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1.
Mahato B  Jash S  Adhya S 《Mitochondrion》2011,11(4):564-574
Mutations in mitochondrial DNA (mtDNA) generate multi-system disorders due to failure of ATP production. A cybrid containing a 1.9-kb mtDNA deletion from a patient with Kearns Sayre Syndrome is respiration-defective and grows glycolytically. When treated with a ribonucleoprotein (RNP) complex of polycistronic RNA 1 (pcRNA1) containing mtDNA-encoded genes and a multi-subunit carrier complex R8, full-length pcRNA1 was transported to mitochondria. Translation of the pcRNA1-encoded mRNAs was observed in mitochondria from RNP-treated cells. Respiration of the cybrid was rescued to approximately 90% of normal within hours, switching the cells to aerobic growth. These findings have implications for the development of effective mitochondrial gene therapy.  相似文献   

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Oxidative stress and mitochondrial dysfunction have been implicated in the pathology of HD; however, the precise mechanisms by which mutant huntingtin modulates levels of oxidative damage in turn resulting in mitochondrial dysfunction are not known. We hypothesize that mutant huntingtin increases oxidative mtDNA damage leading to mitochondrial dysfunction. We measured nuclear and mitochondrial DNA lesions and mitochondrial bioenergetics in the STHdhQ7 and STHdhQ111 in vitro striatal model of HD. Striatal cells expressing mutant huntingtin show higher basal levels of mitochondrial-generated ROS and mtDNA lesions and a lower spare respiratory capacity. Silencing of APE1, the major mammalian apurinic/apyrimidinic (AP) endonuclease that participates in the base excision repair (BER) pathway, caused further reductions of spare respiratory capacity in the mutant huntingtin-expressing cells. Localization experiments show that APE1 increases in the mitochondria of wild-type Q7 cells but not in the mutant huntingtin Q111 cells after treatment with hydrogen peroxide. Moreover, these results are recapitulated in human HD striata and HD skin fibroblasts that show significant mtDNA damage (increased lesion frequency and mtDNA depletion) and significant decreases in spare respiratory capacity, respectively. These data suggest that mtDNA is a major target of mutant huntingtin-associated oxidative stress and may contribute to subsequent mitochondrial dysfunction and that APE1 (and, by extension, BER) is an important target in the maintenance of mitochondrial function in HD.  相似文献   

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The reasons of high level of human mitochondrial DNA (mtDNA) variability remain to be largely unclear. We analyze here three probable mechanisms of mutagenesis leading to generation of mtDNA nucleotide substitutions: (1) deamination of DNA bases; (2) tautomeric migrations of protons in nitrous bases; and (3) hydrolysis of glycoside link between DNA bases and carbohydrate residue on the background of free radical damage of the mitochondrial DNA polymerase gamma. By means of quanto-chemical calculations, it was shown that the most substantiated mechanism of mutation generation is hydrolysis of N-glycoside link. This mechanism is suggestive to be more prominent on the H-strand, which remains to be single-stranded for a long time during the mtDNA replication. It was revealed also that hydrolytic deamination of adenines on the single-stranded H-strand is among of the most probable mechanisms leading to high frequency of T --> C transitions seen in the L-strand mutational spectra of the mtDNA major non-coding region.  相似文献   

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We describe a repetitive DNA region at the 3 end of the mitochondrial DNA (mtDNA) control region and compare it in 21 carnivore species representing eight carnivore families. The sequence and organization of the repetitive motifs can differ extensively between arrays; however, all motifs appear to be derived from the core motif ACGT. Sequence data and Southern blot analysis demonstrate extensive heteroplasmy. The general form of the array is similar between heteroplasmic variants within an individual and between individuals within a species (varying primarily in the length of the array, though two clones from the northern elephant seal are exceptional). Within certain families, notably ursids, the array structure is also similar between species. Similarity between species was not apparent in other carnivore families, such as the mustelids, suggesting rapid changes in the organization and sequence of some arrays. The pattern of change seen within and between species suggests that a dominant mechanism involved in the evolution of these arrays is DNA slippage. A comparative analysis shows that the motifs that are being reiterated or deleted vary within and between arrays, suggesting a varying rate of DNA turnover. We discuss the evolutionary implications of the observed patterns of variation and extreme levels of heteroplasmy.By acceptance of this article, the publisher acknowledges the right of the US Government to retain non-exclusive, royalty-free license in and to any copyright covering the article. Correspondence to: A.R. Hoetzel  相似文献   

8.
We report an 11-year-old boy with exercise-related myopathy, and a novel mutation m.5669G>A in the mitochondrial tRNA Asparagine gene (mt-tRNA(Asn), MTTN). Muscle biopsy studies showed COX-negative, SDH-positive fibers at histochemistry and biochemical defects of oxidative metabolism. The m.5669G>A mutation was present only in patient's muscle resulting in the first muscle-specific MTTN mutation. Mt-tRNA(Asn) steady-state levels and in silico predictions supported the pathogenicity of this mutation. A mitochondrial myopathy should be considered in the differential diagnosis of exercise intolerance in children.  相似文献   

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We report an 11-year-old boy with exercise-related myopathy, and a novel mutation m.5669G>A in the mitochondrial tRNA Asparagine gene (mt-tRNAAsn, MTTN). Muscle biopsy studies showed COX-negative, SDH-positive fibers at histochemistry and biochemical defects of oxidative metabolism. The m.5669G>A mutation was present only in patient’s muscle resulting in the first muscle-specific MTTN mutation. Mt-tRNAAsn steady-state levels and in silico predictions supported the pathogenicity of this mutation. A mitochondrial myopathy should be considered in the differential diagnosis of exercise intolerance in children.  相似文献   

10.
A consanguineous Israeli Bedouin kindred presented with an autosomal-recessive nonlethal phenotype of severe psychomotor retardation and extrapyramidal signs, dystonia, athetosis and ataxia, mild axial hypotonia, and marked global dementia with defects in verbal and expressive communication skills. Metabolic workup was normal except for mildly elevated blood lactate levels. Brain magnetic resonance imaging (MRI) showed increased density in the putamen, with decreased density and size of the caudate and lentiform nuclei. Reduced activity specifically of mitochondrial complex III and variable decrease in complex I activity were evident in muscle biopsies. Homozygosity of affected individuals to UQCRB and to BCSIL, previously associated with isolated complex III deficiency, was ruled out. Genome-wide linkage analysis identified a homozygosity locus of approximately 9 cM on chromosome 5q31 that was further narrowed down to 2.14 cM, harboring 30 genes (logarithm of the odds [LOD] score 8.82 at theta = 0). All 30 genes were sequenced, revealing a single missense (p.Ser45Phe) mutation in UQCRQ (encoding ubiquinol-cytochrome c reductase, complex III subunit VII, 9.5 kDa), one of the ten nuclear genes encoding proteins of mitochondrial complex III.  相似文献   

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Sequence variation in mitochondrial DNA (mtDNA) may cause slight differences both in the functioning of the respiratory chain and in free radical production, and an association between certain mtDNA haplogroups and longevity has been suggested. In order to determine further the role of mtDNA in longevity, we studied the frequencies of mtDNA haplogroups and haplogroup clusters among elderly subjects and controls in a Finnish population. Samples were obtained from 225 persons aged 90-91 years (Vitality 90+) and from 400 middle-aged controls and 257 infants. MtDNA haplogroups were determined by restriction fragment length polymorphism. The haplogroup frequencies of the Vitality 90+ group differed from both those of the middle-aged controls ( P=0.01) and the infants ( P=0.00005), haplogroup H being less frequent than among the middle-aged subjects ( P=0.001) and infants ( P=0.00001), whereas haplogroups U and J were more frequent. Haplogroup clusters also differed between Vitality 90+ and both the middle-aged subjects ( P=0.002) and infants ( P=0.00001), the frequency of haplogroup cluster HV being lower in the former and that of UK and WIX being higher. These data suggest an association between certain mtDNA haplogroups or haplogroup clusters and longevity. Furthermore, our data appear to favour the presence of advantageous polymorphisms and support a role for mitochondria and mtDNA in the degenerative processes involved in ageing.  相似文献   

12.
Previous research has established a discrepancy of nearly anorder of magnitude between pedigree-based and phylogeny-based(human vs. chimpanzee) estimates of the mitochondrial DNA (mtDNA)control region mutation rate. We characterize the time dependencyof the human mitochondrial hypervariable region one mutationrate by generating 14 new phylogeny-based mutation rate estimatesusing within-human comparisons and archaeological dates. Rateestimates based on population events between 15,000 and 50,000years ago are at least 2-fold lower than pedigree-based estimates.These within-human estimates are also higher than estimatesgenerated from phylogeny-based human–chimpanzee comparisons.Our new estimates establish a rapid decay in evolutionary mutationrate between approximately 2,500 and 50,000 years ago and aslow decay from 50,000 to 6 Ma. We then extend this analysisto the mtDNA-coding region. Our within-human coding region mutationrate estimates display a similar, though less rapid, time-dependentdecay. We explore the possibility that multiple hits explainthe discrepancy between pedigree-based and phylogeny-based mutationrates. We conclude that whereas nucleotide substitution modelsincorporating multiple hits do provide a possible explanationfor the discrepancy between pedigree-based and human–chimpanzeemutation rate estimates, they do not explain the rapid declineof within-human rate estimates. We propose that demographicprocesses such as serial bottlenecks prior to the Holocene couldexplain the difference between rates estimated before and after15,000 years ago. Our findings suggest that human mtDNA estimatesof dates of population and phylogenetic events should be adjustedin light of this time dependency of the mutation rate estimates.  相似文献   

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Mitochondrial DNA (mtDNA) mutations are responsible for mitochondrial diseases in numerous patients. But, until now, no rapid method was available for the identification of unknown deleterious point mutations. Here, we describe a new strategy for the rapid identification of heteroplasmic mtDNA mutations using mismatch-specific Surveyor Nuclease. This protocol involves the following three steps: (i) PCR amplification of the entire human mitochondrial genome in 17 overlapping fragments; (ii) localization of mtDNA mismatch(es) after digestion of the 17 amplicons by Surveyor Nuclease; and (iii) identification of the mutation by sequencing the region containing the mismatch. This Surveyor Nuclease-based strategy allows a systematic screening of the entire mtDNA to identify a mutation within 2 days. It represents an important diagnostic approach for mitochondrial diseases that can be routinely used in molecular diagnostic laboratories.  相似文献   

14.
Lee HC  Hsu LS  Yin PH  Lee LM  Chi CW 《Mitochondrion》2007,7(1-2):157-163
Somatic mutations in mitochondrial DNA (mtDNA) have been demonstrated in various human cancers. Many cancers have high frequently of mtDNA with homoplasmic point mutations, and carry less frequently of mtDNA with large-scale deletions as compared with corresponding non-cancerous tissue. Moreover, most cancers harbor a decreased copy number of mtDNA than their corresponding non-cancerous tissue. However, it is unclear whether the process of decreasing in mtDNA content would be involved in an increase in the heteroplasmic level of somatic mtDNA point mutation, and/or involved in a decrease in the proportion of mtDNA with large-scale deletion in cancer cells. In this study, we provided evidence that the heteroplasmic levels of variations in cytidine number in np 303-309 poly C tract of mtDNA in three colon cancer cells were not changed during an ethidium bromide-induced mtDNA depleting process. In the mtDNA depleting process, the proportions of mtDNA with 4977-bp deletion in cybrid cells were not significantly altered. These results suggest that the decreasing process of mtDNA copy number per se may neither contribute to the shift of homoplasmic/heteroplasmic state of point mutation in mtDNA nor to the decrease in proportion of mtDNA with large-scale deletions in cancer cells. Mitochondrial genome instability and reduced mtDNA copy number may independently occur in human cancer.  相似文献   

15.
Recent studies have used a variety of theoretical arguments to show that mitochondrial (mt) DNA rarely evolves as a strictly neutral marker and that selection operates on the mtDNA of many species. However, the vast majority of researchers are not convinced by these arguments because data linking mtDNA variation with phenotypic differences are limited. We investigated sequence variation in the three mtDNA and nine nuclear genes (including all isoforms) that encode the 12 subunits of cytochrome c oxidase of the electron transport chain in Drosophila. We then studied cytochrome c oxidase activity as a key aspect of mitochondrial bioenergetics and four life-history traits. In Drosophila simulans, sequence data from the three mtDNA encoded cytochrome c oxidase genes show that there are 76 synonymous and two nonsynonymous fixed differences among flies harboring siII compared with siIII mtDNA. In contrast, 13 nuclear encoded genes show no evidence of genetic subdivision associated with the mtDNA. Flies with siIII mtDNA had higher cytochrome c oxidase activity and were more starvation resistant. Flies harboring siII mtDNA had greater egg size and fecundity, and recovered faster from cold coma. These data are consistent with a causative role for mtDNA variation in these phenotypic differences, but we cannot completely rule out the involvement of nuclear genes. The results of this study have significant implications for the use of mtDNA as an assumed neutral marker and show that evolutionary shifts can involve changes in mtDNA despite the small number of genes encoded in the organelle genome.  相似文献   

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Human DNA topoisomerase I (hTop1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of the antitumor drug camptothecin (CPT). The X-ray crystal structure of the enzyme covalently joined to DNA and bound to the CPT analog Topotecan suggests that there are two classes of mutations that can produce a CPT-resistant enzyme. The first class includes changes in residues that directly interact with the drug, whereas a second class alters interactions with the DNA and thereby destabilizes the drug binding site. The Thr729Ala, that is part of a hydrophobic pocket in the enzyme C-terminal domain, belongs to a third group of mutations that confer CPT resistance, but do not interact directly with the drug or the DNA. To understand the contribution of this residue in drug resistance, we have studied the effect on hTop1p catalysis and CPT sensitivity of four different substitutions in the Thr729 position (Thr729Ala, Thr729Glu, Thr729Lys and Thr729Pro). Tht729Glu and Thr729Lys mutants show severe CPT resistance and furthermore, Thr729Glu shows a remarkable defect in DNA binding. We postulate that the maintenance of the hydrophobic pocket integrity, where Thr729 is positioned, is crucial for drug sensitivity and DNA binding.  相似文献   

17.
To achieve mitochondrial therapy, we previously reported on the use of an octaarginine (R8) modified Dual Function (DF)-MITO-Porter for delivering molecules to mitochondria in living cells. In this study, using isolated mitochondria, homogenates and living cells, we evaluated the utility of mitochondrial targeting functional peptides as a ligand for delivering carriers. The S2 peptide modified carrier showed a high mitochondrial targeting activity in homogenates and living cells. In addition, the S2 peptide had a lower cell toxicity compared to R8 modified liposomes. The S2 peptide represents a potentially useful moiety for constructing an efficient and safe mitochondrial delivery system.  相似文献   

18.
We describe a lethal mitochondrial disease in a 10-month-old child who presented with encephalomyopathy. Histochemical and electron microscopy examinations of skeletal muscle biopsy revealed abnormal mitochondria associated with a combined deficiency of complexes I and IV. After excluding mitochondrial DNA deletions and depletion, direct sequencing was used to screen for mutation in all transfer RNA (tRNA) genes. A T-to-C substitution at position 5693 in the tRNA(Asn) gene was found in blood and muscle. Microdissection of muscle biopsy and its analysis revealed the highest level of this mutation in cytochrome c oxidase (COX)-negative fibres. We suggest that this novel mutation would affect the anticodon loop structure of the tRNA(Asn) and cause a fatal mitochondrial disease.  相似文献   

19.
We report the nucleotidic mapping of a 4,666 base pairs deletion of the human mitochondrial DNA localized at positions 8571 and 13237 in a Kearns-Sayre syndrome patient. The gene fusion between the 15 N terminal amino acid residues of ATP synthetase subunit 6 and the 303 C terminal aminoacids of NADH dehydrogenase yields a potential protein of 35,000 d MW called A6-ND5. Deletion boundaries show a short inverted repeat ATCXTA. The heteroplasmic deletion mechanism is discussed in view of these data.  相似文献   

20.
The 13 polypeptides encoded in mitochondrial DNA (mtDNA) are synthesized in the mitochondrial matrix on a dedicated protein-translation apparatus that resembles that found in prokaryotes. Here, we have investigated the genetic basis for a mitochondrial protein-synthesis defect associated with a combined oxidative phosphorylation enzyme deficiency in two patients, one of whom presented with encephalomyopathy and the other with hypertrophic cardiomyopathy. Sequencing of candidate genes revealed the same homozygous mutation (C997T) in both patients in TSFM, a gene coding for the mitochondrial translation elongation factor EFTs. EFTs functions as a guanine nucleotide exchange factor for EFTu, another translation elongation factor that brings aminoacylated transfer RNAs to the ribosomal A site as a ternary complex with guanosine triphosphate. The mutation predicts an Arg333Trp substitution at an evolutionarily conserved site in a subdomain of EFTs that interacts with EFTu. Molecular modeling showed that the substitution disrupts local subdomain structure and the dimerization interface. The steady-state levels of EFTs and EFTu in patient fibroblasts were reduced by 75% and 60%, respectively, and the amounts of assembled complexes I, IV, and V were reduced by 35%–91% compared with the amounts in controls. These phenotypes and the translation defect were rescued by retroviral expression of either EFTs or EFTu. These data clearly establish mutant EFTs as the cause of disease in these patients. The fact that the same mutation is associated with distinct clinical phenotypes suggests the presence of genetic modifiers of the mitochondrial translation apparatus.  相似文献   

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