Mitochondrial DNA deletion in human myocardium

Mutation of myocardial mitochondrial DNA was investigated in human left ventricles obtained at autopsy using the polymerase chain reaction (PCR). Seventeen autopsy cases were examined, including patients with diabetes mellitus, myocardial infarction, cardiomyopathy, cancer, and other diseases. Two cases of diabetes mellitus, 2 of myocardial infarction, and 1 of pulmonary fibrosis showed a 7.4 kb deletion of myocardial mitochondrial DNA. Primer shift PCR confirmed that an amplified DNA fragment had not been obtained by misannealing of the primers. It is unclear how much these findings are related to the severity or prognosis of the various diseases, but they indicate that mutation of myocardial mitochondrial DNA can occur in other diseases besides cardiomyopathy, although the influence of aging could not be excluded.     

Mitochondrial DNA mutations and breast tumorigenesis

Breast cancer is a heterogeneous disease and genetic factors play an important role in its genesis. Although mutations in tumor suppressors and oncogenes encoded by the nuclear genome are known to play a critical role in breast tumorigenesis, the contribution of the mitochondrial genome to this process is unclear. Like the nuclear genome, the mitochondrial genome also encodes proteins critical for mitochondrion functions such as oxidative phosphorylation (OXPHOS), which is known to be defective in cancer including breast cancer. Mitochondrial DNA (mtDNA) is more susceptible to mutations due to limited repair mechanisms compared to nuclear DNA (nDNA). Thus changes in mitochondrial genes could also contribute to the development of breast cancer. In this review we discuss mtDNA mutations that affect OXPHOS. Continuous acquisition of mtDNA mutations and selection of advantageous mutations ultimately leads to generation of cells that propagate uncontrollably to form tumors. Since irreversible damage to OXPHOS leads to a shift in energy metabolism towards enhanced aerobic glycolysis in most cancers, mutations in mtDNA represent an early event during breast tumorigenesis, and thus may serve as potential biomarkers for early detection and prognosis of breast cancer. Because mtDNA mutations lead to defective OXPHOS, development of agents that target OXPHOS will provide specificity for preventative and therapeutic agents against breast cancer with minimal toxicity.     

Mitochondrial DNA variation in River Usk brown trout, Salmo trutta

Mitochondrial DNA (mtDNA) polymorphism in the essentially non-anadromous River Usk brown trout Salmo trutta population was investigated by restriction analysis. Following mtDNA extraction and purification on caesium chloride density gradients, monomorphic restriction profiles were obtained with Hae III , Hind III , Sau 3AI and Xbal . However, the restriction endonucleases Ava II and HinfIl proved informative. The distribution of four composite genotypes found within the Usk system was heterogeneous, and a fifth genotype appeared exclusively in an outgroup sample from the adjacent River Wye drainage. The source of the observed genetic variation is discussed in relation to estimated divergence times for Usk mtDNA genotypes and the stocking history of the catchment.     

Mitochondrial DNA and respiratory chain function in spinal cords of ALS patients

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective motor neuron death. In order to address the question of a putative role of mitochondrial dysfunction in the pathogenesis of ALS, we studied the mitochondrial DNA (mtDNA) and mitochondrial respiratory chain enzyme activities in spinal cords of ALS patients and in control subjects without neuropathologic abnormalities. Using a "double PCR and digestion" technique to estimate the levels of randomly distributed point mutations in two small regions of the mtDNA, we found significantly higher levels of mutant mtDNA in the spinal cord of ALS patients compared to controls. No large-scale rearrangements were found, but the amount of mtDNA, measured by Southern blot, was significantly lower in the ALS samples. This reduction correlated well with a decrease of citrate synthase (CS) activity, a mitochondrial marker, as were the activities of respiratory chain complexes I + III, II + III, and IV, suggesting a loss of mitochondria in ALS spinal cords.     

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.     

Mitochondrial DNA mutations in multiple symmetric lipomatosis

Multiple symmetric lipomatosis (MSL) is a rare disorder of middle life characterized by large subcutaneous fat masses around the neck, shoulders and other parts of the trunk. Peripheral neuropathy is a common finding in these predominantly male patients. Employing electrophysiological measures, we found additional signs of central nervous system involvement in a majority of patients. Etiologically, there is an association with mitochondrial dysfunction. In muscle biopsy, we found ragged red fibers in 8 of 12 patients. Molecular genetic analysis revealed multiple deletions of mitochondrial DNA in one patient and the MERRF mutation at nucleotide 8344 in another. In this review, we summarize our clinical, electrophysiological morphological, biochemical and molecular genetic findings in 17 MSL patients, and give a survey of the literature. (Mol Cell Biochem 174: 271–275, 1997)     

A Method for Isolation of Chloroplast DNA and Mitochondrial DNA from Sunflower

We present a method for isolation of chloroplast and mitochondrial DNA from sunflower seedlings. The protocol includes: organelle isolation, deoxyribonuclease treatment, lysis, deproteinisation and a final DNA purification with sodium dodecyl sulphate and potassium acetate. The organelle DNA yield is 5–10 micrograms per gram of tissue and the DNA is fully restrictable. The technique is inexpensive and appropriate for the isolation of multiple samples of organelle DNA from a small amount of tissue.     

Hybrid male sterility is caused by mitochondrial DNA deletion

Although it is known that the hybrid male mouse is sterile just like any other animal’s heterogametic sex, the reason why only the male germ cells are impaired has yet to be discovered. TdT-mediated dUTP nick end labeling assay using a confocal fluorescence microscope and DNA fragmentation assay of hybrid testis indicated destruction of the mitochondrial DNA (mtDNA) rather than the nuclear DNA. Previously we reported that maternal mtDNA inheritance is through selective sperm mtDNA elimination based on the sperm factor and two egg factors, and expression of these three factors was recognized in the hybrid testis. It was thereby assumed that mtDNA destruction caused by the expression of maternal mtDNA inheritance system in male germ cells is implicated in the hybrid male sterility of mice.     

Mitochondrial DNA and RNA isolation from small amounts of potato tissue

We present a fast and simple protocol for purification of mitochondrial DNA and RNA from small amounts of potato tissue including tubers, leaves, flowers, and flower buds. This method uses a high ionic strength medium to isolate mitochondria and extract mitochondrial DNA and RNA from a single preparation and is easily adaptable to other plant species. The mitochondrial DNA was not contaminated by plastid DNA, was fully restrictable and was successfully used for PCR, cloning and Southern analyses. Similarly, the isolated mitochondrial RNA was not contaminated (flower buds) or only slightly contaminated (leaves) by plastid RNA. RNA prepared according to our method was acceptable for northern and RT-PCR analyses.     

Mitochondrial DNA as a genetic marker for brown trout, Salmo trutta L., populations

Mitochondrial DNA was examined in natural and hatchery-reared stocks of brown trout, using different methods of restriction analysis. The methods included the development of a brown trout mt DNA hybridization probe through cloning part of the brown trout mitochondrial genome. In addition, fragments were analysed by ethidium bromide staining and end-labelling. The relative merits of each of these methods in assessing levels of genetic relatedness between the natural and hatchery-reared brown trout stocks were evaluated. In addition, the study revealed a diagnostic mtDNA restriction pattern which could be used as a genetic marker for the discrimination of these two groups of brown trout.     

Mitochondrial DNA deletions and differential mitochondrial DNA content in Rhesus monkeys: Implications for aging

The purpose of this study was to determine the relationship between mitochondrial DNA (mtDNA) deletions, mtDNA content and aging in rhesus monkeys. Using 2 sets of specific primers, we amplified an 8 kb mtDNA fragment covering a common 5.7 kb deletion and the entire 16.5 kb mitochondrial genome in the brain and buffy-coats of young and aged monkeys. We studied a total of 66 DNA samples: 39 were prepared from a buffy-coat and 27 were prepared from occipital cortex tissues. The mtDNA data were assessed using a permutation test to identify differences in mtDNA, in the different monkey groups. Using real-time RT-PCR strategy, we also assessed both mtDNA and nuclear DNA levels for young, aged and male and female monkeys. We found a 5.7 kb mtDNA deletion in 81.8% (54 of 66) of the total tested samples. In the young group of buffy-coat DNA, we found 5.7 kb deletions in 7 of 17 (41%), and in the aged group, we found 5.7 kb deletions in 12 of 22 (54%), suggesting that the prevalence of mtDNA deletions is related to age. We found decreased mRNA levels of mtDNA in aged monkeys relative to young monkeys. The increases in mtDNA deletions and mtDNA levels in aged rhesus monkeys suggest that damaged DNA accumulates as rhesus monkeys age and these altered mtDNA changes may have physiological relevance to compensate decreased mitochondrial function.     

Age-associated damage in mitochondrial DNA in human hearts

Damage to mitochondrial DNA seems to be involved in the etiology of age-associated degenerative diseases. The aim of this study is to elucidate effects of aging on human mitochondrial DNA. 8-Hydroxy-deoxyguanosine, a product of free radical damage to deoxyguanosine, is reported to cause random point mutations. In human mitochondrial DNA, 8-hydroxy-deoxyguanosine increased exponentially with age, and the population of mitochondrial DNA with deletion increased also exponentially with age. Furthermore, a clear correlation existed between the accumulation of 8-hydroxy-deoxyguanosine and that of mitochondrial DNA with deletion. We also determined the effects of aging on rat mitochondrial function together with 8-hydroxy-deoxyguanosine content in mitochondrial DNA. The activities of complexes I and IV of the mitochondrial electron transport chain decreased significantly in rats aged 100 weeks compared with those in rats aged 7 weeks. A concomitant increase in 8-hydroxy-deoxyguanosine was observed in mitochondrial DNA of rats aged 100 weeks. From our results, it is concluded that the age-associated accumulation of somatically acquired oxygen damage together with deletions in mitochondrial DNA might be important contributors to the deterioration of cardiac function associated with age.Abbreviations mtDNA mitochondrial DNA - 8-OH-dG 8-Hydroxy-Deoxyguanosine - dG Deoxyguanosine - HPLC/MS Micro-High Performance Liquid Chromatography/Mass Spectrometry     

A Tunisian patient with Pearson syndrome harboring the 4.977kb common deletion associated to two novel large-scale mitochondrial deletions

Pearson syndrome (PS) is a multisystem disease including refractory anemia, vacuolization of marrow precursors and pancreatic fibrosis. The disease starts during infancy and affects various tissues and organs, and most affected children die before the age of 3 years. Pearson syndrome is caused by de novo large-scale deletions or, more rarely, duplications in the mitochondrial genome. In the present report, we described a Pearson syndrome patient harboring multiple mitochondrial deletions which is, in our knowledge, the first case described and studied in Tunisia. In fact, we reported the common 4.977 kb deletion and two novel heteroplasmic deletions (5.030 and 5.234 kb) of the mtDNA. These deletions affect several protein-coding and tRNAs genes and could strongly lead to defects in mitochondrial polypeptides synthesis, and impair oxidative phosphorylation and energy metabolism in the respiratory chain in the studied patient.     

动物线粒体DNA分析技术及其应用

结合动物线粒体基因组的最新研究进展,对目前动物线粒体基因序列的分析技术进行了概述,分析了应用较广的限制性片段长度多态性(RFLP)、序列特异性寡核苷酸分析(SSO)、DNA芯片、单链构象多态性(SSCP)、变性梯度凝胶电泳(DGGE)和竞争性寡核苷酸引物延伸(COP)等技术的原理、方法和适用范围,并对它们在线粒体基因的遗传、起源和进化等热点问题中的应用进行了阐述。     

Mitochondrial DNA and its Inheritance in Pleurotus ostreatus and P. pulmonarius

Six stocks each of the basidiomycetes Pleurotus ostreatus and P. pulmonarius were checked for intraspecific polymorphism and inheritance of mitochondrial DNA (mtDNA). Polymorphisms were extensive in both species after digestion with the endonucleases Hae III and Hind III, and they were stock-specific with one exception. The range of mtDNA sizes was similar for both taxa, lying between approximately 56 kb and 70 kb for P. ostreatus and 60 kb and 78 kb for P. pulmonarius. The inheritance of mtDNA was uniparental throughout. In pairings of monokaryons exhibiting specific mtDNA types, only one parental mtDNA type was recovered in the resulting dikaryons; while the mitochondria of the donor mycelium spread out, the mitochondria of the recipient mycelium apparently are lost. Certain mtDNA types were more often inherited than others. By examining this dominance of mtDNA types one could establish parentage for both species.     

Mitochondrial DNA alterations in blood of the humans exposed to N,N-dimethylformamide

N,N-Dimethylformamide (DMF) has been widely used in industries because of its extensive miscibility with water and solvents. Its health effects include hepatotoxicity and male reproductoxicity, possibly linked with mitochondrial DNA (mtDNA) alterations including mtDNA common deletion (DeltamtDNA(4977)) and mtDNA copy number. The relationship between DMF exposure and mtDNA alterations, however, has not been postulated yet. The purposes of this study were to investigate whether the DMF exposure is associated with DeltamtDNA(4977) and mtDNA copy number and to evaluate the DMF-derived mtDNA alterations are more associated with exposure to the airborne DMF concentrations or to the levels of two urinary DMF biomarkers of N-methylformamide (NMF) and N-acetyl-S-(N-methylcarbamoryl) cysteine(AMCC). Thirteen DMF-exposed workers and 13 age and seniority-matched control workers in a synthetic leather factory were monitored on their airborne DMF, NMF and AMCC in the urine as well as DeltamtDNA(4977) and mtDNA copy number in blood cells. We found that the frequencies of relative DeltamtDNA(4977) in DMF-exposed group were significantly higher than those in the control group. Moreover, elevation in the proportion of DeltamtDNA(4977) of individuals with high urine AMCC (U-AMCC) and airborne DMF levels were significantly higher than those without. We conclude that long-term exposure to DMF is highly associated with the alterations of mtDNA in urine and blood cells. The DeltamtDNA(4977) was more significantly related to repeated exposure to DMF and mtDNA copy number was more closely related to short-term DMF exposure. We also confirmed that U-AMCC is more appropriate to serve as a toxicity biomarker for DMF exposure than U-NMF. Further study with a larger number of subjects is warranted.     

Mitochondrial DNA variation in North American Oedipodinae

Restriction fragment analysis of mitochondrial DNA (mtDNA) was used to examine genetic variation and population structure in 13 species of banded-winged grasshoppers (subfamily Oedipodinae). Total DNA of 246 individuals was digested with 11 restriction enzymes and probed with three clonedEcoRI fragments representing the entire mitochondrial genome ofMelanoplus sanguinipes. On average, members of this subfamily were five times more variable than those in another subfamily, Melanoplinae, previously examined. This would appear to lend support to Vickery's claim that the time of origin of Nearctic oedipodines is more ancient than that of melanoplines. With respect to population structure, a few different patterns were exhibited. Species such asCamnula pellucida had populations containing a mixture of haplotypes, some widespread and some geographically restricted. In contrast,Chortophaga viridifasciata populations were characterized by unique assemblages of diverse haplotypes. Phylogeographic hypotheses are advanced to account for these observations.     

Mitochondrial DNA variation in the Viking age population of Norway

The medieval Norsemen or Vikings had an important biological and cultural impact on many parts of Europe through raids, colonization and trade, from about AD 793 to 1066. To help understand the genetic affinities of the ancient Norsemen, and their genetic contribution to the gene pool of other Europeans, we analysed DNA markers in Late Iron Age skeletal remains from Norway. DNA was extracted from 80 individuals, and mitochondrial DNA polymorphisms were detected by next-generation sequencing. The sequences of 45 ancient Norwegians were verified as genuine through the identification of damage patterns characteristic of ancient DNA. The ancient Norwegians were genetically similar to previously analysed ancient Icelanders, and to present-day Shetland and Orkney Islanders, Norwegians, Swedes, Scots, English, German and French. The Viking Age population had higher frequencies of K*, U*, V* and I* haplogroups than their modern counterparts, but a lower proportion of T* and H* haplogroups. Three individuals carried haplotypes that are rare in Norway today (U5b1b1, Hg A* and an uncommon variant of H*). Our combined analyses indicate that Norse women were important agents in the overseas expansion and settlement of the Vikings, and that women from the Orkneys and Western Isles contributed to the colonization of Iceland.     

西南地区家猪和野猪mtDNA遗传多样性研究

本实验用ＡｐａＩ、ＡｖａＩ、ＢａｍＨＩ、ＢｃｌＩ、ＢｇｌＩＩ、ＢｇｌＩＩ、ＣｌａＩ、ＥｃｏＲＩ、ＥｃｏＲＶ、ＨｉｎｄＩＩＩ、Ｈｐａｌ、ＫｐｎＩ、ＰｓｔＩ、ＰｖｕＩＩ、ＳａｃＩ、ＳａｌＩ、ＳｃａＩ、ＳｍａＩ、ＸｂａＩ和ＸｈｏＩ等２０种限制性内切酶分析来自中国西南地区的家猪和野猪的ｍｔＤＮＡ群体遗传多样性。结果表明：在全部２８只个体中,共检出２６种限制性态型（ｍｏｒｐｈ）,归结为６种不同的限制性类型,限制性类型的差异主要来源于少数几个限制性位点的偶然突变。利用现代分子群体遗传学方法,对这些猪的遗传多样性进行评估,结果表明中国西南地区猪的ｍｔＤＮＡ变异度很低,遗传多样性贫乏,提示西南地区的猪起源于一个共同的祖先,在品种形成的早期可能受到创立者效应的制约。     

An integrase of endogenous retrovirus is involved in maternal mitochondrial DNA inheritance of the mouse

The mechanism of maternal mitochondrial DNA (mtDNA) inheritance in animals can be said to be the selective elimination of sperm mtDNA via the elimination factor of the egg and a sperm mitochondria-specific factor. In 2005, we clarified that t-tpis (Spag1 isoform 1) is a mitochondria-specific translocator and the sperm factor, and furthermore estimated that the elimination factors of the egg are the divalent cation-dependent endonuclease and s-tpis (Spag1 isoform 2 and isoform 3) as the elimination system-specific chaperone [K. Hayashida, K. Omagari, J. Masuda, H. Hazama, Y. Kadokawa, K. Ohba, S. Kohno, The sperm mitochondria-specific translocator has a key role in maternal mitochondrial inheritance, Cell Biol. Int. 29 (2005) 472-481]. This time, using a recombinant Spag1 isoform 1 protein, a pull-down assay of ovary cytosol was performed and the elimination factors searched for. Surprisingly, an endogenous retroviral integrase fragment (Eri15) was identified using mass spectrometry of the electrophoresis band of the pull-down protein. Eri15 was detected as a complex of ∼500 kDa with Spag1 isoform 2 or isoform 3 in native PAGE of the ovary cytosol. This strongly suggested that Eri15 is selectively transported into the sperm mitochondria matrix by Spag1 isoform 2 and 3 via Spag1 isoform 1 and that sperm mtDNA is destroyed, thus causing the establishment of maternal mtDNA inheritance.