PCR amplification using a single cell allows the detection of the mtDNA lesion associated with Leber's hereditary optic neuropathy

The development of the polymerase chain reaction (PCR), which routinely can amplify specific target sequences more than one billion-fold, has made it possible to produce readily detectable amounts of DNA from a few copies of very rare sequences. We have begun a study of mitochondrial myopathies with the purpose of developing a diagnostic test using PCR to amplify appropriate mitochondrial DNA (mtDNA) target sequences from small amounts of sample. We have developed a 15-min procedure for recovering mtDNA which can be amplified by PCR to detectable levels, from as little as 30 μl of blood or 5 μl of amniotic fluid. We have microscopically selected HL60 cells, and have found that 28 cycles of PCR allows the detection of mitochondrial targets from a single cell. Using micromanipulation techniques, we utilized this approach to analyze mtDNA from a single cell isolated from an 8-cell stage mouse blastocyst. Finally, a single cell cultured from a patient with Leber's hereditary optic neuropathy, a mitochondrial myopathy, provided sufficient mtDNA for detection of the single base substitution that leads to loss of a restriction endonuclease recognition site for SfaNI and generation of a site for MaeIII.     

No evidence for an mtDNA role in sperm motility: data from complete sequencing of asthenozoospermic males

The first complete mitochondrial DNA (mtDNA) sequences (approximately 16,569 bp) in 20 patients with asthenozoospermia and a comparison with 23 new complete mtDNA sequences in teratoasthenozoospermic individuals, confirmed no sharing of specific polymorphisms or specific mitochondrial lineages between these individuals. This is strong evidence against the accepted claim of a major role played by mtDNA in male fertility, once supported by haplogroup association studies based on the screening of hypervariable region I. The hypothesis of maternally driven selection acting in male reproductive success must thus be treated with caution.     

Obtaining high-quality DNA from elusive small mammals using low-tech hair snares

Noninvasive sampling approaches are becoming increasingly important for enabling genetic studies of wildlife populations. While a number of methods have been described to noninvasively sample hair from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. Here we describe a novel and inexpensive noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We explore the quality of the sample by assessing PCR amplification success of mitochondrial and nuclear DNA fragments across four commercially available DNA isolation kits and two different quantities of hair in a factorial design. Additionally, we determined the sex of the individual samples using PCR–RFLP of ZFX/ZFY loci. We found that our snare is effective in obtaining hair that yield DNA of sufficient quality and quantity to successfully amplify a range of mitochondrial and nuclear fragment sizes. Specifically, we found the greatest success in amplifying mitochondrial DNA, nuclear microsatellites and ZFX/ZFY loci using at least 25 hairs as starting material and the DNA IQ™ system. The hair snares thus provide a cost-effective and minimally intrusive approach to sample elusive or rare small mammals. We anticipate that this approach will be useful to obtain samples for molecular studies of the ecology, evolution and conservation of small, elusive mammals.     

Non-invasive genetic identification of small mammal species using real-time polymerase chain reaction

DNA identification of non-invasive samples is a potentially useful tool for monitoring small mammal species. Here we describe a novel method for identifying five small mammal species: wood mouse, bank vole, common shrew, pygmy shrew and water shrew. Species-specific real-time polymerase chain reaction primers were designed to amplify fragments of the mitochondrial cytochrome b gene from hair and scat samples. We also amplified nuclear DNA from scats, demonstrating their potential as a source of DNA for population genetic studies.     

Characterization of the structure and DNA complexity of mung bean mitochondrial nucleoids

Electron microscopic images of mitochondrial nucleoids isolated from mung bean seedlings revealed a relatively homogeneous population of particles, each consisting of a chromatin-like structure associated with a membrane component. Association of F-actin with mitochondrial nucleoids was also observed. The mitochondrial nucleoid structure identified in situ showed heterogeneous genomic organization. After pulsed-field gel electrophoresis (PFGE), a large proportion of the mitochondrial nucleoid DNA remained in the well, whereas the rest migrated as a 50–200 kb smear zone. This PFGE migration pattern was not affected by high salt, topoisomerase I or latrunculin B treatments; however, the mobility of a fraction of the fastmoving DNA decreased conspicuously following an in-gel ethidium-enhanced UV-irradiation treatment, suggesting that molecules with intricately compact structures were present in the 50-200 kb region. Approximately 70% of the mitochondrial nucleoid DNA molecules examined via electron microscopy were open circles, supercoils, complex forms, and linear molecules with interspersed sigma-shaped structures and/or loops. Increased sensitivity of mtDNA to DNase I was found after mitochondrial nucleoids were pretreated with high salt. This result indicates that some loosely bound or peripheral DNA binding proteins protected the mtDNA from DNase I degradation.     

(鱼兆)科鱼类细胞色素b基因片段的序列测定及其系统发育的初步研究

从9种(鱼兆)科鱼类的福尔马林标本中获得了333 bp的细胞色素b基因片段的序列.这9个种分别代表(鱼兆)科鱼类的8个属.333 bp的DNA序列经MUST软件排序后,有101个变异位点,其中有39个信息位点.序列在成对物种间的距离为8～48.平均遗传距离为2.4%～14.4%.简约分析产生了最大简约系统树,其步长是162(CI=0.735,RI=0.494).在该系统树上,Bagarius是最原始的属,并与所有其他的物种形成姊妹群.其余8个属形成一个单系类群并分为二个姊妹群.尽管在形态上具有13个离征,但在分子系统树上,(鱼宴)(鱼兆)鱼类并未形成一个单系类群.可能的原因是333 bp序列中的星系信息位点太少;另外单从福尔马林浸制标本获得的DNA序列的可靠性尚有待进一步验证.     

Improved COI barcoding primers for Southeast Asian perching birds (Aves: Passeriformes)

The All Birds Barcoding Initiative aims to assemble a DNA barcode database for all bird species, but the 648-bp 'barcoding' region of cytochrome c oxidase subunit I (COI) can be difficult to amplify in Southeast Asian perching birds (Aves: Passeriformes). Using COI sequences from complete mitochondrial genomes, we designed a primer pair that more reliably amplifies and sequences the COI barcoding region of Southeast Asian passerine birds. The 655-bp region amplified with these primers overlaps the COI region amplified with other barcoding primer pairs, enabling direct comparison of sequences with previously published DNA barcodes.     

Fine characterization of the Iceman's mtDNA haplogroup

Starting from specimens of the intestinal contents of the so-called Tyrolean Iceman or Otzi (5,350-5,100 years before present), it was possible by polymerase chain reaction to amplify fragments of the human mitochondrial DNA (mtDNA) control region that correspond to the sequence found in 1994 at the Munich and Oxford laboratories and which had been attributed to the original DNA of the mummy. The particularly favorable condition of the specimens, showing very low contamination levels, made it easier to extend the analyses to the coding region, which had not previously been considered. The mtDNA of the European population is currently divided into nine (H, T, U, V, W, X, I, J, and K) main groups (haplogroups). The K haplogroup, in particular, is composed of two (K1 and K2) subclusters. The results demonstrate that the Iceman's mtDNA belongs to the K1 subcluster, yet it does not fit any of the three known branches (a, b, and c) into which the K1 subcluster is presently divided. In addition, some other sites, reported to be linked to environmental adaptation or pathologies, were investigated.     

Amassing diversity in an ancient lake: evolution of a morphologically diverse parthenogenetic gastropod assemblage in Lake Malawi

Exceptional ecological niche diversity, clear waters and unique divergent selection pressures have often been invoked to explain high morphological and genetic diversity of taxa within ancient lakes. However, it is possible that in some ancient lake taxa high diversity has arisen because these historically stable environments have allowed accumulation of lineages over evolutionary timescales, a process impossible in neighbouring aquatic habitats undergoing desiccation and reflooding. Here we examined the evolution of a unique morphologically diverse assemblage of thiarid gastropods belonging to the Melanoides polymorpha'complex' in Lake Malawi. Using mitochondrial DNA sequences, we found this Lake Malawi complex was not monophyletic, instead sharing common ancestry with Melanoides anomala and Melanoides mweruensis from the Congo Basin. Fossil calibrations of molecular divergence placed the origins of this complex to within the last 4 million years. Nuclear amplified fragment length polymorphism markers revealed sympatric M. polymorpha morphs to be strongly genetically differentiated lineages, and males were absent from our samples indicating that reproduction is predominantly parthenogenetic. These results imply the presence of Lake Malawi as a standing water body over the last million years or more has facilitated accumulation of clonal morphological diversity, a process that has not taken place in more transient freshwater habitats. As such, the historical stability of aquatic environments may have been critical in determining present spatial distributions of biodiversity.     

Mitochondrial genome in animal cells

In the past decade, the development of new DNA, RNA, and protein technologies has greatly incremented the knowledge about the organization and expression of mitochondrial DNA. The complete base sequence of mitochondrial DNA of several animals is known and many data are rapidly accumulating on the mitochondrial genomes of other systems. Here we discuss the results so far obtained that disclosed unexpected features of mitochondrial genetics. Furthermore, mitochondrial DNA has become established as a powerful tool for evolutionary studies in animals. Evidences are preented demonstrating that the evolution of mitochondrial DNA has proceeded in different ways in the various taxonomic groups. Data on heteroplasmic animals, which demonstrate the rapid evolution of mitochondrial DNA, are also presented.     

High resolution analysis and phylogenetic network construction using complete mtDNA sequences in sardinian genetic isolates

For mitochondrial phylogenetic analysis, the best result comes from complete sequences. We therefore decided to sequence the entire mitochondrial DNA (mtDNA) (coding and D-loop regions) of 63 individuals selected in 3 small Ogliastra villages, an isolated area of eastern Sardinia: Talana, Urzulei, and Perdasdefogu. We studied at least one individual for each of the most frequent maternal genealogical lineages belonging to haplogroups H, V, J, K, T, U, and X. We found in our 63 samples, 172 and 69 sequence changes in the coding and in the D-loop region, respectively. Thirteen out of 172 sequence changes in the coding region are novel. It is our hypothesis that some of them are characteristic of the Ogliastra region and/or Sardinia. We reconstructed the phylogenetic network of the 63 complete mtDNA sequences for the 3 villages. We also drew a network including a large number of European sequences and calculated various indices of genetic diversity in Ogliastra. It appears that these small populations remained extremely isolated and genetically differentiated compared with other European populations. We also identified in our samples a never previously described subhaplogroup, U5b3, which seems peculiar to the Ogliastra region.     

Noncoding mitochondrial loci for corals

We developed five degenerate primer pairs for the amplification and sequencing of two noncoding regions found in the mitochondrial genome of corals. These primers amplify products ranging from 380 to 950 bp, and work in a wide variety of scleractinian taxa from both the Pacific and Caribbean. Based on our initial analysis of ～300 sequences from 13 scleractinian taxa, both these noncoding regions appear to have equivalent levels of variability to the most variable of previously published coral mitochondrial loci, but work in a wider variety of taxa. We believe these primers will be of use to coral biologists studying questions above the level of species; as with other mithochondrial DNA markers in corals, these loci will likely provide little resolution for within‐species studies.     

Complete mitochondrial DNA sequence of Aulopus japonicus (Teleostei: Aulopiformes), a basal Eurypterygii: longer DNA sequences and higher-level relationships

For the first step toward resolution of the higher-level relationships of the order Aulopiformes (Teleostei: Eurypterygii) using longer DNA sequences, we determined the complete mitochondrial DNA sequence for Aulopus japonicus (Aulopodidae). The entire genome was purified by gene amplification using a long PCR technique, and the products were subsequently used as templates for PCR with 63 fish-versatile and 3 species-specific primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products demonstrated that the genome (16 653 base pairs [bp]) contained the same 37 mitochondrial genes (2 ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as found in other vertebrates, with the gene order identical to that in typical vertebrates. Maximum-parsimony analysis using nucleotide sequences from the concatenated 12 protein-coding genes (no third codon positions and excluding the ND6 gene) plus 22 tRNA genes (stem regions only) from eight teleosts placed A. japonicus in a reasonable phylogenetic position; those from individual protein-coding genes and the concatenated 22 tRNA genes alone, however, did not reproduce the expected phylogeny with few exceptions, probably owing to insufficient phylogenetic information in these smaller data sets. This result suggests that further taxonomic sampling and sequencing efforts may clarify limits and intra- and interrelationships of this morphologically and ecologically diverse group of fishes using mitochondrial genomic (mitogenomic) data. Received: August 31, 2000 / Revised: December 20, 2000 / Accepted: January 23, 2001     

PRELIMINARY STUDY ON MITOCHONDRIAL CYTOCHROME b DNA SEQUENCES AND PHYLOGENY OF FORMALIN FIXED SISORID FISHES (Teleostei:Siluriformes)

从９种科鱼类的福尔马林标本中获得了３３３ｂｐ的细胞色素ｂ基因片段的序列。这９个种分别代表科鱼类的８个属。３３３ｂｐ的ＤＮＡ序列经ＭＵＳＴ软件排序后,有１０１个变异位点,其中有３９个信息位点。序列在成对物种间的距离为８～４８。平均遗传距离为２４％～１４４％。简约分析产生了最大简约系统树,其步长是１６２（ＣＩ＝０７３５,ＲＩ＝０４９４）。在该系统树上,Ｂａｇａｒｉｕｓ是最原始的属,并与所有其他的物种形成姊妹群。其余８个属形成一个单系类群并分为二个姊妹群。尽管在形态上具有１３个离征,但在分子系统树上,鱼类并未形成一个单系类群。可能的原因是３３３ｂｐ序列中的星系信息位点太少;另外单从福尔马林浸制标本获得的ＤＮＡ序列的可靠性尚有待进一步验证     

鱼类线粒体DNA研究新进展

线粒体DNA是分子生物学研究中的一个热门领域,已成为鱼类进化生物学和群体遗传学研究的重要分子遗传标记。本文对鱼类线粒体DNA分子生物学的最新研究进展进行了较详细的阐述。重点介绍鱼类线粒体DNA全序列的研究进展、组成及特征,鱼类线粒体DNA非编码区结构研究进展,鱼类线粒体DNA多态性及其主要的检测方法;综述了最近有关鱼类线粒体DNA在鱼类系统学、种间杂交渐渗、种群识别、起源和进化、地理分化等研究中的应用情况。     

Population structure of the Indonesian giant tiger shrimp Penaeus monodon: a window into evolutionary similarities between paralogous mitochondrial DNA sequences and their genomes

Here we used both microsatellites and mtCR (mitochondrial DNA control region) sequences as genetic markers to examine the genetic diversity and population structure of Penaeus monodon shrimp from six Indonesian regions. The microsatellite data showed that shrimp from the Indian and the Pacific Ocean were genetically distinct from each other. It has been reported previously that P. monodon mtCR sequences from the Indo‐Pacific group into two major paralogous clades of unclear origin. Here we show that the population structure inferred from mtCR sequences matches the microsatellite‐based population structure for one of these clades. This is consistent with the notion that this mtCR clade shares evolutionary history with nuclear DNA and may thus represent nuclear mitochondrial pseudogenes (Numts).     

The mitochondrial genome of the Basidiomycete fungus Pleurotus ostreatus (oyster mushroom)

In this study, the full mitochondrial genome of a basidiomycete fungus, Pleurotus ostreatus, was sequenced and analyzed. It is a circular DNA molecule of 73 242 bp and contains 44 known genes encoding 18 proteins and 26 RNA genes. The protein-coding genes include 14 common mitochondrial genes, one ribosomal small subunit protein 3 gene, one RNA polymerase gene and two DNA polymerase genes. In addition, one RNA and one DNA polymerase genes were identified in a mitochondrial plasmid. These two genes show relatively low similarities to their homologs in the mitochondrial genome but they are nearly identical to the known mitochondrial plasmid genes from another Pleurotus ostreatus strain. This suggests that the plasmid may mediate the horizontal gene transfer of the DNA and RNA polymerase genes into mitochondrial genome, and such a transfer may be an ancient event. Phylogenetic analysis based on the cox1 ORFs verified the traditional classification of Pleurotus ostreatus among fungi. However, the discordances were observed in the phylogenetic trees based on the six cox1 intronic ORFs of Pleurotus ostreatus and their homologs in other species, suggesting that these intronic ORFs are foreign DNA sequences obtained through HGT. In summary, this analysis provides valuable information towards the understanding of the evolution of fungal mtDNA.     

Locating the sources of an invasive pest,grape phylloxera,using a mitochondrial DNA gene genealogy

Range expansions through human introductions have increased with global commerce and have led to the extinction of native species, alterations in community structure and pest status of the invasive species. Inferring the evolutionary history of invasive species can help to build a firmer footing for management tactics. This study used mitochondrial DNA (mtDNA) sequence comparisons of samples collected from the native and introduced ranges of a pest herbivore of cultivated grapes, grape phylloxera (Daktulosphaira vitifoliae Fitch, Phylloxeridae) to infer the sources and pattern of introductions into worldwide viticulture. Introductions into viticulture from its native North American range first occurred in the mid-19th century. The pattern of spread has suggested a focus of introduction into France, but independent introductions may have occurred elsewhere. The results show that the introduced population represents a limited subsample of the native genetic diversity. The data suggest that most grape phylloxera in viticulture, including all European, have originated in the northeastern USA where the grape species Vitis riparia dominates. There was evidence for independent introductions into South Africa and California. Most California haplotypes were most closely related to native grape phylloxera from the Atlantic Coast on V. vulpina. It is likely that subsequent spread from California into Australia, New Zealand and Peru has occurred.     

Multiple origins and rapid evolution of duplicated mitochondrial genes in parthenogenetic geckos (Heteronotia binoei; Squamata, Gekkonidae)

Accumulating evidence for alternative gene orders demonstrates that vertebrate mitochondrial genomes are more evolutionarily dynamic than previously thought. Several lineages of parthenogenetic lizards contain large, tandem duplications that include rRNA, tRNA, and protein-coding genes, as well as the control region. Such duplications are hypothesized as intermediate stages in gene rearrangement, but the early stages of their evolution have not been previously studied. To better understand the evolutionary dynamics of duplicated segments of mitochondrial DNA, we sequenced 10 mitochondrial genomes from recently formed ( approximately 300,000 years ago) hybrid parthenogenetic geckos of the Heteronotia binoei complex and 1 from a sexual form. These genomes included some with an arrangement typical of vertebrates and others with tandem duplications varying in size from 5.7 to 9.4 kb, each with different gene contents and duplication endpoints. These results, together with phylogenetic analyses, indicate independent and frequent origins of the duplications. Small, direct repeats at the duplication endpoints imply slipped-strand error as a mechanism generating the duplications as opposed to a false initiation/termination of DNA replication mechanism that has been invoked to explain duplications in other lizard mitochondrial systems. Despite their recent origin, there is evidence for nonfunctionalization of genes due primarily to deletions, and the observed pattern of gene disruption supports the duplication-deletion model for rearrangement of mtDNA gene order. Conversely, the accumulation of mutations between these recent duplicates provides no evidence for gene conversion, as has been reported in some other systems. These results demonstrate that, despite their long-term stasis in gene content and arrangement in some lineages, vertebrate mitochondrial genomes can be evolutionary dynamic even at short timescales.