected in the species. The results indicate that the structures of these parts are similar to those of most marine organisms. Two conserved regions and many stable conserved boxes are found in the extended TAS area, central sequences blocks, and conserved sequences blocks (CSBs). This is the special character of F. chinensis. All the mtDNA control region sequences do not have CSB2 and CSB3 blocks, which is quite different from most vertebrates. In addition, the complete mtDNA control region sequences are used to analyze the phylogenetic relationships of F. chinensis. The phylogenetic trees show a lack of genetic structure among populations, which is similar to many previous studies.     

Structure and evolution of the avian mitochondrial control region

The structural and evolutionary characteristics of the mitochondrial control region were studied by using control region sequences of 68 avian species. The distribution of the variable nucleotide positions within the control region was found to be genus specific and not dependant on the level of divergence, as suggested before. Saturation was shown to occur at the level of divergence of 10% in pairwise comparisons of the control region sequences, as has also been reported for the third codon positions in ND2 and cytochrome b genes of mtDNA. The ratio of control region vs cytochrome b divergence in pairwise comparisons of the sequences was shown to vary from 0.13 to 21.65, indicating that the control region is not always the most variable region of the mtDNA, but also that there are differences in the rate of divergence among the lineages. Only two of the conserved sequence blocks localized earlier for other species, D box and CSB-1, were found to show a considerable amount of sequence conservation across the avian and mammalian sequences. Additionally, a novel avian-specific sequence block was found.     

Evidence that the large noncoding sequence is the main control region of maternally and paternally transmitted mitochondrial genomes of the marine mussel (Mytilus spp.)

Both the maternal (F-type) and paternal (M-type) mitochondrial genomes of the Mytilus species complex M. edulis/galloprovincialis contain a noncoding sequence between the l-rRNA and the tRNA(Tyr) genes, here called the large unassigned region (LUR). The LUR, which is shorter in M genomes, is capable of forming secondary structures and contains motifs of significant sequence similarity with elements known to have specific functions in the sea urchin and the mammalian control region. Such features are not present in other noncoding regions of the F or M Mytilus mtDNA. The LUR can be divided on the basis of indels and nucleotide variation in three domains, which is reminiscent of the tripartite structure of the mammalian control region. These features suggest that the LUR is the main control region of the Mytilus mitochondrial genome. The middle domain has diverged by only 1.5% between F and M genomes, while the average divergence over the whole molecule is approximately 20%. In contrast, the first domain is among the most divergent parts of the genome. This suggests that different parts of the LUR are under different selection constraints that are also different from those acting on the coding parts of the molecule.