Numerical chromosomal abnormalities in equine embryos produced in vivo and in vitro

Alkaline gel electrophoresis, pulsed field gel electrophoresis, and quantitative PCR analyses (QPCR) of the nuclear (nDNA) and mitochondrial (mtDNA) genomes were used to assess DNA integrity in the spermatozoa of three species exposed to oxidative stress. In human and murine spermatozoa, the mtDNA was significantly more susceptible to H2O2-mediated damage than nDNA. In both eutherian species, exposure to 250 microM H2O2 induced around 0.6 lesions/10 kb of mtDNA. The mtDNA of human spermatozoa was particularly vulnerable to oxidative stress; 0.25, 1, and 5 mM H2O2 inducing DNA damage equivalent to 0.62, 1.34, and 1.42 lesions/10 kb, respectively. Such results emphasize the diagnostic significance of mtDNA as a biomarker of oxidative stress in the male germ line. In contrast, no damage could be detected by QPCR in the nDNA of either eutherian species, on exposure to H2O2 at doses as high as 5 mM. However, electrophoretic analysis indicated that severe oxidative stress could induce detectable nDNA fragmentation in human, but not murine spermatozoa. The mtDNA of tammar wallaby spermatozoa was relatively resistant to oxidative stress, only exhibiting damage (0.6 lesions/10 kb DNA) on exposure to 5 mM H2O2. By contrast, the nDNA of wallaby spermatozoa was significantly more susceptible to this oxidant than the other species. Such vulnerability is consistent with the lack of disulfide cross-linking in marsupial sperm chromatin and suggests that chromatin condensation during epididymal maturation may be important in establishing the resistance of these cells to the genotoxic effects of reactive oxygen species.     

The long amplicon quantitative PCR for DNA damage assay as a sensitive method of assessing DNA damage in the environmental model, Atlantic killifish (Fundulus heteroclitus)

DNA damage is an important mechanism of toxicity for a variety of pollutants, and therefore, is often used as an indicator of pollutant effects in ecotoxicological studies. Here, we adapted a PCR-based assay for nuclear and mitochondrial DNA damage for use in an important environmental model, the Atlantic killifish (Fundulus heteroclitus). We refer to this assay as the long amplicon quantitative PCR (LA-QPCR) assay. To validate this method in killifish, DNA damage was measured in liver, brain, and muscle of fish dosed with 10 mg/kg benzo[a]pyrene. This exposure caused 0.4-0.8 lesions/10 kb. We also measured DNA damage in liver and muscle tissues from killifish inhabiting a Superfund site, confirming the utility of this method for biomonitoring. In both cases, damage levels were comparable in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Since extensive nDNA sequence data are not readily available for many environmentally relevant species, but mitochondrial genomes are frequently fully sequenced, this assay can be adapted to examine mtDNA damage in virtually any species with little development. Therefore, we argue that this assay will be a valuable tool in assessing DNA damage in ecotoxicological studies.     

Paternal mitochondrial DNA transmission during nonhuman primate nuclear transfer

Offspring produced by nuclear transfer (NT) have identical nuclear DNA (nDNA). However, mitochondrial DNA (mtDNA) inheritance could vary considerably. In sheep, homoplasmy is maintained since mtDNA is transmitted from the oocyte (recipient) only. In contrast, cattle are heteroplasmic, harboring a predominance of recipient mtDNA along with varying levels of donor mtDNA. We show that the two nonhuman primate Macaca mulatta offspring born by NT have mtDNA from three sources: (1) maternal mtDNA from the recipient egg, (2) maternal mtDNA from the egg contributing to the donor blastomere, and (3) paternal mtDNA from the sperm that fertilized the egg from which the donor blastomere was isolated. The introduction of foreign mtDNA into reconstructed recipient eggs has also been demonstrated in mice through pronuclear injection and in humans through cytoplasmic transfer. The mitochondrial triplasmy following M. mulatta NT reported here forces concerns regarding the parental origins of mtDNA in clinically reconstructed eggs. In addition, mtDNA heteroplasmy might result in the embryonic stem cell lines generated for experimental and therapeutic purposes ("therapeutic cloning").     

Nuclear and mitochondrial DNA repair: similar pathways?

Mitochondrial DNA (mtDNA) alterations are implicated in a broad range of human diseases and alterations of the mitochondrial genome are assumed to be a result of its high susceptibility to oxidative damage and its limited DNA repair compared to nuclear DNA (nDNA). Characterization of DNA repair mechanisms has generally focused on these processes in nDNA but increasing interest and research effort have contributed to our knowledge of the mechanisms underlying DNA repair in mitochondria. In this review, we make comparisons between nDNA and mtDNA repair pathways and propose a model for how these pathways interact in mitochondria.     

Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals.

DNA damage is considered of paramount importance in aging. Among causes of this damage, free radical attack, particularly from mitochondrial origin, is receiving special attention. If oxidative damage to DNA is involved in aging, long-lived animals (which age slowly) should show lower levels of markers of this kind of damage than short-lived ones. However, this possibility has not heretofore been investigated. In this study, steady-state levels of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) referred to deoxyguanosine (dG) were measured by high performance liquid chromatography (HPLC) in the mitochondrial (mtDNA) and nuclear (nDNA) DNA from the heart of eight and the brain of six mammalian species ranging in maximum life span (MLSP) from 3.5 to 46 years. Exactly the same digestion of DNA to deoxynucleosides and HPLC protocols was used for mtDNA and nDNA. Significantly higher (three- to ninefold) 8-oxodG/dG values were found in mtDNA than in nDNA in all the species studied in both tissues. 8-oxodG/dG in nDNA did not correlate with MLSP across species either in the heart (r=-0.68; P<0.06) or brain (r = 0.53; P<0.27). However, 8-oxodG/dG in mtDNA was inversely correlated with MLSP both in heart (r=-0.92; P<0.001) and brain (r=-0.88; P<0.016) tissues following the power function y = a(.)x(b), where y is 8-oxodG/dG and x is the MLSP. This agrees with the consistent observation that mitochondrial free radical generation is also lower in long-lived than in short-lived species. The results obtained agree with the notion that oxygen radicals of mitochondrial origin oxidatively damage mtDNA in a way related to the aging rate of each species.-Barja, G., Herrero, A. Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals.     

DNA extraction procedures meaningfully influence qPCR-based mtDNA copy number determination

Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA:nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively impact mtDNA copy number determination via qPCR. To test this we measured mtDNA:nDNA ratios in genomic DNA samples prepared using organic solvent (phenol–chloroform–isoamyl alcohol) extraction and two different silica-based column methods, and found mtDNA:nDNA ratio estimates were not uniform. We further evaluated whether different genomic DNA preparation methods could influence outcomes of experiments that use mtDNA:nDNA ratios as endpoints, and found the method of genomic DNA extraction can indeed alter experimental outcomes. We conclude genomic DNA sample preparation can meaningfully influence mtDNA copy number determination by qPCR.     

Mitochondrial DNA-depleted A549 cells are resistant to bleomycin

Alveolar epithelial cells are considered to be the primary target of bleomycin-induced lung injury, leading to interstitial fibrosis. The molecular mechanisms by which bleomycin causes this damage are poorly understood but are suspected to involve generation of reactive oxygen species and DNA damage. We studied the effect of bleomycin on mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) in human alveolar epithelial A549 cells. Bleomycin caused an increase in reactive oxygen species production, DNA damage, and apoptosis in A549 cells; however, bleomycin induced more mtDNA than nDNA damage. DNA damage was associated with activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and cleavage and activation of protein kinase D1 (PKD1), a newly identified mitochondrial oxidative stress sensor. These effects appear to be mtDNA-dependent, because no caspase-3 or PKD1 activation was observed in mtDNA-depleted (ρ(0)) A549 cells. Survival rate after bleomycin treatment was higher for A549 ρ(0) than A549 cells. These results suggest that A549 ρ(0) cells are more resistant to bleomycin toxicity than are parent A549 cells, likely in part due to the depletion of mtDNA and impairment of mitochondria-dependent apoptotic pathways.     

线粒体DNA与DNA甲基化的关系

线粒体DNA（mitochondrial DNA,mtDNA）遗传信息量虽小,却控制着线粒体一些最基本的性质,对细胞及其功能有着重要影响。mtDNA的损伤与衰老、肿瘤等疾病的发生有关。DNA甲基化是调节基因表达的重要方式之一。mtDNA基因的表达受核DNA（nuclear DNA,nDNA）的调控,mtDNA和nDNA协同作用参与机体代谢调节和发病。本文就近年来mtDNA与DNA甲基化的关系作一综述。     

Curcumin induces apoptosis through mitochondrial hyperpolarization and mtDNA damage in human hepatoma G2 cells

Curcumin, a major pigment of turmeric, is a natural antioxidant possessing a variety of pharmacological activities and therapeutic properties. But its mechanisms are unknown. In our previous study, we found that a 2-h exposure to curcumin induced DNA damage to both the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA) in HepG2 cells and that mtDNA damage was more extensive than nDNA damage. Therefore, experiments were initiated to evaluate the role of mtDNA damage in curcumin-induced apoptosis. The results demonstrated that HepG2 cells challenged with curcumin for 1 h showed a transient elevation of the mitochondrial membrane potential (DeltaPsim), followed by cytochrome c release into the cytosol and disruption of DeltaPsim after 6 h exposure to curcumin. Apoptosis was detected by Hoechst 33342 and annexin V/PI assay after 10 h treatment. Interestingly, the expression of Bcl-2 remained unchanged. A resistance to apoptosis for the corresponding rho0 counterparts confirmed a critical dependency for mitochondria during the induction of apoptosis in HepG2 cells mediated by curcumin. The effects of PEG-SOD in protecting against curcumin-induced cytotoxicity suggest that curcumin-induced cytotoxicity is directly dependent on superoxide anion O2- production. These data suggest that mitochondrial hyperpolarization is a prerequisite for curcumin-induced apoptosis and that mtDNA damage is the initial event triggering a chain of events leading to apoptosis in HepG2 cells.     

Nuclear mitochondrial pseudogenes

As has been demonstrated recently, the transfer of genetic material from mitochondria to the nucleus and its integration into the nuclear genome is a continuous and dynamic process. Fragments of mitochondrial DNA (mtDNA) are incorporated in the nuclear genome as noncoding sequences, which are called nuclear mitochondrial pseudogenes (NUMT pseudogenes or NUMT inserts). In various eukaryotes, NUMT pseudogenes are distributed through different chromosomes to form a “library” of mtDNA fragments, providing important information on genome evolution. The escape of mtDNA from mitochondria is mostly associated with mitochondrial damage and mitophagy. Fragments of mtDNA may be integrated into nuclear DNA (nDNA) during repair of double-strand breaks (DSBs), which are caused by endogenous or exogenous agents. DSB repair of nDNA with a capture of mtDNA fragments may occur via nonhomologous end joining or a similar mechanism that involves microhomologous terminal sequences. An analysis of the available data makes it possible to suppose that the NUMT pseudogene formation rate depends on the DSB rate in nDNA, the activity of the repair systems, and the number of mtDNA fragments leaving organelles and migrating into the nucleus. Such situations are likely after exposure to damaging agents, first and foremost, ionizing radiation. Not only do new NUMT pseudogenes change the genome structure in the regions of their integration, but they may also have a significant impact on the actualization of genetic information. The de novo integration of NUMT pseudogenes in the nuclear genome may play a role in various pathologies and aging. NUMT pseudogenes may cause errors in PCR-based analyses of free mtDNA as a component of total cell DNA because of their coamplification.     

Formalin removal from archival tissue by critical point drying

The extraction of high-quality nucleic acid may be problematic in formalin-fixed tissues because of cross-linking between proteins and DNA. Old fixed tissue specimens do produce fragmented DNA (<1.2 kb), which is only used for PCR amplification. Here we show that high molecular weight DNA (>194 kb) can be successfully extracted from fixed tissue samples (16-70 years old) by gradual dehydration and critical point drying. The reliability of extracted DNA was measured by its ability to serve as a template for the amplification of mtDNA fragments (403 and 1198 bp) and an nDNA fragment (1844 bp). In addition, fingerprinting analysis was performed using DNA from fixed human tissue to ensure the ability of extracted DNA to hybridize with the DNA probe. DNA derived by this method can be subject to amplification, complete digestion by restriction endonuclease, and hybridization.     

Simultaneous extraction of nuclear and mitochondrial DNA from human blood

A method of simultaneous isolation of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) from human blood has been proposed by improvising Lahiri's method of isolation of nuclear DNA. The approach presented here provides selectively enriched fractions and eliminates the need for two different methods or separate reagent sets for the extraction of nDNA and mtDNA. It employs an initial nuclear/ cytoplasm partitioning, followed by the similar procedural steps for the two fractions separately. It gives good quality and quantity of the nDNA as well as the mtDNA, suitable for processes like PCR amplification and sequencing and may prove to be useful for people studying population genetics and evolution using molecular markers maximizing the available resources, especially in cases where a large database needs to be generated from limited amount of blood sample. From 3 ml of blood, the yields of mtDNA salvaged from the supernatant were sufficient to set approximately 4x10(5) reactions (starting with 250 fg DNA per reactions) of mtDNA loci which otherwise would have been discarded as per original Lahiri's procedure. The quality of mtDNA from the mitochondrial fraction was suitable for all major downstream processes as confirmed by locus specific PCR amplifications and sequencing. Through this procedure, the wastage of nDNA can be avoided when mtDNA loci is studied.     

Isolation of two subpopulations of Mycobacterium avium within human macrophages.

Mycobacterium avium is an intracellular pathogen that is associated with disseminated infection in acquired immunodeficiency syndrome (AIDS) patients. Human monocyte-derived macrophages were infected with M. avium strain 101 and a quinolone (Bay y 3118) was used at 8 micrograms ml-1, a concentration that kills growing bacteria but fails to eliminate static organisms. Infected monolayers were treated with Bay y 3118 for 4 days and viable bacteria obtained from the lysis of macrophages were used to infect other macrophages without passage in media. The procedure was repeated five times, after which seven different subpopulations that failed to grow within macrophages were identified. While the DNA fingerprinting confirmed that all came from the same strain, three protein profiles were observed. Static subpopulations were not killed by cytokine-stimulated macrophages, in contrast to the replicating subpopulation. Three of the static subpopulation strains were shown to be auxotrophic for glutamic acid or methionine. All seven non-duplicating subpopulation strains grew well in complete 7H10 agar. The importance of a static subpopulation of M. avium within macrophages is presently unknown. It is possible, however, that the non-growing bacteria would persist within macrophages.     

Multiple SNP Markers Reveal Fine-Scale Population and Deep Phylogeographic Structure in European Anchovy (Engraulis encrasicolus L.)

Geographic surveys of allozymes, microsatellites, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) have detected several genetic subdivisions among European anchovy populations. However, these studies have been limited in their power to detect some aspects of population structure by the use of a single or a few molecular markers, or by limited geographic sampling. We use a multi-marker approach, 47 nDNA and 15 mtDNA single nucleotide polymorphisms (SNPs), to analyze 626 European anchovies from the whole range of the species to resolve shallow and deep levels of population structure. Nuclear SNPs define 10 genetic entities within two larger genetically distinctive groups associated with oceanic variables and different life-history traits. MtDNA SNPs define two deep phylogroups that reflect ancient dispersals and colonizations. These markers define two ecological groups. One major group of Iberian-Atlantic populations is associated with upwelling areas on narrow continental shelves and includes populations spawning and overwintering in coastal areas. A second major group includes northern populations in the North East (NE) Atlantic (including the Bay of Biscay) and the Mediterranean and is associated with wide continental shelves with local larval retention currents. This group tends to spawn and overwinter in oceanic areas. These two groups encompass ten populations that differ from previously defined management stocks in the Alboran Sea, Iberian-Atlantic and Bay of Biscay regions. In addition, a new North Sea-English Channel stock is defined. SNPs indicate that some populations in the Bay of Biscay are genetically closer to North Western (NW) Mediterranean populations than to other populations in the NE Atlantic, likely due to colonizations of the Bay of Biscay and NW Mediterranean by migrants from a common ancestral population. Northern NE Atlantic populations were subsequently established by migrants from the Bay of Biscay. Populations along the Iberian-Atlantic coast appear to have been founded by secondary waves of migrants from a southern refuge.     

Regulation of energy metabolism in human cells in aging and diabetes: FoF(1), mtDNA, UCP, and ROS

Recent advances in bioenergetics consist of discoveries related to rotational coupling in ATP synthase (FoF(1)), uncoupling proteins (UCP), reactive oxygen species (ROS) and mitochondrial DNA (mtDNA). As shown in cloned sheep, mammalian genomes are composed of both nuclear DNA (nDNA) and maternal mtDNA. Oxidative phosphorylation (oxphos) varies greatly depending on cellular activities, and is regulated by both gene expression and the electrochemical potential difference of H(+) (Delta muH(+)). The expression of both mtDNA (by mtTFA) and nDNA for oxphos and UCP (by NRFs, etc.) is coordinated by a factor called PGC-1. The Delta muH(+) rotates an axis in FoF(1) that is regulated by inhibitors and ATP-sensitive K(+)-channels. We cultured human rho(o) cells (cells without mtDNA) in synthetic media and elucidated relationships among mtDNA, nDNA, Delta muH(+), UCPs, ROS, and apoptosis. These cells lack oxphos-dependent ROS formation and survive under conditions of high O(2). Cells cultured in the absence of ROS scavengers have proliferated for 40 years. UCPs lower Delta muH(+) and prevent ROS formation and resulting apoptosis. These results were applied to diabetology and gerontology. The pancreatic rho(o) cells did not secrete insulin, and mtDNA mutations caused diabetes, owing to the deficient Delta muH(+). Insulin resistance was closely related to UCPs and other energy regulators. The resulting high-glucose environment caused glycation of proteins and ROS-mediated apoptosis in vascular cells involved in diabetic complications. Telomeres, oxphos, and ROS are determinants in cellular aging. Cell division and ROS shortened telomeres and accelerated aging. In aged cells, Delta muH(+) was reduced by the slow respiration, and this change induced apoptosis. Cybrids made from aged cytoplasts and rho(o) cells showed that both decreased expression of nDNA, and somatic mutations of mtDNA are involved in the slowing of respiration in aged cells.     

Quality assessment of human mitochondrial DNA quantification: MITONAUTS, an international multicentre survey

Mitochondrial DNA quantification by qPCR is used in the context of many diseases and toxicity studies but comparison of results between laboratories is challenging. Through two multigroup distributions of DNA samples from human cell lines, the MITONAUTS group anonymously compared mtDNA/nDNA quantification across nine laboratories involved in HIV research worldwide. Eight of the nine sites showed significant correlation between them (mean raw data R(2)=0.664; log(10)-transformed data R(2)=0.844). Although mtDNA/nDNA values were well correlated between sites, the inter-site variability on the absolute measurements remained high with a mean (range) coefficient of variation of 71 (37-212) %. Some variability appeared cell line-specific, probably due to chromosomal alterations or pseudogenes affecting the quantification of certain genes, while within cell line variability was likely due to differences in calibration of the standard curves. The use of two mtDNA and two single copy nDNA genes with highly specific primers to quantify each genome would help address copy number variants. Our results indicate that sample shipment must be done frozen and that absolute mtDNA/nDNA ratio values cannot readily be compared between laboratories, especially if assessing cultured cell mtDNA content. However, within laboratory and relative mtDNA/nDNA comparisons between laboratories should be reliable.