Obtaining mice that carry human mitochondrial DNA transmitted to the progeny

To study human diseases associated with mutations in mitochondrial DNA one needs an animal model in which the distribution of abnormal mtDNA and its impact on the phenotype might be followed. We isolated human mitochondria from HepG2 cell culture and microinjected them into murine zygotes, upon which those were transplanted to the pseudopregnant mice. PCR with species-specific primers allowed detecting human mtDNA in the tissues of 7-13-day embryos. No serious alterations in the development of transmitochondrial embryos were noticed. Among various organs/tissues of the 13-day embryos, human mtDNA was detected only in the heart, skeletal muscles, and stomach, which is in line with its uneven distribution among the blastomeres of an early mouse embryo that we described previously. In four recipient females, the microinjected zygotes were allowed to develop to term, the four neonate males of their joint litter were sacrificed, and in three of them human mtDNA was detected in the heart, skeletal muscles, stomach, brain, testes, and bladder. Six females of that joint litter were grown and mated to intact males. In the progeny (F1) of one of the females two mice were carrying human mtDNA in the heart, skeletal muscles, stomach, brain, lungs, uterus, ovaries, and kidneys. The study confirms the possibility to obtain transmitochondrial mice carrying human mtDNA that is transmitted to the animals of the next generation. Our results also indicate that among the organs to which human mtDNA is distributed some are more likely to receive it than others.     

Paternal transmission of mitochondrial DNA as an integral part of mitochondrial inheritance in metapopulations of Drosophila simulans

Maternal inheritance is one of the hallmarks of animal mitochondrial DNA (mtDNA) and central to its success as a molecular marker. This mode of inheritance and subsequent lack of heterologous recombination allows us to retrace evolutionary relationships unambiguously down the matriline and without the confounding effects of recombinant genetic information. Accumulating evidence of biparental inheritance of mtDNA (paternal leakage), however, challenges our current understanding of how this molecule is inherited. Here, using Drosophila simulans collected from an East African metapopulation exhibiting recurring mitochondrial heteroplasmy, we conducted single fly matings and screened F1 offspring for the presence of paternal mtDNA using allele-specific PCR assays (AS–PCR). In all, 27 out of 4092 offspring were identified as harboring paternal mtDNA, suggesting a frequency of 0.66% paternal leakage in this species. Our findings strongly suggest that recurring mtDNA heteroplasmy as observed in natural populations of Drosophila simulans is most likely caused by repeated paternal leakage. Our findings further suggest that this phenomenon to potentially be an integral part of mtDNA inheritance in these populations and consequently of significance for mtDNA as a molecular marker.     

Single-cell A3243G mitochondrial DNA mutation load assays for segregation analysis.

Segregation of mitochondrial DNA (mtDNA) is an important underlying pathogenic factor in mtDNA mutation accumulation in mitochondrial diseases and aging, but the molecular mechanisms of mtDNA segregation are elusive. Lack of high-throughput single-cell mutation load assays lies at the root of the paucity of studies in which, at the single-cell level, mitotic mtDNA segregation patterns have been analyzed. Here we describe development of a novel fluorescence-based, non-gel PCR restriction fragment length polymorphism method for single-cell A3243G mtDNA mutation load measurement. Results correlated very well with a quantitative in situ Padlock/rolling circle amplification-based genotyping method. In view of the throughput and accuracy of both methods for single-cell A3243G mtDNA mutation load determination, we conclude that they are well suited for segregation analysis.     

Evidence for extreme sequence divergence between the male‐ and female‐transmitted mitochondrial genomes in the bivalve mollusc,Modiolus modiolus (Mytilidae)

Marine mussels of the family Mytilidae, as well as a number of other bivalves, have a unique system of mitochondrial DNA inheritance called doubly uniparental inheritance (DUI). DUI is characterized by the presence of an ‘F’ mitochondrial genome that is transmitted through mothers to daughters and sons, and an ‘M’ mitochondrial genome that is transmitted only from fathers to sons. In this paper, we demonstrate that DUI exists in the horse mussel, Modiolus modiolus (Linnaeus, 1758) and compare the pattern of molecular evolution of the M and F types in this species. Total DNA was isolated from M. modiolus male and female gonad tissues, as well as from spawned sperm cells. From these DNA samples, partial mitochondrial DNA fragments were amplified from both cytochrome c oxidase subunit I (cox1), and 16S ribosomal RNA (rrnL) genes. Based on cox1 and rrnL sequences, heteroplasmy was observed in M. modiolus and characterized by the resolution of two mitotypes: an F mitotype present in tissues of both males and females, and an M mitotype present in spawned sperm. Using standardized p‐distance and Tamura‐Nei values, M. modiolus is found to display the highest M/F conspecific sequence divergence for any member of the family Mytilidae (i.e. 38% M/F sequence divergence, which is 9% higher than any other intraspecific M/F comparison for the family Mytilidae when standardized using p‐distances across all taxa observed). Sequence analysis also indicated that the M. modiolus M mitotype evolves significantly faster than its conspecific F type. The findings discussed herein broaden the range of mytilid species known to exhibit DUI and they also establish a new threshold for the genetic divergence of male mytilid mitochondrial genomes.     

PATERNAL LEAKAGE OF MITOCHONDRIAL DNA IN A FUCUS (PHAEOPHYCEAE) HYBRID ZONE1

Eukaryotic mitochondria are mostly uniparentally (maternally) inherited, although mtDNA heteroplasmy has been reported in all major lineages. Heteroplasmy, the presence of more than one mitochondrial genome in an individual, can arise from recombination, point mutations, or by occasional transmission of the paternal mtDNA (=paternal leakage). Here, we report the first evidence of mtDNA paternal leakage in brown algae. In Denmark, where Fucus serratus L. and Fucus evanescens C. Agardh have hybridized for years, we found eight introgressed individuals that possessed the very distinct haplotypes of each parental species. The finding of heteroplasmy in individuals resulting from several generations of backcrosses suggests that paternal leakage occurred in earlier generations and has persisted through several meiotic bottlenecks.     

Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome

So‐called ρ0 cells lack mitochondrial DNA and are therefore incapable of aerobic ATP synthesis. How cells adapt to survive ablation of oxidative phosphorylation remains poorly understood. Complexome profiling analysis of ρ0 cells covered 1,002 mitochondrial proteins and revealed changes in abundance and organization of numerous multiprotein complexes including previously not described assemblies. Beyond multiple subassemblies of complexes that would normally contain components encoded by mitochondrial DNA, we observed widespread reorganization of the complexome. This included distinct changes in the expression pattern of adenine nucleotide carrier isoforms, other mitochondrial transporters, and components of the protein import machinery. Remarkably, ablation of mitochondrial DNA hardly affected the complexes organizing cristae junctions indicating that the altered cristae morphology in ρ0 mitochondria predominantly resulted from the loss of complex V dimers required to impose narrow curvatures to the inner membrane. Our data provide a comprehensive resource for in‐depth analysis of remodeling of the mitochondrial complexome in response to respiratory deficiency.     

Site heteroplasmy in the mitochondrial cytochrome b gene of the sterlet sturgeon Acipenser ruthenus

Sturgeons are fish species with a complex biology. They are also characterized by complex aspects including polyploidization and easiness of hybridization. As with most of the Ponto-Caspian sturgeons, the populations of Acipenser ruthenus from the Danube have declined drastically during the last decades. This is the first report on mitochondrial point heteroplasmy in the cytochrome b gene of this species. The 1141 bp sequence of the cytb gene in wild sterlet sturgeon individuals from the Lower Danube was determined, and site heteroplasmy evidenced in three of the 30 specimens collected. Two nucleotide sequences were identified in these heteroplasmic individuals. The majority of the heteroplasmic sites are synonymous and do not modify the sequence of amino acids in cytochrome B protein. To date, several cases of point heteroplasmy have been reported in animals, mostly due to paternal leakage of mtDNA. The presence of specific point heteroplasmic sites might be interesting for a possible correlation with genetically distinct groups in the Danube River.     

Tissue segregation of mitochondrial haplotypes in heteroplasmic Hawaiian bees: implications for DNA barcoding

The issue of mitochondrial heteroplasmy has been cited as a theoretical problem for DNA barcoding but is only beginning to be examined in natural systems. We sequenced multiple DNA extractions from 20 individuals of four Hawaiian Hylaeus bee species known to be heteroplasmic. All species showed strong differences at polymorphic sites between abdominal and muscle tissue in most individuals, and only two individuals had no obvious segregation. Two specimens produced completely clean sequences from abdominal DNA. The fact that these differences are clearly visible by direct sequencing indicates that substantial intra-individual mtDNA diversity may be overlooked when DNA is taken from small tissue fragments. At the same time, differences in haplotype distribution among individuals may result in incorrect recognition of cryptic species. Because DNA barcoding studies typically use only a small fragment of an organism, they are particularly vulnerable to sequencing bias where heteroplasmy and haplotype segregation are present. It is important to anticipate this possibility prior to undertaking large-scale barcoding projects to reduce the likelihood of haplotype segregation confounding the results.     

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.     

Frequency and Pattern of Heteroplasmy in the Control Region of Human Mitochondrial DNA

In this work, we present the results of the screening of human mitochondrial DNA (mtDNA) heteroplasmy in the control region of mtDNA from 210 unrelated Spanish individuals. Both hypervariable regions of mtDNA were amplified and sequenced in order to identify and quantify point and length heteroplasmy. Of the 210 individuals analyzed, 30% were fully homoplasmic and the remaining presented point and/or length heteroplasmy. The prevalent form of heteroplasmy was length heteroplasmy in the poly(C) tract of the hypervariable region II (HVRII), followed by length heteroplasmy in the poly(C) tract of hypervariable region I (HVRI) and, finally, point heteroplasmy, which was found in 3.81% of the individuals analyzed. Moreover, no significant differences were found in the proportions of the different kinds of heteroplasmy in the population when blood and buccal cell samples were compared. The pattern of heteroplasmy in HVRI and HVRII presents important differences. Moreover, the mutational profile in heteroplasmy seems to be different from the mutational pattern detected in population. The results suggest that a considerable number of mutations and, particularly, transitions that appear in heteroplasmy are probably eliminated by drift and/or by selection acting at different mtDNA levels of organization. Taking as a whole the results reported in this work, it is mandatory to perform a broad-scale screening of heteroplasmy to better establish the heteroplasmy profile which would be important for medical, evolutionary, and forensic proposes.     

Evidence for a two membrane-spanning autonomous mitochondrial DNA replisome

The unit of inheritance for mitochondrial DNA (mtDNA) is a complex nucleoprotein structure termed the nucleoid. The organization of the nucleoid as well as its role in mtDNA replication remain largely unknown. Here, we show in Saccharomyces cerevisiae that at least two populations of nucleoids exist within the same mitochondrion and can be distinguished by their association with a discrete proteinaceous structure that spans the outer and inner mitochondrial membranes. Surprisingly, this two membrane-spanning structure (TMS) persists and self-replicates in the absence of mtDNA. We tested whether TMS functions to direct the replication of mtDNA. By monitoring BrdU incorporation, we observed that actively replicating nucleoids are associated exclusively with TMS. Consistent with TMS's role in mtDNA replication, we found that Mip1, the mtDNA polymerase, is also a stable component of TMS. Taken together, our observations reveal the existence of an autonomous two membrane-spanning mitochondrial replisome as well as provide a mechanism for how mtDNA replication and inheritance may be physically linked.     

Geographically specific heteroplasmy of mitochondrial DNA in the seaweed, Fucus serratus (Heterokontophyta: Phaeophyceae, Fucales)

The presence of more than one type of mitochondrial DNA within the same organism (mtDNA heteroplasmy) has been reported in vertebrates, invertebrates, basidiomycetes and some angiosperms, but never in marine (macro)algae. We examined sequence differences in a 135‐base pair (bp) region of the nad11 gene in mitochondria of the intertidal rockweed, Fucus serratus, using single‐strand conformation polymorphism (SSCP). Each of 70 and 22 individuals from Blushøj (Denmark) and Oskarshamn (Sweden), respectively, displayed haplotypes 2, 3, and 4 (= mtDNA heteroplasmy), whereas only haplotype 2 was found in each of 24 individuals from locations in Spain, France, Ireland, Iceland and Norway. As Blushøj and Oskarshamn were among the last areas to emerge from ice cover during the Last Glacial Maximum (18 000–20 000 years bp ), the geographically specific heteroplasmy may represent a founder effect and therefore, a valuable marker for understanding the role of post‐Ice Age recolonization. Geographically specific heteroplasmy also has important implications in phylogeographical studies based on mtDNA sequences.     

Mitochondrial DNA somatic mutation burden and heteroplasmy are associated with chronological age,smoking, and HIV infection

The gradual accumulation of mitochondrial DNA (mtDNA) mutations is implicated in aging and may contribute to the accelerated aging phenotype seen with tobacco smoking and HIV infection. mtDNA mutations are thought to arise from oxidative damage; however, recent reports implicate polymerase γ errors during mtDNA replication. Investigations of somatic mtDNA mutations have been hampered by technical challenges in measuring low‐frequency mutations. We use primer ID‐based next‐generation sequencing to quantify both somatic and heteroplasmic blood mtDNA point mutations within the D‐loop, in 164 women and girls aged 2–72 years, of whom 35% were smokers and 56% were HIV‐positive. Somatic mutations and the occurrence of heteroplasmic mutations increased with age. While transitions are theorized to result from polymerase γ errors, transversions are believed to arise from DNA oxidative damage. In our study, both transition and transversion mutations were associated with age. However, transition somatic mutations were more prevalent than transversions, and no heteroplasmic transversions were observed. We also measured elevated somatic mutations, but not heteroplasmy, in association with high peak HIV viremia. Conversely, heteroplasmy was higher among smokers, but somatic mutations were not, suggesting that smoking promotes the expansion of preexisting mutations rather than de novo mutations. Taken together, our results are consistent with blood mtDNA mutations increasing with age, inferring a greater contribution of polymerase γ errors in mtDNA mutagenesis. We further suggest that smoking and HIV infection both contribute to the accumulation of mtDNA mutations, though in different ways.     

Human mitochondrial function during cardiac growth and development

Little information is presently available concerning mitochondrial respiratory and oxidative phosphorylation function in the normal human heart during growth and development. We investigated the levels of specific mitochondrial enzyme activities and content during cardiac growth and development from the early neonatal period (10-20 days) to adulthood (67 years). Biochemical analysis of enzyme specific activities and content and mitochondrial DNA (mtDNA) copy number was performed with left ventricular tissues derived from 30 control individuals. The levels of cytochrome c oxidase (COX) and complex V specific activity, mtDNA copy number and COX subunit II content remained unchanged in contrast to increased citrate synthase (CS) activity and content. The developmental increase in CS activity paralleled increasing CS polypeptide content, but was neither related to overall increases in mitochondrial number nor coordinately regulated with mitochondrial respiratory enzyme activities. Our findings of unchanged levels of cardiac mitochondrial respiratory enzyme activity during the progression from early childhood to older adult contrasts with the age-specific regulation found with CS, a Krebs cycle mitochondrial enzyme.     

Evidence of doubly uniparental inheritance of the mitochondrial DNA in Polititapes rhomboides (Bivalvia,Veneridae): Evolutionary and population genetic analysis of F and M mitotypes

Doubly uniparental inheritance (DUI) is a particular mitochondrial DNA inheritance mode reported in a number of bivalves. DUI species show two types of mtDNA, one transmitted from females to daughters and sons (F mitotype) and another one from males to sons (M mitotype). In Veneridae, the existence of DUI has been investigated in several species but it was found in only two of them. In this study, we obtained partial sequences of rrnL, cytb and cox1 genes of males and females of Polititapes rhomboides from NW Spain and we demonstrated the existence of heteroplasmy in males, as expected under DUI. F and M mitotypes showed a taxon-specific phylogenetic pattern and similar evolutionary rates. We focused on cox1 for population genetic analysis, examining separately F and M mitotypes, but also F mitotypes from females (F_♀) and males (F_♂). In all cases, cox1 bears signs of strong purifying selection, with no apparent evidence of relaxed selection in the M genome, while the divergence between F and M genomes is in agreement with the neutral model of evolution. The cox1 polymorphism, higher at the M than at the F genome, also shows clear footprints of genetic hitchhiking with favourable mutations at other mtDNA loci, except for F_♂. In terms of population structure, results suggest that the pattern depends on the examined mitotype (F, F_♀, F_♂ or M).     

In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice

In mtDNA mutator mice, mtDNA mutations accumulate leading to a rapidly aging phenotype. However, there is little evidence of oxidative damage to tissues, and when analyzed ex vivo, no change in production of the reactive oxygen species (ROS) superoxide and hydrogen peroxide by mitochondria has been reported, undermining the mitochondrial oxidative damage theory of aging. Paradoxically, interventions that decrease mitochondrial ROS levels in vivo delay onset of aging. To reconcile these findings, we used the mitochondria‐targeted mass spectrometry probe MitoB to measure hydrogen peroxide within mitochondria of living mice. Mitochondrial hydrogen peroxide was the same in young mutator and control mice, but as the mutator mice aged, hydrogen peroxide increased. This suggests that the prolonged presence of mtDNA mutations in vivo increases hydrogen peroxide that contributes to an accelerated aging phenotype, perhaps through the activation of pro‐apoptotic and pro‐inflammatory redox signaling pathways.     

Of circles, forks and humanity: Topological organisation and replication of mammalian mitochondrial DNA

The organisation of mammalian mitochondrial DNA (mtDNA) is more complex than usually assumed. Despite often being depicted as a simple circle, the topology of mtDNA can vary from supercoiled monomeric circles over catenanes and oligomers to complex multimeric networks. Replication of mtDNA is also not clear cut. Two different mechanisms of replication have been found in cultured cells and in most tissues: a strand-asynchronous mode involving temporary RNA coverage of one strand, and a strand-coupled mode rather resembling conventional nuclear DNA replication. In addition, a recombination-initiated replication mechanism is likely to be associated with the multimeric mtDNA networks found in human heart. Although an insight into the general principles and key factors of mtDNA organisation and maintenance has been gained over the last few years, there are many open questions regarding replication initiation, termination and physiological factors determining mtDNA organisation and replication mode. However, common themes in mtDNA maintenance across eukaryotic kingdoms can provide valuable lessons for future work.     

Analysis of sequence homology between human and mouse mitochondrial DNA

The base sequence homology between human and mouse mitochondrial DNA has been investigated by hybridization of highly labelled mitochondrial DNA probes with restriction fragments of mitochondrial DNA blotted according to the Southern technique. By this analysis, the homologous regions have been found to be widely distributed along the mitochondrial genome. Competition hybridization experiments with unlabelled HeLa mitochondrial RNAs have shown that most of the cross-hybridization involves the ribosomal and 4 S RNA genes.     

Quantitative Changes in Gimap3 and Gimap5 Expression Modify Mitochondrial DNA Segregation in Mice

Mammalian mitochondrial DNA (mtDNA) is a high-copy maternally inherited genome essential for aerobic energy metabolism. Mutations in mtDNA can lead to heteroplasmy, the co-occurence of two different mtDNA variants in the same cell, which can segregate in a tissue-specific manner affecting the onset and severity of mitochondrial dysfunction. To investigate mechanisms regulating mtDNA segregation we use a heteroplasmic mouse model with two polymorphic neutral mtDNA haplotypes (NZB and BALB) that displays tissue-specific and age-dependent selection for mtDNA haplotypes. In the hematopoietic compartment there is selection for the BALB mtDNA haplotype, a phenotype that can be modified by allelic variants of Gimap3. Gimap3 is a tail-anchored member of the GTPase of the immunity-associated protein (Gimap) family of protein scaffolds important for leukocyte development and survival. Here we show how the expression of two murine Gimap3 alleles from Mus musculus domesticus and M. m. castaneus differentially affect mtDNA segregation. The castaneus allele has incorporated a uORF (upstream open reading frame) in-frame with the Gimap3 mRNA that impairs translation and imparts a negative effect on the steady-state protein abundance. We found that quantitative changes in the expression of Gimap3 and the paralogue Gimap5, which encodes a lysosomal protein, affect mtDNA segregation in the mouse hematopoietic tissues. We also show that Gimap3 localizes to the endoplasmic reticulum and not mitochondria as previously reported. Collectively these data show that the abundance of protein scaffolds on the endoplasmic reticulum and lysosomes are important to the segregation of the mitochondrial genome in the mouse hematopoietic compartment.