Species-Specific Restriction Site Polymorphism in Root-knot Nematode Mitochondrial DNA

Research was initiated to physically characterize the mitochondrial genomes of several Meloidogyne spp. and host-races, to address questions regarding their systematics and dispersal, and to assess the possibility of developing molecular diagnostics for these nematodes. Techniques were developed for purification and rapid detection of mitochondrial DNA from root-knot nematodes. Mitochondrial DNAs among Meloidogyne spp. were demonstrated to exhibit extensive divergence. The potential for using the rapidly diverging mitochondrial genomes as a diagnostic assay for M. incognita, M. hapla, M. arenaria, and M. javanica is discussed.     

Disruption of Mitochondrial DNA Replication in Drosophila Increases Mitochondrial Fast Axonal Transport In Vivo

Mutations in mitochondrial DNA polymerase (pol γ) cause several progressive human diseases including Parkinson''s disease, Alper''s syndrome, and progressive external ophthalmoplegia. At the cellular level, disruption of pol γ leads to depletion of mtDNA, disrupts the mitochondrial respiratory chain, and increases susceptibility to oxidative stress. Although recent studies have intensified focus on the role of mtDNA in neuronal diseases, the changes that take place in mitochondrial biogenesis and mitochondrial axonal transport when mtDNA replication is disrupted are unknown. Using high-speed confocal microscopy, electron microscopy and biochemical approaches, we report that mutations in pol γ deplete mtDNA levels and lead to an increase in mitochondrial density in Drosophila proximal nerves and muscles, without a noticeable increase in mitochondrial fragmentation. Furthermore, there is a rise in flux of bidirectional mitochondrial axonal transport, albeit with slower kinesin-based anterograde transport. In contrast, flux of synaptic vesicle precursors was modestly decreased in pol γ−α mutants. Our data indicate that disruption of mtDNA replication does not hinder mitochondrial biogenesis, increases mitochondrial axonal transport, and raises the question of whether high levels of circulating mtDNA-deficient mitochondria are beneficial or deleterious in mtDNA diseases.     

Listeria monocytogenes Dampens the DNA Damage Response

The DNA damage response (DDR) is an essential signaling pathway that detects DNA lesions, which constantly occur upon either endogenous or exogenous assaults, and maintains genetic integrity. An infection by an invading pathogen is one such assault, but how bacteria impact the cellular DDR is poorly documented. Here, we report that infection with Listeria monocytogenes induces host DNA breaks. Strikingly, the signature response to these breaks is only moderately activated. We uncover the role of the listerial toxin listeriolysin O (LLO) in blocking the signaling response to DNA breaks through degradation of the sensor Mre11. Knocking out or inactivating proteins involved in the DDR promotes bacterial replication showing the importance of this mechanism for the control of infection. Together, our data highlight that bacterial dampening of the DDR is critical for a successful listerial infection.     

Staphylococcus aureus Sepsis Induces Early Renal Mitochondrial DNA Repair and Mitochondrial Biogenesis in Mice

Acute kidney injury (AKI) contributes to the high morbidity and mortality of multi-system organ failure in sepsis. However, recovery of renal function after sepsis-induced AKI suggests active repair of energy-producing pathways. Here, we tested the hypothesis in mice that Staphyloccocus aureus sepsis damages mitochondrial DNA (mtDNA) in the kidney and activates mtDNA repair and mitochondrial biogenesis. Sepsis was induced in wild-type C57Bl/6J and Cox-8 Gfp-tagged mitochondrial-reporter mice via intraperitoneal fibrin clots embedded with S. aureus. Kidneys from surviving mice were harvested at time zero (control), 24, or 48 hours after infection and evaluated for renal inflammation, oxidative stress markers, mtDNA content, and mitochondrial biogenesis markers, and OGG1 and UDG mitochondrial DNA repair enzymes. We examined the kidneys of the mitochondrial reporter mice for changes in staining density and distribution. S. aureus sepsis induced sharp amplification of renal Tnf, Il-10, and Ngal mRNAs with decreased renal mtDNA content and increased tubular and glomerular cell death and accumulation of protein carbonyls and 8-OHdG. Subsequently, mtDNA repair and mitochondrial biogenesis was evidenced by elevated OGG1 levels and significant increases in NRF-1, NRF-2, and mtTFA expression. Overall, renal mitochondrial mass, tracked by citrate synthase mRNA and protein, increased in parallel with changes in mitochondrial GFP-fluorescence especially in proximal tubules in the renal cortex and medulla. Sub-lethal S. aureus sepsis thus induces widespread renal mitochondrial damage that triggers the induction of the renal mtDNA repair protein, OGG1, and mitochondrial biogenesis as a conspicuous resolution mechanism after systemic bacterial infection.     

Oxidative Damage and Fragmentation of Mitochondrial DNA in Cellular Apoptosis

The molecular genetics and bioenergetics of oxidative damage, fragmentation, and fragility of mitochondrial DNA in cellular apoptosis is reviewed in connection with the redox mechanism of ageing.     

Comparisons of Mitochondrial DNA from the Sibling Species Heterodera glycines and H. schachtii

Restriction fragment patterns of mitochondrial DNA from sibling species of cyst nematodes Heterodera glycines and H. schachtii were examined. Fourteen restriction endonucleases recognizing four, five, and six base-pair sequences yielded a total of 90 scorable fragments of which 10% were shared by both species. Mitochondrial genome sizes for H. glycines and H. schachtii were estimated to be 22.5-23.5 kb and 23.0 kb, respectively. A single wild type mitochondrial genome was identified in all populations of H. glycines examined, although other mitochondrial genomes were present in some populations. The H. schachtii genome exhibited 57 scorable fragments, compared with 33 identified in the H. glycines wild type genome. The estimated nucleotide sequence divergence between the two species was p = 0.145. This estimate suggests these species diverged from a common ancestor 7.3-14.8 million years ago.     

Population Polymorphism of Nuclear Mitochondrial DNA Insertions Reveals Widespread Diploidy Associated with Loss of Heterozygosity in Debaryomyces hansenii

Debaryomyces hansenii, a yeast that participates in the elaboration of foodstuff, displays important genetic diversity. Our recent phylogenetic classification of this species led to the subdivision of the species into three distinct clades. D. hansenii harbors the highest number of nuclear mitochondrial DNA (NUMT) insertions known so far for hemiascomycetous yeasts. Here we assessed the intraspecific variability of the NUMTs in this species by testing their presence/absence first in 28 strains, with 21 loci previously detected in the completely sequenced strain CBS 767^T, and second in a larger panel of 77 strains, with 8 most informative loci. We were able for the first time to structure populations in D. hansenii, although we observed little NUMT insertion variability within the clades. We determined the chronology of the NUMT insertions, which turned out to correlate with the previously defined taxonomy and provided additional evidence that colonization of nuclear genomes by mitochondrial DNA is a dynamic process in yeast. In combination with flow cytometry experiments, the NUMT analysis revealed the existence of both haploid and diploid strains, the latter being heterozygous and resulting from at least four crosses among strains from the various clades. As in the diploid pathogen Candida albicans, to which D. hansenii is phylogenetically related, we observed a differential loss of heterozygosity in the diploid strains, which can explain some of the large genetic diversity found in D. hansenii over the years.Debaryomyces hansenii is a ubiquist, hemiascomycetous yeast that can be found in soil, fruits, and various manufactured foodstuff in which it participates by contributing to the maturation or as a contaminant. Its ability to grow at low temperatures and in high salinity environments makes it the most common yeast in cheeses, to which it brings a number of proteolytic and lipolytic activities and aromas in the course of maturation. D. hansenii has also been implicated as an emerging pathogen, sometimes under the name of Candida famata var. famata (see reference 17). Taxonomic classification of the species related to D. hansenii has always been subject to debate. Recent analyses have reinstated D. hansenii (previously D. hansenii var. hansenii), Debaryomyces fabryi (previously D. hansenii var. fabryi), and Debaryomyces subglobosus (previously Candida famata var. flareri) (13, 25). Phylogenetic analysis using conserved spliceosomal intron sequence comparison has shown that D. hansenii is a complex of species, which comprises at least four members: D. hansenii, Debaryomyces tyrocola, D. fabryi, and Candida flareri (previously Candida famata var. flareri) (18). In addition, our study has revealed the existence of at least three populations (clades 1 to 3) in D. hansenii, with the first one containing the strain CBS 767^T, which has been entirely sequenced (8), and the last one containing Candida famata var. famata CBS 1795.Most eukaryotic nuclear genomes contain pieces of mitochondrial sequences (designated NUMT [nuclear mitochondrial DNA] for nuclear sequences of mitochondrial origin) that result from the transfer of fragments of mitochondrial DNA (mtDNA) to the chromosomes. The number and size of the NUMTs varies greatly between eukaryotic genomes (33). A recent investigation of six hemiascomycetous yeasts has shown that even within this monophyletic group, the number of NUMTs varies greatly, from 1 in Kluyveromyces thermotolerans CBS 6340^T to 145 in D. hansenii CBS 767^T (36). The mtDNA is thought to invade nuclear genomes during the repair of chromosomal DNA double-strand breaks (DSB) by nonhomologous end joining (NHEJ), as shown experimentally in the yeast Saccharomyces cerevisiae (31, 44). The colonization of nuclear genomes by mtDNA is a dynamic evolutionary process, as observed in yeast and humans (3, 32).D. hansenii harbors the highest number of NUMTs known so far for hemiascomycetous yeasts, making it of particular interest for NUMT studies. Conversely, NUMTs are potentially interesting markers to differentiate strains of this species. The 145 NUMTs of type strain CBS 767^T are distributed in 86 loci (61 single NUMTs and 25 clusters). Most clusters (23, 25) are mosaics of NUMTs formed from noncontiguous mtDNA fragments inserted in random orientation at the same chromosomal locus. In the other two clusters, the NUMTs are all in the same orientation and order, as in the mitochondrial genome. These clusters (designated “processions”) correspond to a single ancient mtDNA insertion, followed by mutational decay, leaving recognizable mtDNA segments separated by more diverged sequences (36).Few studies have attempted to evaluate the variability of NUMTs within the same species (2, 23, 32). Here, we have studied natural isolates to assess the intraspecific variability of the NUMT insertions in the nuclear genome of the yeast species D. hansenii. We were able to structure populations in this species, to determine the chronology of the NUMT insertions, and to correlate this chronology to the taxonomy of the D. hansenii complex species. Moreover, NUMT analysis revealed the existence of both haploid and diploid strains, the latter resulting from crosses between different D. hansenii clades.     

Stochastic Drift in Mitochondrial DNA Point Mutations: A Novel Perspective Ex Silico

The mitochondrial free radical theory of aging (mFRTA) implicates Reactive Oxygen Species (ROS)-induced mutations of mitochondrial DNA (mtDNA) as a major cause of aging. However, fifty years after its inception, several of its premises are intensely debated. Much of this uncertainty is due to the large range of values in the reported experimental data, for example on oxidative damage and mutational burden in mtDNA. This is in part due to limitations with available measurement technologies. Here we show that sample preparations in some assays necessitating high dilution of DNA (single molecule level) may introduce significant statistical variability. Adding to this complexity is the intrinsically stochastic nature of cellular processes, which manifests in cells from the same tissue harboring varying mutation load. In conjunction, these random elements make the determination of the underlying mutation dynamics extremely challenging. Our in silico stochastic study reveals the effect of coupling the experimental variability and the intrinsic stochasticity of aging process in some of the reported experimental data. We also show that the stochastic nature of a de novo point mutation generated during embryonic development is a major contributor of different mutation burdens in the individuals of mouse population. Analysis of simulation results leads to several new insights on the relevance of mutation stochasticity in the context of dividing tissues and the plausibility of ROS ”vicious cycle” hypothesis.     

Mitochondrial Telomerase Protects Cancer Cells from Nuclear DNA Damage and Apoptosis

Most cancer cells express high levels of telomerase and proliferate indefinitely. In addition to its telomere maintenance function, telomerase also has a pro-survival function resulting in an increased resistance against DNA damage and decreased apoptosis induction. However, the molecular mechanisms for this protective function remain elusive and it is unclear whether it is connected to telomere maintenance or is rather a non-telomeric function of the telomerase protein, TERT. It was shown recently that the protein subunit of telomerase can shuttle from the nucleus to the mitochondria upon oxidative stress where it protects mitochondrial function and decreases intracellular oxidative stress. Here we show that endogenous telomerase (TERT protein) shuttles from the nucleus into mitochondria upon oxidative stress in cancer cells and analyzed the nuclear exclusion patterns of endogenous telomerase after treatment with hydrogen peroxide in different cell lines. Cell populations excluded TERT from the nucleus upon oxidative stress in a heterogeneous fashion. We found a significant correlation between nuclear localization of telomerase and high DNA damage, while cells which excluded telomerase from the nucleus displayed no or very low DNA damage. We modeled nuclear and mitochondrial telomerase using organelle specific localization vectors and confirmed that mitochondrial localization of telomerase protects the nucleus from inflicted DNA damage and apoptosis while, in contrast, nuclear localization of telomerase correlated with higher amounts of DNA damage and apoptosis. It is known that nuclear DNA damage can be caused by mitochondrially generated reactive oxygen species (ROS). We demonstrate here that mitochondrial localization of telomerase specifically prevents nuclear DNA damage by decreasing levels of mitochondrial ROS. We suggest that this decrease of oxidative stress might be a possible cause for high stress resistance of cancer cells and could be especially important for cancer stem cells.     

The Role of the DNA Damage Response Kinase Ataxia Telangiectasia Mutated in Neuroprotection

It has been estimated that a human cell is confronted with 1 million DNA lesions every day, one fifth of which may originate from the activity of Reactive Oxygen Species (ROS) alone [1,2]. Terminally differentiated neurons are highly active cells with, if any, very restricted regeneration potential [3]. In addition, genome integrity and maintenance during neuronal development is crucial for the organism. Therefore, highly accurate and robust mechanisms for DNA repair are vital for neuronal cells. This requirement is emphasized by the long list of human diseases with neurodegenerative phenotypes, which are either caused by or associated with impaired function of proteins involved in the cellular response to genotoxic stress [4-8]. Ataxia Telangiectasia Mutated (ATM), one of the major kinases of the DNA Damage Response (DDR), is a node that links DDR, neuronal development, and neurodegeneration [2,9-12]. In humans, inactivating mutations of ATM lead to Ataxia-Telangiectasia (A-T) disease [11,13], which is characterized by severe cerebellar neurodegeneration, indicating an important protective function of ATM in the nervous system [14]. Despite the large number of studies on the molecular cause of A-T, the neuroprotective role of ATM is not well established and is contradictory to its general proapoptotic function. This review discusses the putative functions of ATM in neuronal cells and how they might contribute to neuroprotection.     

Influence of the Presence of B Chromosomes on DNA Damage in Crepis capillaris

The sensitivity of different plant species to mutagenic agents is related to the DNA content and organization of the chromatin, which have been described in ABCW and bodyguard hypotheses, respectively. Plant species that have B chromosomes are good models for the study of these hypotheses. This study presents an analysis of the correlation between the occurrence of B chromosomes and the DNA damage that is induced by the chemical mutagen, maleic hydrazide (MH), in Crepis capillaris plants using comet assay. The presence of B chromosomes has a detectable impact on the level of DNA damage. The level of DNA damage after MH treatment was correlated with the number of B chromosomes and it was observed that it increased significantly in plants with 3B chromosomes. We did not find evidence of the protective role from chemical mutagens of the constitutive heterochromatin for euchromatin in relation to DNA damage. The DNA damage involving the 25S rDNA sequences was analyzed using the comet-FISH technique. Fragmentation within or near the 25S rDNA involved the loci on the A and B chromosomes. The presence of B chromosomes in C. capillaris cells had an influence on the level of DNA damage that involves the 25S rDNA region.     

Aristolochic Acid Causes Albuminuria by Promoting Mitochondrial DNA Damage and Dysfunction in Podocyte

Aristolochic acid nephropathy, initially found in patients intaking of slimming herbs containing aristolochic acid (AA), was previously considered as a progressive renal interstitial fibrosis and urothelial malignancy. However, the presence of albuminuria in some patients with AAN suggests that AA may also damage the glomerular filtration barrier. In this study, mice AAN model was generated by daily administration of aristolochic acid I sodium salt intraperitoneally at a dose of 6 mg/kg body weight for 3 days. All of the mice developed heavy albuminuria at day 3 and 7 after receiving AA. In the mice received AA, morphologic change of glomeruli was minor under light microscopy but podocyte foot-process effacement was evident under electron microscopy. In mitochondria isolated from kidney, prominent mitochondrial DNA (mtDNA) damage was accompanied with marked decrease of mtDNA copy number and mitochondrial protein expression level. Similar to those in vivo results, AA treatment impaired the filtration barrier function of cultured podocytes. AA promoted mtDNA damage, decreased mtDNA copy number and mitochondrial protein expression in cultured podocytes. In addition, AA treatment also decreased ATP content, oxygen consumption rate and mitochondrial membrane potential as well as increased cellular reactive oxygen species in cultured podocytes. This study highlighted that AA could induce podocyte damage and albuminuria, which may be mediated by promoting mtDNA damage and mitochondrial dysfunction in podocytes.     

Optimization of Mitochondrial DNA-based Hybridization Assays to Diagnostics in Soil

Nucleic acid hybridization among root-knot nematode mitochondrial DNAs can be used to identify several Meloidogyne species. Research was initiated to optimize mitochondrial DNA-based molecular diagnostics for the demanding environments likely to be encountered in field isolates. DNA hybridization using reconstituted DNA-soil mixtures revealed a loss of assay sensitivity ranging from 34% to 92% with four agronomic soils tested. This problem was alleviated by the addition of exogenously added DNA. Variation in nematode egg lysis procedures also affected hybridization efficiency, with NaOC1 treatment most effective at disrupting Meloidogyne eggs. These optimized conditions permit detection of mtDNA released from one to five Meloidogyne eggs using standard nucleic acid hybridization procedures.     

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.     

Prenatal Stress Causes Oxidative Damage to Mitochondrial DNA in Hippocampus of Offspring Rats

Mitochondrion, the primary source of reactive oxygen species (ROS), is also the target of ROS. 8-Hydroxy-2′-deoxyguanosine (8-OH-dG) is the major end-product of damaged DNA caused by ROS. In our previous studies, we showed that prenatal stress (PNS) preferentially caused cognitive dysfunction and increased ROS in the hippocampus of female offspring rats. The present study aimed to determine 8-OH-dG level of mitochondria in order to elucidate the mechanism of hippocampal pyramidal neuronal damage and cognitive dysfunction induced by PNS. Pregnant rats were divided into two groups: control group (undisturbed) and PNS group (exposed to a restraint stress for 7 days at the late stage of gestation). Offspring rats were divided into four groups: female-control group, male-control group, female-stress group, male-stress group and used at 30-day-old after their birth. The content of 8-OH-dG was determined by high performance liquid chromatography-electrochemical detection (HPLC-ECD). The results showed that the contents of 8-OH-dG in female and male prenatal stressed offspring were significantly higher than that in their respective controls (P < 0.001). 8-OH-dG level was significantly higher in the female-stress group than in the male-stress group (P < 0.05), whereas there was no any gender-dependent difference in the control groups. These results suggest that accumulation of oxidative mitochondrial DNA damage may play an important role in PNS-induced cognitive dysfunction in female offspring rats. Special issue article in honor of Dr. Akitane Mori.     

Mitochondrial DNA variants help monitor the dynamics of Wolbachia invasion into host populations

Wolbachia is the most widespread endosymbiotic bacterium of insects and other arthropods that can rapidly invade host populations. Deliberate releases of Wolbachia into natural populations of the dengue fever mosquito, Aedes aegypti, are used as a novel biocontrol strategy for dengue suppression. Invasion of Wolbachia through the host population relies on factors such as high fidelity of the endosymbiont transmission and limited immigration of uninfected individuals, but these factors can be difficult to measure. One way of acquiring relevant information is to consider mitochondrial DNA (mtDNA) variation alongside Wolbachia in field-caught mosquitoes. Here we used diagnostic mtDNA markers to differentiate infection-associated mtDNA haplotypes from those of the uninfected mosquitoes at release sites. Unique haplotypes associated with Wolbachia were found at locations outside Australia. We also performed mathematical and qualitative analyses including modelling the expected dynamics of the Wolbachia and mtDNA variants during and after a release. Our analyses identified key features in haplotype frequency patterns to infer the presence of imperfect maternal transmission of Wolbachia, presence of immigration and possibly incomplete cytoplasmic incompatibility. We demonstrate that ongoing screening of the mtDNA variants should provide information on maternal leakage and immigration, particularly in releases outside Australia. As we demonstrate in a case study, our models to track the Wolbachia dynamics can be successfully applied to temporal studies in natural populations or Wolbachia release programs, as long as there is co-occurring mtDNA variation that differentiates infected and uninfected populations.