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At least 19 recordsLinked to original sources
PubMed1999

Introduction of heteroplasmic mitochondrial DNA (mtDNA) from a patient with NARP into two human rho degrees cell lines is associated either with selection and maintenance of NARP mutant mtDNA or failure to maintain mtDNA.

Mitochondria from a patient heteroplasmic at nucleo-tide position 8993 of mitochondrial DNA (mtDNA) were introduced into two human tumour cell lines lacking mtDNA. The donor mitochondria contained between 85 and 95% 8993G:C mtDNA. All detectable mtDNA in the mitochondrially transformed cells contained the pathological 8993G:C mutation 3 months after transformation. These results suggest that 8993G:C mtDNA had a selective advantage over 8993T:A mtDNA in both lung carcinoma and osteo-sarcoma cell backgrounds. In contrast, two other presumed pathological mtDNA variants were lost in favour of 'wild-type' mtDNA molecules in the same lung carcinoma cell background. Taken together, these findings suggest that the transmission bias of mtDNA variants is dependent upon a combination of nuclear background and mtDNA genotype. A second phenomenon observed was a marked decrease in the growth rate of many putative transformed cell lines after 6 weeks of culturing in selective medium, and in these cell lines mtDNA was not readily detectable by Southern blotting. Restriction endonuclease analysis and sequencing of amplified mtDNA demonstrated that the slow growing cells contained little or no mtDNA. It is concluded that these cells represented transient mitochondrial transformants.

Ataxia↗
PubMed1998

Maternal inheritance of mouse mtDNA in interspecific hybrids: segregation of the leaked paternal mtDNA followed by the prevention of subsequent paternal leakage.

The transmission profiles of sperm mtDNA introduced into fertilized eggs were examined in detail in F1 hybrids of mouse interspecific crosses by addressing three aspects. The first is whether the leaked paternal mtDNA in fertilized eggs produced by interspecific crosses was distributed stably to all tissues after the eggs' development to adults. The second is whether the leaked paternal mtDNA was transmitted to the subsequent generations. The third is whether paternal mtDNA continuously leaks in subsequent backcrosses. For identification of the leaked paternal mtDNA, we prepared total DNA samples directly from tissues or embryos and used PCR techniques that can detect a few molecules of paternal mtDNA even in the presence of 10(8)-fold excess of maternal mtDNA. The results showed that the leaked paternal mtDNA was not distributed to all tissues in the F1 hybrids or transmitted to the following generations through the female germ line. Moreover, the paternal mtDNA leakage was limited to the first generation of an interspecific cross and did not occur in progeny from subsequent backcrosses. These observations suggest that species-specific exclusion of sperm mtDNA in mammalian fertilized eggs is extremely stringent, ensuring strictly maternal inheritance of mtDNA.

Animals↗
PubMed2003

Pathogenic role of mtDNA duplications in mitochondrial diseases associated with mtDNA deletions.

We estimated the frequency of multiple mtDNA rearrangements by Southern blot in 32 patients affected by mitochondrial disorders associated with single deletions in order to assess genotype-phenotype correlations and elucidate the pathogenic significance of mtDNA duplications. Muscle in situ hybridization studies were performed in patients showing mtDNA duplications at Southern blot. We found multiple rearrangements in 12/32 (37.5%) patients; in particular, mtDNA duplications were detected in 4/4 Kearns-Sayre syndrome (KSS), in 1 Pearson's syndrome, in 1/3 encephalomyopathies with progressive external ophthalmoplegia (PEO), and in 2/23 PEO. In situ studies documented an exclusive accumulation of deleted mtDNAs in cytochrome c oxidase negative fibers of patients with mtDNA duplications. The presence of mtDNA duplications significantly correlated with onset of symptoms before age 15 and occurrence of clinical multisystem involvement. Analysis of biochemical data documented a predominant reduction of complex III in patients without duplications compared to patients with mtDNA duplications. Our data indicate that multiple mtDNA rearrangements are detectable in a considerable proportion of patients with single deletions and that mtDNA duplications do not cause any oxidative impairment. They more likely play a pathogenic role in the determination of clinical expression of mitochondrial diseases associated with single mtDNA deletions, possibly generating deleted mtDNAs in embryonic tissues by homologous recombination.

Adolescent↗
PubMed2000

Rearrangements of human mitochondrial DNA (mtDNA): new insights into the regulation of mtDNA copy number and gene expression.

Mitochondria from patients with Kearns-Sayre syndrome harboring large-scale rearrangements of human mitochondrial DNA (mtDNA; both partial deletions and a partial duplication) were introduced into human cells lacking endogenous mtDNA. Cytoplasmic hybrids containing 100% wild-type mtDNA, 100% mtDNA with partial duplications, and 100% mtDNA with partial deletions were isolated and characterized. The cell lines with 100% deleted mtDNAs exhibited a complete impairment of respiratory chain function and oxidative phosphorylation. In contrast, there were no detectable respiratory chain or protein synthesis defects in the cell lines with 100% duplicated mtDNAs. Unexpectedly, the mass of mtDNA was identical in all cell lines, despite the fact that different lines contained mtDNAs of vastly different sizes and with different numbers of replication origins, suggesting that mtDNA copy number may be regulated by tightly controlled mitochondrial dNTP pools. In addition, quantitation of mtDNA-encoded RNAs and polypeptides in these lines provided evidence that mtDNA gene copy number affects gene expression, which, in turn, is regulated at both the post-transcriptional and translational levels.

Cell Division↗
PubMed2005

Double-strand breaks of mouse muscle mtDNA promote large deletions similar to multiple mtDNA deletions in humans.

Mitochondrial DNA (mtDNA) deletions are a common cause of mitochondrial disorders and have been found to accumulate during normal aging. Despite the fact that hundreds of deletions have been characterized at the molecular level, their mechanisms of genesis are unknown. We tested the effect of double-strand breaks of muscle mtDNA by developing a mouse model in which a mitochondrially targeted restriction endonuclease (PstI) was expressed in skeletal muscle of mice. Because mouse mtDNA harbors two PstI sites, transgenic founders developed a mitochondrial myopathy associated with mtDNA depletion. The founders showed a chimeric pattern of transgene expression and their residual level of wild-type mtDNA in muscle was approximately 40% of controls. We were able to identify the formation of large mtDNA deletions in muscle of transgenic mice. A family of mtDNA deletions was identified, and most of these rearrangements involved one of the PstI sites and the 3' end of the D-loop region. The deletions had no or small direct repeats at the breakpoint region. These features are essentially identical to the ones observed in humans with multiple mtDNA deletions in muscle, suggesting that double-strand DNA breaks mediate the formation of large mtDNA deletions.

Animals↗
PubMed2000

Cloning of neuronal mtDNA variants in cultured cells by synaptosome fusion with mtDNA-less cells.

Synaptosome cybrids were used to confirm the presence of heteroplasmic mtDNA sequence variants in the human brain. Synaptosomes contain one to several mitochondria, and when fused to mtDNA-deficient (rho degrees ) mouse or human cell lines result in viable cybrid cell lines. The brain origin of mouse synaptosome cybrid mtDNAs was confirmed using sequence polymorphisms in the mtDNA COIII, ND3 and tRNA(Arg)genes. The brain origin of the human synaptosome cybrids was confirmed using a rare mtDNA Mbo I polymorphism. Fusion of synaptosomes from the brain of a 35-year-old woman resulted in 71 synaptosome cybrids. Sequencing the mtDNA control region of these cybrid clones revealed differences in the number of Cs in a poly C track between nucleotide pairs (nps) 301 and 309. Three percent of the cybrid clones had mtDNAs with 10 Cs, 76% had nine, 18% had eight and 3% had seven Cs. Comparable results were obtained by PCR amplification, cloning and sequencing of mtDNA control regions directly from the patient's brain tissue, but not when the control region was amplified and cloned from a synaptosome cybrid homoplasmic for a mtDNA with nine Cs. Thus, we have clonally recovered mtDNA control region length variants from an adult human brain without recourse to PCR, and established the variant mtDNAs within living cultured cells. This confirms that some mtDNA heteroplasmy can exist in human neurons, and provides the opportunity to study its functional significance.

Adult↗
PubMed2006

Does premature aging of the mtDNA mutator mouse prove that mtDNA mutations are involved in natural aging?

Recent studies have demonstrated that transgenic mice with an increased rate of somatic point mutations in mitochondrial DNA (mtDNA mutator mice) display a premature aging phenotype reminiscent of human aging. These results are widely interpreted as implying that mtDNA mutations may be a central mechanism in mammalian aging. However, the levels of mutations in the mutator mice typically are more than an order of magnitude higher than typical levels in aged humans. Furthermore, most of the aging-like features are not specific to the mtDNA mutator mice, but are shared with several other premature aging mouse models, where no mtDNA mutations are involved. We conclude that, although mtDNA mutator mouse is a very useful model for studies of phenotypes associated with mtDNA mutations, the aging-like phenotypes of the mouse do not imply that mtDNA mutations are necessarily involved in natural mammalian aging. On the other hand, the fact that point mutations in aged human tissues are much less abundant than those causing premature aging in mutator mice does not mean that mtDNA mutations are not involved in human aging. Thus, mtDNA mutations may indeed be relevant to human aging, but they probably differ by origin, type, distribution, and spectra of affected tissues from those observed in mutator mice.

Adult↗
PubMedDate not supplied

[Synthesis of mtDNA and proteins coded by mtDNA in liver mitochondrial fractions of rats of varying age].

The in vivo synthesis of mtDNA and mtDNA-coded proteins was studied in the whole population and in separate fractions (heavy, middle and light) of liver mitochondria from young (3-4 months), adult (6-8 months) and old (24-26 months) rats. The synthesis rate was estimated from the value of relative specific radioactivity which represented a ratio of specific radioactivity of acid insoluble fraction to that of acid soluble fraction in suspension aliquot of isolated mitochondria. It was found that the synthesis rate of mtDNA in the whole mitochondrial population was increased in the adult rats and decreased in the old animals. The same direction of changes was noticed in heavy and light mitochondrial fractions. Moreover, at any age the synthesis rate of mtDNA was higher in light fractions as compared to other fractions. In the whole mitochondrial population, the synthesis rate of proteins coded by mtDNA was also increased in adult rats and decreased in old animals. However, this decrease was less expressed than in mtDNA case. The increase in the synthesis rate of mitochondrial proteins was noted in all mitochondrial fractions too, whereas its decrease in the old age was marked in the light mitochondria only. Owing to the above peculiarities of age-related changes of the synthesis rate of mtDNA and proteins coded by mtDNA, there takes place the prevalence with age of protein synthesis over mtDNA synthesis in the whole population and in the fractions of rat liver mitochondria.

Aging↗
PubMed1993

Accumulation of mtDNA with a mutation at position 3271 in tRNA(Leu)(UUR) gene introduced from a MELAS patient to HeLa cells lacking mtDNA results in progressive inhibition of mitochondrial respiratory function.

A new mitochondrial DNA (mtDNA) mutation of tRNA(Leu)(UUR) at nucleotide position 3271 (MELAS3271) was determined to be involved in the pathogenic process of mitochondrial diseases MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) using intercellular transfer of patient-derived mtDNA to mtDNA-less HeLa cells (rho 0 HeLa cells). Cybrid clones containing imported mtDNA exclusively from a MELAS patient with MELAS3271 mtDNA were isolated, and the influence of MELAS3271 mtDNA on mitochondrial translation activity and mitochondrial respiratory complex I enzyme activity were examined. Accumulation of more than 87% MELAS3271 mutant mtDNA in the cybrid clones induced both low complex I activity and abnormal mtDNA-encoded polypeptide synthesis including at least complex I subunit ND6. suggesting involvement of the new MELAS-associated mutation in the pathogenesis.

Blotting, Southern↗
PubMed2001

Cytoplasmic transfer of platelet mtDNA from elderly patients with Parkinson's disease to mtDNA-less HeLa cells restores complete mitochondrial respiratory function.

For determination of whether platelet mtDNA in patients with Parkinson's disease (PD) possesses some lesions to reduce respiratory enzyme activities, platelet mtDNA was transferred into mtDNA-less (rho0) HeLa cells from aged PD patients and age-matched normal subjects, since their activities were controlled by both mitochondrial and nuclear genomes. The resultant mtDNA-repopulated cybrid clones containing the HeLa nuclear genome as a common background were used for comparison of respiratory enzyme activities. Remarkable variations of the enzyme activities were observed in the cybrid clones, irrespective of whether their mtDNA was transferred from normal subjects or PD patients, and some of them showed 20% reduction of average activities. Thus, the mtDNA mutations responsible for inducing 20% reduction should be polymorphic rather than pathogenic. On the other hand, pathogenic control cybrid clones possessing mtDNA mutations from patients with mitochondrial disorders showed significant and specific decline of respiratory enzyme complex I activity beyond the normal range of the variations. These observations warrant reassessment of the conventional concept that complex I activity in platelets of PD patients is defective due to mtDNA mutations.

Aged↗
PubMed2004

No evidence for paternal inheritance of mtDNA in patients with sporadic mtDNA mutations.

With the publication of a patient with severe exercise intolerance, in whom the mutated mtDNA in muscle was shown to be paternally inherited, the strict maternal inheritance of mtDNA was challenged. Paternal mtDNA inheritance may have gone unrecognized in cases of mitochondrial disease with no clear maternal pattern of inheritance because mitochondrial haplotypes are rarely investigated in diagnostic analyses. To find further evidence for a paternal inheritance of mtDNA, we reinvestigated 12 patients with mitochondrial myopathy, in whom the pathogenic mutation was known to be sporadic. We compared the mtDNA haplotypes from the patient's muscle with that of the mtDNA haplotypes in blood from either the mother or the patient. No evidence of paternal inheritance of mtDNA was found in this small study. Although these findings indicate that the paternal inheritance of mtDNA is rare, they do not rule out that the phenomenon may occur at a rate that could still affect genetic counselling and anthropological research.

DNA, Mitochondrial↗
PubMed1980

Uniqueness of sperm mtDNA as compared to somatic mtDNA in ram.

Sperm mtDNA exhibits unique physicochemical properties compared with mtDNA from somatic tissues in the ram. The buoyant density of sperm mtDNA was 1,6983 g/cm3 while the brain, heart and liver mtDNAs was about 1.7005 g/cm3. The Tm of the liver and sperm mtDNA was 71.0 degrees C and 69.5 degrees C, respectively. The G + C content of sperm mtDNA was 3.0 moles % lower than the liver mtDNA. The contour length of the circular sperm mtDNA was 5.01 micrometers compared with the liver mtDNA with contour length of 5.33 micrometers.

Animals↗
PubMed2005

A modified procedure for quantitative analysis of mtDNA, detecting mtDNA depletion.

Quantitative analysis of mitochondrial DNA (mtDNA) is crucial for proper diagnosis of diseases that are caused by or associated with mtDNA depletion. However, such a quantitative characterization of mtDNA is not a simple procedure and requires several laboratory steps at which potential errors can accumulate. Here, we describe a modified procedure for quantitative human mtDNA analysis. The procedure is based on using two PCR-amplified, fluorescein-labeled DNA probes, complementary to mtDNA (detection probe) and chromosomal 18S rDNA (reference probe), both of similar length. Thus, equal amounts of these probes can be used and, contrary to previously published procedures, no mtDNA purification (apart from total DNA isolation) or 18S rDNA cloning is necessary for probe preparation. Two separate hybridizations (each with one probe) are suggested instead of one hybridization with both probes; this decreases background signals and enables adjustment of the strength of specific signals from both probes, which is useful in the subsequent densitometric analysis after superimposing of both pictures. Using different DNA amounts for reactions, we have proved that the procedure is quantitative in a broad range of sample DNA concentrations. Moreover, we were able to detect mtDNA depletion unambiguously in tissue samples from patients suffering from diseases caused by dysfunction of mtDNA.

DNA Primers↗
PubMed1995

Intracellular heteroplasmy for disease-associated point mutations in mtDNA: implications for disease expression and evidence for mitotic segregation of heteroplasmic units of mtDNA.

Studies in vitro have shown that a respiratory-deficient phenotype is expressed by cells when the proportion of mtDNA with a disease-associated mutation exceeds a threshold level, but analysis of tissues from patients with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) have failed to show a consistent relationship between the degree of heteroplasmy and biochemical expression of the defect. One possible explanation for this phenomenon is that there is variation of heteroplasmy between individual cells that is not adequately reflected by the mean heteroplasmy for a tissue. We have confirmed this by study of fibroblast clones from subjects heteroplasmic for the MELAS 3243 (A-->G) mtDNA mutation. Similar observations were made with fibroblast clones derived from two subjects heteroplasmic for the 11778 (G-->A) mtDNA mutation of Leber's hereditary optic neuropathy. For the MELAS 3243 mutation, the distribution of mutant mtDNA between different cells was not randomly distributed about the mean, suggesting that selection against cells with high proportions of mutant mtDNA had occurred. To explore the way in which heteroplasmic mtDNA segregates in mitosis we followed the distribution of heteroplasmy between clones over approximately 15 generations. There was either no change or a decrease in the variance of intercellular heteroplasmy for the MELAS 3243 mutation, which is most consistent with segregation of heteroplasmic units of multiple mtDNA molecules in mitosis. After mitochondria from one of the MELAS 3243 fibroblast cultures were transferred to a mitochondrial DNA-free (rho0) cell line derived from osteosarcoma cells by cytoplast fusion, the mean level and intercellular distribution of heteroplasmy was unchanged. We interpret this as evidence that somatic segregation (rather than nuclear background or cell differentiation state) is the primary determinant of the level of heteroplasmy.

Adolescent↗
PubMed2004

mtDNA mutations in tumors of the central nervous system reflect the neutral evolution of mtDNA in populations.

Mitochondrial DNA (mtDNA) instability has been observed in different types of cancer, including colorectal, breast, and gastric cancer. The relationship between the occurrence of such alteration and the nuclear microsatellite instability (nMI) status of the neoplastic cells remains controversial. In an attempt to clarify this issue, we sequenced the first and second mtDNA hypervariable regions, and typed the mitochondrial (CA)(n) dinucleotide polymorphism in 69 patients with primary tumors of the central nervous system (CNS), previously screened for nMI. Tumor samples showed 27 D-loop sequence changes (39.1%) compared to the corresponding blood samples. Microsatellite homoplasmic allele mutations were detected in four cases (5.8%). We did not find significant associations of the mtDNA instability status with clinicopathological parameters including sex, age, tumor size, and duration of clinical course. Neither did we find any association between mtDNA and nuclear instabilities, indicating that, at least in CNS tumors, they respond to different DNA repair mechanisms. We have also compiled the mtDNA instabilities previously reported by other authors for several types of tumors, comparing them to mtDNA polymorphisms in human populations. Most of the tumor-associated changes are common human polymorphisms and mutational hotspots. To explain the molecular behavior of mtDNA instability in tumors, we propose a model also common to other biological situations.

Central Nervous System Neoplasms↗
PubMed2004

Twinkle helicase is essential for mtDNA maintenance and regulates mtDNA copy number.

Mechanisms of mitochondrial DNA (mtDNA) maintenance have recently gained wide interest owing to their role in inherited diseases as well as in aging. Twinkle is a new mitochondrial 5'-3' DNA helicase, defects of which we have previously shown to underlie a mitochondrial disease, progressive external ophthalmoplegia with multiple mtDNA deletions. Mouse Twinkle is highly similar to the human counterpart, suggesting conserved function. Here, we have characterized the mouse Twinkle gene and expression profile and report that the expression patterns are not conserved between human and mouse, but are synchronized with the adjacent gene MrpL43, suggesting a shared promoter. To elucidate the in vivo role of Twinkle in mtDNA maintenance, we generated two transgenic mouse lines overexpressing wild-type Twinkle. We could demonstrate for the first time that increased expression of Twinkle in muscle and heart increases mtDNA copy number up to 3-fold higher than controls, more than any other factor reported to date. Additionally, we utilized cultured human cells and observed that reduced expression of Twinkle by RNA interference mediated a rapid drop in mtDNA copy number, further supporting the in vivo results. These data demonstrate that Twinkle helicase is essential for mtDNA maintenance, and that it may be a key regulator of mtDNA copy number in mammals.

Animals↗
PubMed1992

Heteroplasmic mtDNA mutation (T----G) at 8993 can cause Leigh disease when the percentage of abnormal mtDNA is high.

A female infant showing lacticacidemia, hypotonia, and neurodegenerative disease died at 7 mo of age. Autopsy revealed lesions typical of Leigh disease, both in the basal ganglia and in the brain stem. A maternal aunt and uncle died 1 year and 5 mo, respectively, after following a similar clinical course, while another uncle, presently 33 years of age, has retinitis pigmentosa and ataxia and is mentally retarded. PCR restriction-digest analysis of mtDNA isolated from the proband revealed a T-to-G change at position 8993, creating a new AvaI restriction site. The mutation present in the ATP 6 gene results in the substitution of an arginine residue for a leucine. The indexed patient had greater than 95% abnormal mtDNA in her skin fibroblasts, brain, kidney, and liver tissues, as measured by laser densitometry. The maternal aunt who died at age 1 year had greater than 95% abnormal mtDNA in her lymphoblasts. The uncle with retinitis pigmentosa had 78% and 79% abnormal mtDNA in his skin fibroblasts and lymphoblasts, respectively, while an asymptomatic maternal aunt and her son had no trace of this mutation. The mother of the index case had 71% and 39% abnormal mtDNA in her skin fibroblasts and lymphoblasts, respectively, showing that the heteroplasmy can be variable, on a tissue-specific basis, within one individual. This shows that mtDNA mutations at 8993 can produce the clinical phenotype of Leigh disease in addition to the phenotype of ataxia and retinitis pigmentosa described by Holt et al.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Mitochondrial↗
PubMed2002

Segregation of mitochondrial DNA (mtDNA) in human oocytes and in animal models of mtDNA disease: clinical implications.

Mitochondrial DNA (mtDNA) is almost entirely maternally inherited. Thousands of copies of mtDNA are present in every nucleated cell and in most normal individuals these are virtually identical (homoplasmy). mtDNA diseases may be caused by mutations in either mitochondrial or nuclear genes and, hence, give rise to maternal or autosomal patterns of inheritance. Antenatal diagnosis of mitochondrial diseases based on chorionic villous sampling is available for Mendelian disorders and the syndromes caused by mutations at bp 8993 (associated with Leigh's syndrome and neurogenic weakness, ataxia and retinitis pigmentosa (NARP)). However, prenatal diagnosis of many other maternally inherited mtDNA diseases is less reliable because it is not possible to predict with confidence the way in which heteroplasmic mtDNA mutations segregate within tissues and find clinical expression. This review focuses on the substantial progress in genetics that has been made recently, and on the management options that clinicians can offer to families.

Animals↗