Variation of Human Mitochondrial DNA: Distribution of Hot Spots in Hypervariable Segment I of the Major Noncoding Region

Mitochondrial DNA (mtDNA) samples belonging to fifteen phylogenetically related mtDNA types specific to the populations of Europe (H, V, J, T, U, K, I, W, and X) and Northern Asia (A, C, D, G, Y, and Z) were typed for sequence variation in hypervariable segment I (HVSI). The approach used allowed to distinguish several hypervariable sites at nucleotide positions 16093, 16129, 16189, 16311, and 16362. Identical mutations at these sites were found in 10–11 out of 15 mtDNA groups examined. Positions 16126, 16172, 16192, 16256, 16261, 16291, 16293, and 16298 appeared to be less variable, since parallel mutations at these sites were found in 6–8 European and Asian mtDNA groups. The examples of the effects of mutations in hypervariable positions at the major noncoding mtDNA region on the frequency of reverse mutations in other mtDNA regions are presented. It was shown that such effects of nucleotide context on the mutation rate could be observed in phylogenetic mtDNA networks such as cyclic structures like rhombs and cubes. Analogous structures in the networks could be seen also in the case of the appearance of recombinant mtDNA types resulted from homologous recombination between mtDNA molecules in heteroplasmic mixture. The problem of the effect of polynucleotide context on the intensity of mtDNA mutagenesis is discussed. Recombination processes along with site-directed mutagenesis caused by action of genetic factors (of nuclear genome) and/or of the environment are considered as possible mechanisms of mitochondrial genome evolution.     

Some physicochemical properties of rice mitochondrial DNA

Summary Certain physicochemical properties of rice mitochondrial DNA (mtDNA) were determined. Certain low-molecular-weight mtDNA bands were found in addition to the major mtDNA band. Rice mtDNA appeared in the electron microscope as a collection of linear molecules with heterogeneous length in the range of 1–156 kb. The major distribution area was 60–105 kb. A small fraction (less than 5%) of rice mtDNA was found in the form of a circular molecule. Some molecules had the appearance of being supercoiled. Replication fork structures were found in both circular and linear mtDNA molecules. In one rice species, Jin Nante, 15 different circular molecules were found. Rice mtDNA was digested with different restriction enzymes. The total molecular weight of rice mtDNA was calculated to be about 300 kb according to the data of restriction enzyme digestion and electron microscopy.     

Variation of human mitochondrial DNA: distribution of hot spots in hypervariable segment I of the major noncoding region

Mitochondrial DNA (mtDNA) samples belonging to fifteen phylogenetically related mtDNA types specific to the populations of Europe (H, V, J, T, U, K, I, W, and X) and Northern Asia (A, C, D, G, Y, and Z) were typed for sequence variation in hypervariable segment I (HVSI). The approach used allowed to distinguish several hypervariable sites at nucleotide positions 16093, 16129, 16189, 16311, and 16362. Identical mutations at these sites were found in 10-11 out of 15 mtDNA groups examined. Positions 16126, 16172, 16192, 16256, 16261, 16291, 16293, and 16298 appeared to be less variable, since parallel mutations at these sites were found in 6-8 European and Asian mtDNA groups. The examples of the effects of mutations in hypervariable positions at the major noncoding mtDNA region on the frequency of reverse mutations in other mtDNA regions are presented. It was shown that such effects of nucleotide context on the mutation rate could be observed in phylogenetic mtDNA networks such as cyclic structures like rhombs and cubes. Analogous structures in the networks could be seen also in the case of the appearance of recombinant mtDNA types resulted from homologous recombination between mtDNA molecules in heteroplasmic mixture. The problem of the effect of polynucleotide context on the intensity of mtDNA mutagenesis is discussed. Recombination processes along with site-directed mutagenesis caused by action of genetic factors (of nuclear genome) and/or of the environment are considered as possible mechanisms of mitochondrial genome evolution.     

Mitochondrial DNA recombination in Brassica campestris

The structure of the mitochondrial genome in plants is unclear, but appears to consist of mostly linear DNA with some other structures, including branched molecules and subgenomic circles. Mitochondrial DNA (mtDNA) recombination was analyzed in Brassica campestris, which has one of the smallest mitochondrial genomes (218 kb) in higher plants. Field-inversion gel electrophoresis (FIGE) separated mtDNA into discrete populations that each represents the entire genome. Electron microscopy revealed large, mostly linear molecules trapped in the wells, slower migrating populations with mostly linear DNA and a low level of circular and networked mtDNA molecules of 10–140 kbp, and a fast migrating population of 10–50 kbp linear mtDNA. Some smaller than genome size circular molecules and circles with tails were observed, and may represent recombination or rolling circle replication intermediates. Hybridization of end-labeled mtDNA suggests there may be specific ends (or recombination hotspots) for some linear molecules. Analysis of mtDNA enriched by BND-cellulose and separated by two-dimensional agarose gel electrophoresis shows the presence of complex recombination structures and the presence of significant single-stranded regions in mtDNA. These findings provide further evidence that DNA recombination contributes to the complex structure of mtDNA in plants.     

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。     

Resolution of mitochondrial DNA structures in the large yeast Wickerhamia fluorescens

DAPI (4′6 Diamidino-2-phenylindole) staining of the large yeast Wickerhamia fluorescens revealed extensive cytoplasmic staining material attributable to mitochondrial DNA (mtDNA), often present in large network-like structures. The maintenance of this mtDNA appears to be insensitive to a variety of mitochondrial specific mutagens, suggesting that W. fluorescens may be classified as a petite negative yeast. Restriction enzyme analysis generated a unit genome size of 42(10⁶) D for this mtDNA which, together with determinations of the average mtDNA per cell, allowed an estimate of the cellular copy number of mitochondrial genomes. A physical map of this mtDNA was also derived. These experiments suggested models which might reflect the cytological structures resolved by DAPI staining in W. fluorescens relative to other yeasts.     

"Cold" spots in hypervariable segment 1 of human mitochondrial DNA

Analysis of distribution of mutations in hypervariable segment 1 (HVS1) of mitochondrial DNA (mtDNA) in data set of more than 37,000 individuals, representing different regions of the world, allowed one to estimate the mutation processes intensity and peculiarity of mtDNA "cold" spots distribution. Results of analysis of structure-function organization and variability of the mtDNA fragment under study revealed that decrease of variability is characteristic for HVS1 sequences with probable functional importance. Analysis of distribution of "cold" spots CAT in secondary structures testified against the correlation between "cold" spot location and mtDNA structure type.     

区域人群mtDNA序列聚类分析

人类mtDNA序列是遵循母系遗传的重要生物信息学资源,利用遗传算法和k-modes模型结合的聚类算法,对西安和长沙两个区域人群mtDNA序列进行聚类分析,在分子层次上阐明了西安和长沙两地区人口结构特点.发现西安地区人口是发散性分布,而长沙地区人口具有主导性类群.     

Mitochondrial genomes of two demosponges provide insights into an early stage of animal evolution

Mitochondrial DNA (mtDNA) of multicellular animals (Metazoa) is typically a small ( approximately 16 kbp), circular-mapping molecule that encodes 37 tightly packed genes. The structures of mtDNA-encoded transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) are usually highly unorthodox, and proteins are translated with multiple deviations from the standard genetic code. In contrast, mtDNA of the choanoflagellate Monosiga brevicollis, the closest unicellular relative of animals, is four times larger, contains 1.5 times as many genes, and lacks mentioned peculiarities of animal mtDNA. To investigate the evolutionary transition that led to the specific organization of metazoan mtDNA, we determined complete mitochondrial sequences from the demosponges Geodia neptuni and Tethya actinia, two representatives of the most basal animal phylum, the Porifera. We found that poriferan mtDNAs resemble those of other animals in their compact organization, lack of introns, and a well-conserved animal-like gene order. Yet, they contain several extra genes, encode bacterial-like rRNAs and tRNAs, and use a minimally derived genetic code. Our findings suggest that the evolution of the typical metazoan mtDNA has been a multistep process in which the compact genome organization and the reduced gene content were established prior to the reduction of tRNA and rRNA structures and the introduction of multiple changes of the translation code.     

Cold spots of human mitochondrial DNA hypervariable segment 1

The distribution of mutations in hypervariable segment 1 (HVS1) of mitochondrial DNA (mtDNA) was analyzed for more than 37000 individuals from various regions of the world. The results were used to estimate the intensity of mutation processes and the features of the cold spot distribution in mtDNA. Analysis of the structural-functional organization and variation of HVS1 made it possible to associate a lower variation with functionally important HVS1 regions. The distribution of CAT cold spots in secondary DNA structures revealed a lack of correlation between the cold spot location and the structural type of the mtDNA region.     

Disturbed population genetics: suspected introgressive hybridization between two Mnais damselfly species (Odonata)

Mnais costalis and M. pruinosa are damselflies (Odonata: Calopterygidae) with low dispersal abilities, both during their aquatic stream-living immature stage and their flying adult stage. A previous nuclear DNA (nDNA) sequencing and morphology study showed that these two species are very closely related, and cohabit widely in western Japan. The two species, however, segregate microhabitats along a stream: M. costalis lives in the lower reaches, and M. pruinosa in the upper reaches. In this study, our analyses were based on mitochondrial DNA (mtDNA), which usually mutates faster and is more variable among individuals than nDNA, and which is inherited maternally. We found that most COI haplotypes were shared between the two species, and that for most study sites interspecific riverine genetic structures were not clarified by mtDNA analysis. Incongruent population genetic structures based on nDNA and mtDNA suggested hybridization and introgression of mtDNA between the two species.     

Large scale mtDNA sequencing reveals sequence and functional conservation as major determinants of homoplasmic mtDNA variant distribution

The mitochondrial DNA (mtDNA) is highly variable, containing large numbers of pathogenic mutations and neutral polymorphisms. The spectrum of homoplasmic mtDNA variation was characterized in 730 subjects and compared with known pathogenic sites. The frequency and distribution of variants in protein coding genes were inversely correlated with conservation at the amino acid level. Analysis of tRNA secondary structures indicated a preference of variants for the loops and some acceptor stem positions. This comprehensive overview of mtDNA variants distinguishes between regions and positions which are likely not critical, mainly conserved regions with pathogenic mutations and essential regions containing no mutations at all.     

Characterization of DNA-Binding Proteins Involved in the Assembly of Mitochondrial Nucleoids in the Yeast Saccharomyces cerevisiae

Mitochondrial nucleoids (mt-nucleoids) isolated from the yeastSaccharomyces cerevisiae were analyzed to identify the proteincomponents that are involved in the compact packaging of mtDNA.The isolated mt-nucleoids were disassembled by the additionof 2 M NaCl and the disassembled mt-nucleoids were reassembledonce again into compact structures by dialysis against a bufferthat contained NaCl at concentrations below 0.1 M, as monitoredby staining of the DNA with 4',6-diamidino-2-phenylindole. DNA-binding proteins with molecular masses of 67 kDa, 52 kDa,50 kDa, 38 kDa, 30 kDa and 20 kDa were separated from isolatedmt-nucleoids by column chromatography on DNA cellulose afterdigestion of mt-nucleoids by DNase I in the presence or absenceof 2 M NaCl. Purified mtDNA was compactly packaged into nucleoid-likestructures upon the addition of fractions that contained DNA-bindingproteins and subsequent dialysis to reduce the concentrationof NaCl. Five proteins, with molecular masses of 67 kDa, 52kDa, 50 kDa, 38 kDa and 30 kDa, respectively, had lower affinityfor double-stranded DNA than that of the 20-kDa protein. Thefraction that contained the five DNA-binding proteins otherthan the 20-kDa protein was also able to fold mtDNA compactlyinto nucleoid-like structures. By contrast, the combinationof the 20-kDa protein and mtDNA resulted in formation of lesstightly packed, string-of-bead structures. These results suggestthat at least six different DNA-binding proteins are involvedin the organization of the mt-nucleoids. (Received April 7, 1995; Accepted July 10, 1995)     

A single mitochondrial lineage is shared by morphologically and allozymatically distinct freshwater Corbicula clones

Despite that the exotic invasion and rapid range expansion of Asian freshwater Corbicula into new environments have been of intensive research topic in freshwater ecology, the genetic structures of freshwater Corbicula in its native range remain poorly understood. In this study, the genetic structures of two Korean freshwater Corbicula clonal lineages were characterized by cross-referencing the nuclear genomic structures with mtDNA sequence analysis. In spite of substantial genetic differences (Nei's D = 0.363-0.372) and a pronounced level of fixed allelic distinctions (in six of 20 allozyme loci) between Corbicula lineages, no lineage-specific mtDNA differentiation was observed. The evident disjunction between mtDNA sequences and nuclear genomes is a compelling evidence for the existence of interspecific nuclear hybrid genome structures, comprising different combinations of paternal and maternal contributions. This unusual novel finding is the first case demonstrating that morphologically and allozymatically distinct, yet mitochondrially identical clonal lineages exist in the genus Corbicula. However, we could not find the ancestral species for these two clonal lineages in the present study, and the answer for this question must wait until the genetic structure of Asian Corbicula taxa is fully characterized.     

Association of G-quadruplex forming sequences with human mtDNA deletion breakpoints

Background

Mitochondrial DNA (mtDNA) deletions cause disease and accumulate during aging, yet our understanding of the molecular mechanisms underlying their formation remains rudimentary. Guanine-quadruplex (GQ) DNA structures are associated with nuclear DNA instability in cancer; recent evidence indicates they can also form in mitochondrial nucleic acids, suggesting that these non-B DNA structures could be associated with mtDNA deletions. Currently, the multiple types of GQ sequences and their association with human mtDNA stability are unknown.

Results

Here, we show an association between human mtDNA deletion breakpoint locations (sites where DNA ends rejoin after deletion of a section) and sequences with G-quadruplex forming potential (QFP), and establish the ability of selected sequences to form GQ in vitro. QFP contain four runs of either two or three consecutive guanines (2G and 3G, respectively), and we identified four types of QFP for subsequent analysis: intrastrand 2G, intrastrand 3G, duplex derived interstrand (ddi) 2G, and ddi 3G QFP sequences. We analyzed the position of each motif set relative to either 5'' or 3'' unique mtDNA deletion breakpoints, and found that intrastrand QFP sequences, but not ddi QFP sequences, showed significant association with mtDNA deletion breakpoint locations. Moreover, a large proportion of these QFP sequences occur at smaller distances to breakpoints relative to distribution-matched controls. The positive association of 2G QFP sequences persisted when breakpoints were divided into clinical subgroups. We tested in vitro GQ formation of representative mtDNA sequences containing these 2G QFP sequences and detected robust GQ structures by UV–VIS and CD spectroscopy. Notably, the most frequent deletion breakpoints, including those of the "common deletion", are bounded by 2G QFP sequence motifs.

Conclusions

The potential for GQ to influence mitochondrial genome stability supports a high-priority investigation of these structures and their regulation in normal and pathological mitochondrial biology. These findings emphasize the potential importance of helicases that subsequently resolve GQ to maintain the stability of the mitochondrial genome.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-677) contains supplementary material, which is available to authorized users.     

A Rolling Circle Replication Mechanism Produces Multimeric Lariats of Mitochondrial DNA in Caenorhabditis elegans

Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans.     

Analysis of Plasmodium falciparum mitochondrial six-kilobase DNA by pulse-field electrophoresis

The 6-kb mtDNA of Plasmodium falciparum is thought to replicate by a recombination-dependent mechanism generating large complex branched structures. For technical reasons, including shearing caused by DNA extraction methods, a meaningful quantitative comparison of large complex mtDNA forms has not been feasible. With the use of pulse-field gel electrophoresis, which minimizes any loss or shearing of DNA, we were able to identify an unusually slow migrating population of mtDNA that was resolved from the 6-23-kb population of linear concatemers. Levels of this slow-migrating population of mtDNA were highest during early schizont stage, suggesting that these forms represent replication intermediates. This approach provides a convenient means to monitor the presence of large mtDNA structures in P. falciparum.