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PubMed1992

The mitochondrial tRNA(Leu(UUR)) mutation in mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS): genetic, biochemical, and morphological correlations in skeletal muscle.

Mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) has recently been associated with an A----G transition at position 3243 within the mitochondrial tRNA(Leu(UUR)) gene. Besides altering the tRNA(Leu(UUR)) sequence, this point mutation lies within a DNA segment responsible for transcription termination of the rRNA genes. We have studied the distribution and expression of mutant mtDNAs in muscle biopsies from MELAS patients. Histochemical, immunohistochemical, and single-fiber PCR analysis showed that ragged-red fibers (RRF) are associated both with high levels of mutant mitochondrial genomes (greater than 85% mutant mtDNA) and with a partial cytochrome c oxidase deficiency. By quantitative in situ hybridization, the steady-state ratios of mRNAs:rRNAs were found to be similar to controls in six of eight patients studied. In two other patients the relative levels of heavy-strand mRNAs were slightly increased, but a patient with myoclonic epilepsy and RRF also exhibited a similar increase. These results directly correlate the A----G transition at mtDNA position 3243 with muscle mitochondrial proliferation, partial respiratory-chain impairment, decreased mitochondrially synthesized protein content, and no specific alterations in mitochondrial ratios of mRNAs:rRNAs.

Acidosis, Lactic↗
PubMed2004

Attenuation of free radical production and paracrystalline inclusions by creatine supplementation in a patient with a novel cytochrome b mutation.

Mitochondrial cytopathies are associated with increased free radical generation and paracrystalline inclusions. Paracrystalline inclusions were serendipitously found in a young male athlete with a very high respiratory exchange ratio during steady-state exercise; he also had an unusually low aerobic capacity. Direct sequencing of the mitochondrial DNA (mtDNA) coding regions revealed a novel missense mutation (G15497A) resulting in a glycine-->serine conversion at a highly conserved site in the cytochrome b gene in the subject, his mother, and sister. Cybrids, prepared by fusion of the subject's platelets with either U87MG rho degrees or SH-SY5Y rho degrees cells, generated higher basal levels of reactive oxygen species (ROS), had a lower adenosine triphosphate (ATP) content, and were more sensitive to oxygen and glucose deprivation and peroxynitrite generation compared to control cybrids with wild-type mtDNA. Cell survival was significantly enhanced with 50 mmol/L creatine monohydrate (CM) administration. The subject was also treated with CM (10 g/d) for a period of 5 weeks and a repeat muscle biopsy showed no paracrystalline inclusions. The results suggest that the development of exercise-induced paracrystalline inclusions may be influenced by the G15497A mtDNA mutation, and that CM mitigates against the pathological consequences of this mutation.

Adenosine Triphosphate↗
PubMed2005

The complete maternal and paternal mitochondrial genomes of the Mediterranean mussel Mytilus galloprovincialis: implications for the doubly uniparental inheritance mode of mtDNA.

The maternal (F) and paternal (M) mitochondrial genomes of the mussel Mytilus galloprovincialis have diverged by about 20% in nucleotide sequence but retained identical gene content and gene arrangement and similar nucleotide composition and codon usage bias. Both lack the ATPase8 subunit gene, have two tRNAs for methionine and a longer open-reading frame for cox3 than seen in other mollusks. Between the F and M genomes, tRNAs are most conserved followed by rRNAs and protein-coding genes, even though the degree of divergence varies considerably among the latter. Divergence at nad3 is exceptionally low most likely because this gene includes the origin of transcription of the lagging strand (O(L)). Noncoding regions are the least conserved with the notable exception of the central domain of the main control region and a segment of another noncoding region immediately following nad3. The amino acid divergence (14%) of the two genomes is smaller than in two other pairs of conspecific genomes that are available in GenBank, that of the clam Venerupis philippinarum (34%) and of the fresh water mussel Inversidens japanensis (50%), suggesting that doubly uniparental inheritance of mtDNA emerged at different times in the three species or that there has been a relatively recent replacement of the male genome by the female in the Mytilus line. The latter hypothesis is supported from phylogenetic and population studies of Mytilidae. That the M genome contains a full complement of genes with no premature termination codons argues against it being a selfish element that rides with the sperm. It is shorter than the F by 118 bp, which apparently cannot account for the postulated replicative advantage of this genome over the F in male gonads. The high similarity of the two genomes explains why the F genome may assume the role of the M genome, but it does not exclude the possibility that for this to happen some M-specific sequences must be transferred on to the F genome by means of recombination. If such sequences exist they would most likely be located in noncoding regions.

Animals↗
PubMed2002

Mitochondrial DNA in metazoa: degree of freedom in a frozen event.

The mitochondrial genome (mtDNA), due to its peculiar features such as exclusive presence of orthologous genes, uniparental inheritance, lack of recombination, small size and constant gene content, certainly represents a major model system in studies on evolutionary genomics in metazoan. In 800 million years of evolution the gene content of metazoan mitochondrial genomes has remained practically frozen but several evolutionary processes have taken place. These processes, reviewed here, include rearrangements of gene order, changes in base composition and arising of compositional asymmetry between the two strands, variations in the genetic code and evolution of codon usage, lineage-specific nucleotide substitution rates and evolutionary patterns of mtDNA control regions.

Animals↗
PubMed2000

The "SUN" family: UTH1, an ageing gene, is also involved in the regulation of mitochondria biogenesis in Saccharomyces cerevisiae.

Since it was shown in previous work that NCA3 (one of the four genes of the SUN family) is involved in mitochondrial protein synthesis regulation, the effect of the other members of this gene family was tested. UTH1 (but not SUN4 or SIM1) was also shown to interfere with mitochondria biogenesis. In Deltauth1 cells, cytochromes aa(3), c, and b were lowered by 25 and 15%, respectively. In the double-null mutant Deltauth1Deltanca3, only cytochrome aa(3) was lowered by 50% relative to the wild type. However, the ratio of cellular respiration to cytochrome oxidase was greatly enhanced in the double-null mutant. Measurements on whole lysed cells showed that another mitochondrial enzyme, citrate synthase, was also lowered in Deltauth1 and Deltauth1Deltanca3 whereas hexokinase was not. Electron micrographs showed no difference in global mitochondria content in Deltauth1Deltanca3, but mitochondria appeared less dense to electrons compared to the wild type. Cardiolipin and mtDNA were equivalent in parental and mutant strains. Measurements on isolated mitochondria showed that the cyt aa(3)/cyt b ratio was also lowered in Deltauth1Deltanca3, but the control exerted by the oxidase on the respiratory flux was higher. The activity of other mitochondrial complexes versus oxidase was equivalent in mutants compared to the wild type. These results suggest that the protein equipment could be lowered in mitochondria from strains inactivated for UTH1.

Basic Helix-Loop-Helix Proteins↗
PubMed1986

Structure of a Cephalosporium acremonium mtDNA replicator.

We have investigated the ARS (autonomously replicating sequence) activity of a 1.94 kb mitochondrial DNA fragment of Cephalosporium acremonium and found that several subfragments of this piece of mtDNA conferred the ARS phenotype. The nucleotide sequence of the fragment shows: (i) a high A + T content (72.5%); (ii) a perfect consensus ARS sequence (ATTTATATTTA) in the subfragment with the highest ARS activity; (iii) a large number of ARS consensus-related sequences in the other subfragments, even in one lacking ARS activity; (iv) several potential hairpin structures. One of them contains the perfect consensus ARS sequence.

Acremonium↗
PubMed2007

Comparative genomics of large mitochondria in placozoans.

The first sequenced mitochondrial genome of a placozoan, Trichoplax adhaerens, challenged the conventional wisdom that a compact mitochondrial genome is a common feature among all animals. Three additional placozoan mitochondrial genomes representing highly divergent clades have been sequenced to determine whether the large Trichoplax mtDNA is a shared feature among members of the phylum Placozoa or a uniquely derived condition. All three mitochondrial genomes were found to be very large, 32- to 37-kb, circular molecules, having the typical 12 respiratory chain genes, 24 tRNAs, rnS, and rnL. They share with the Trichoplax mitochondrial genome the absence of atp8, atp9, and all ribosomal protein genes, the presence of several cox1 introns, and a large open reading frame containing an intron group I LAGLIDADG endonuclease domain. The differences in mtDNA size within Placozoa are due to variation in intergenic spacer regions and the presence or absence of long open reading frames of unknown function. Phylogenetic analyses of the 12 respiratory chain genes support the monophyly of Placozoa. The similarities in composition and structure between the three mitochondrial genomes reported here and that of Trichoplax's mtDNA suggest that their uncompacted state is a shared ancestral feature to other nonmetazoans while their gene content is a derived feature shared only among the Metazoa.

Amino Acid Sequence↗
PubMed2004

Induction of the photoaging-associated mitochondrial common deletion in vivo in normal human skin.

Mutations of mitochondrial (mt) DNA such as the 4977 base-pair large-scale deletion, also called common deletion, are increased in photoaged skin. Direct evidence for their induction by chronic exposure to ultraviolet (UV) radiation in vivo in human skin has remained elusive however. Furthermore, their fate after induction is unclear. Previously unirradiated skin of 52 normal human individuals was repetitively exposed to physiological doses of UVA light. Skin and blood specimens were investigated for the presence of mtDNA mutations employing semiquantitative nested PCR, as well as real-time PCR, after 2 weeks of UV exposure and the content of the common deletion was followed up for up to 16 mo after cessation of irradiation. As assessed by both methods, repetitive UV exposure led to an approximately 40% increase in the levels of the common deletion in normal human skin. The majority of deletions were detectable in the dermis also showing the biggest increase, whereas in the epidermis only residual levels and no increase were found. Nine individuals were examined up to 16 mo after cessation of UV exposure and some showed accumulation up to 32-fold. Thus, mtDNA mutations are induced in the human skin by repetitive UV exposure. In addition, these mutations seem to represent long-term in-vivo biomarkers for actinic damage in the human skin.

Adolescent↗
PubMed1998

Mitochondrial DNA transmission of the mitochondrial defect in Parkinson's disease.

Several groups have identified mitochondrial complex I deficiency in Parkinson's disease (PD) substantia nigra and in platelets. A search for any mitochondrial DNA (mtDNA) mutation underlying this defect has not yet produced any consistent result. We have made use of a mtDNA-less (p0) cell line to determine if the complex I deficiency follows the genomic transplantation of platelet mtDNA. From a preselected group of PD patients with low platelet complex I activity, 7 patients were used for detailed study. All 7 patients were used for mixed cybrid analysis and demonstrated a selective 25% deficiency of complex I activity. Individual clonal analysis of A549 p0/PD platelet fusion cybrids from 1 of the patients expressed combined complex I and IV deficiencies with 25% and 20% decreased activities in the PD clones, respectively. Histocytochemical, immunocytochemical, and cellular functional imaging studies of these clones showed the cells within the clones were heterogeneous with respect to cytochrome c oxidase (COX) function, COX I content, and mitochondrial respiratory chain activity. These results are in agreement with a previous study and support the proposition that an mtDNA abnormality may underlie the mitochondrial defect in at least a proportion of PD patients. This p0 technology may serve as a means to identify the subgroup of PD patients in whom an mtDNA defect may contribute to development of the disease.

Benzimidazoles↗
PubMed2004

Evolution of the mitochondrial genome: protist connections to animals, fungi and plants.

The past decade has seen the determination of complete mitochondrial genome sequences from a taxonomically diverse set of organisms. These data have allowed an unprecedented understanding of the evolution of the mitochondrial genome in terms of gene content and order, as well as genome size and structure. In addition, phylogenetic reconstructions based on mitochondrial DNA (mtDNA)-encoded protein sequences have firmly established the identities of protistan relatives of the animal, fungal and plant lineages. Analysis of the mtDNAs of these protists has provided insight into the structure of the mitochondrial genome at the origin of these three, mainly multicellular, eukaryotic groups. Further research into mtDNAs of taxa ancestral and intermediate to currently characterized organisms will help to refine pathways and modes of mtDNA evolution, as well as provide valuable phylogenetic characters to assist in unraveling the deep branching order of all eukaryotes.

Animals↗
PubMed2002

Mitochondrial cardiomyopathy--case report.

A male infant developed progressive neuromuscular disease, hypertrophic cardiomyopathy and brain atrophy since the birth. Increased level of lactate with increased lactate/pyruvate ratio suggested a disturbance in the mitochondrial energy metabolism. The activities of respiratory chain complexes III, IV and II + III, of pyruvate dehydrogenase complex and of citrate synthase in isolated muscle mitochondria were low in comparison with controls, with parallel decrease in the content of protein amount of respiratory chain complexes III and IV. No large scale deletions of mitochondrial DNA (mtDNA) and mtDNA point mutations A3243G, A8344G or T8993G indicating syndromes MELAS, MERRF or NARP were detected. The boy died at the age of 7 weeks. The autopsy revealed typical changes of mitochondrial cardiomyopathy-marked myocardial hypertrophy with muscle pallor, histological finding of diffuse fine granularity of the cytoplasm in the perinuclear regions, and ultrastructural findings of mitochondrial hyperplasia, enlargement (megamitochondria) and abnormal shape.

Adult↗
PubMed2001

Human cells are protected from mitochondrial dysfunction by complementation of DNA products in fused mitochondria.

Extensive complementation between fused mitochondria is indicated by recombination of 'parental' mitochondrial (mt) DNA (ref. 1,2) of yeast and plant cells. It has been difficult, however, to demonstrate the occurrence of complementation between fused mitochondria in mammalian species through the presence of recombinant mtDNA molecules, because sequence of mtDNA throughout an individual tends to be uniform owing to its strictly maternal inheritance. We isolated two types of respiration-deficient cell lines, with pathogenic mutations in mitochondrial tRNAIle or tRNALeu(UUR) genes from patients with mitochondrial diseases. The coexistence of their mitochondria within hybrids restored their normal morphology and respiratory enzyme activity by 10-14 days after fusion, indicating the presence of an extensive and continuous exchange of genetic contents between the mitochondria. This complementation between fused mitochondria may represent a defence of highly oxidative organelles against mitochondrial dysfunction caused by the accumulation of mtDNA lesions with age.

DNA, Mitochondrial↗
PubMed2002

A novel defense system of mitochondria in mice and human subjects for preventing expression of mitochondrial dysfunction by pathogenic mutant mtDNAs.

Recently, we generated mtDNA-based disease mice (mito mice) by introduction of respiration-deficient mitochondria possessing pathogenic mutant mtDNA with a 4696 bp deletion (deltamtDNA4696) from somatic cells into mouse zygotes. Mito mice and cytochrome c oxidase (COX) electronmicrographs, that could identify the respiration enzyme activity at individual mitochondrial levels, enabled precise investigation of the pathogenesis of deltamtDNA4696. All the observations represented unambiguous evidence for the presence of extensive and continuous exchange of genetic contents between mitochondria. Thus, the inter-mitochondrial interaction could correspond to a very unique and effective defense system of the highly oxidative organelles for preventing mice and human subjects from expressing mitochondrial dysfunction caused by mtDNA lesions, which have been continuously created by oxidative stresses during aging. Here, we would like to propose a new hypothesis on mitochondrial biogenesis, 'the interaction theory of mammalian mitochondria': mitochondria exchange genetic contents, and thus lose individuality and function as a single dynamic cellular unit.

Journal Article↗
PubMedDate not supplied

[Simple procedure for the isolation of animal mitochondrial deoxyribonucleic acid].

A simple procedure for the isolation of animal mitochondrial DNA is described. It includes deproteinization of mitochondrial lysates with phenol, precipitation of total nucleic acids with polyethylene glycol, RNA precipitation by saet and final purification of DNA using gel chromatography on a Sepharose 4B column. MtDNA preparations obtained were free from protein and RNA and were represented predimonantly by the covalently closed molecules. The content of D-looped molecules in these preparations was equal to 45%.

Animals↗
PubMed2000

Glial cell type-specific responses to menadione-induced oxidative stress.

Glial cell types in the central nervous system are continuously exposed to reactive oxygen species (ROS) due to their high oxygen metabolism and demonstrate differential susceptibility to certain pathological conditions believed to involve oxidative stress. The purpose of the current studies was to test the hypothesis that mtDNA damage could contribute to the differential susceptibility of glial cell types to apoptosis induced by oxidative stress. Primary cultures of rat astrocytes, oligodendrocytes, and microglia were utilized, and menadione was used to produce the oxidative stress. Apoptosis was detected and quantitated in menadione-treated oligodendrocytes and microglia (but not astrocytes) using either positive annexin-V staining or positive staining for 3'-OH groups in DNA. The apoptotic pathway that was activated involved the release of cytochrome c from the intermitochondrial space and activation of caspase 9. Caspase 8 was not activated after exposure to menadione in any of the cells. Using equimolar concentrations of menadione, more initial damage was observed in mtDNA from oligodendrocytes and microglia. Additionally, using concentrations of menadione that resulted in comparable initial mtDNA damage, more efficient repair was observed in astrocytes compared to either oligodendrocytes or microglia. The differential susceptibility of glial cell types to oxidative damage and apoptosis did not appear related to cellular antioxidant capacity, because under the current culture conditions astrocytes had lower total glutathione content and superoxide dismutase activity than oligodendrocytes and microglia. These results show that the differential susceptibility of glial cell types to menadione-induced oxidative stress and apoptosis appears to correlate with increased oxidative mtDNA damage and support the hypothesis that mtDNA damage could participate in the initiation of apoptosis through the enhanced release of cytochrome c and the activation of caspase 9.

Animals↗
PubMed2004

Cadmium-induced nephropathy in rats is mediated by expression of senescence-associated beta-galactosidase and accumulation of mitochondrial DNA deletion.

Long-term exposure to cadmium (Cd) induces perturbation of kidney proximal tubular epithelial cells. Mitochondrial dysfunction in renal cortical cells may contribute to the pathogenesis of Cd-induced nephropathy. In this study, we examined the accumulation of mitochondrial DNA (mtDNA) with a large deletion and cellular senescence in the renal cortex. Wistar rats at 8 weeks of age were intraperitoneally injected with 1 mL of 1 mM CdCl(2) or saline, 3 times/week for 5, 20, 40, or 80 weeks. Mitochondrial Cd content in the renal cortex was quantified by atomic absorption analysis. Cytochrome c oxidase (CCO) and senescence-associated beta-galactosidase (SA-beta-gal) activity were determined in renal cortex by enzyme-histochemistry. mtDNA in total DNA extracted from the renal cortex was amplified by PCR, and mtDNA deletions, including 4,834-bp (nt8118-nt12937) deletion, were determined and semiquantified. After 40 weeks of Cd injection, Cd levels in the renal cortex reached a saturation level, and 30% of the level of the whole-cell fraction was found in the mitochondria. CCO activity in the renal cortex, which was predominantly found in proximal tubular cells, decreased after 40 weeks of Cd exposure. Expression of SA-beta-gal was detected primarily in the proximal tubular cells and significantly increased after 80 weeks of Cd exposure. After 40 weeks of study, accumulation of 4,834-bp deletion in mtDNA was evident in both groups of rats; however, the amount of the deletion was significantly greater in Cd-treated rats than in control rats. Our results indicate that long-term Cd exposure induced a post-regenerative state of proximal tubular cells, which accelerated accumulation of 4,834-bp mtDNA deletions in the renal cortex, suggesting that Cd may be a senescence acceleration factor for kidney proximal tubular epithelial cells, which results in Cd-induced nephropathy.

Animals↗
PubMedDate not supplied

[Mitochondrial DNA sequencing of human hair shafts stored for long time].

Mitochondrial DNA (mtDNA) sequencing is commonly used for forensic genetic identification of relation and personality identification based on analysis of tooth and skeletal rudiments. We demonstrated the possibility of DNA extraction and subsequent enzymatic amplification of fragments of a hypervariable segment I of mtDNA control region from hair shafts after long storage (up to 75 years). Shed hairs are the most common biological material evidence in forensic investigations. Low content of DNA and its possible degradation in hair shafts without bulbs may cause artifacts in polymerase chain reaction. However comparative analysis of amplified nucleotide sequences of amplified fragments from hair stored for 75 years was identical to the sequence from hairs cut immediately before experiment. This indicates high quality of the resultant matrices, stability of results, and hence, the possibility of using DNA extracted from hair shafts without bulbs stored for a long time for expert genetic analysis. Theoretical and methodological prospects of using mtDNA polymorphism analysis for forensic expert evaluations are discussed.

Adult↗
PubMed2004

Mitochondrial DNA-like sequences in the nucleus (NUMTs): insights into our African origins and the mechanism of foreign DNA integration.

Nuclear mitochondrial DNA sequences (NUMTs) are common in eukaryotes. However, the mechanism by which they integrate into the nuclear genome remains a riddle. We analyzed 247 NUMTs in the human nuclear DNA (nDNA), along with their flanking regions. This analysis revealed that some NUMTs have accumulated many changes, and thus have resided in the nucleus a long time, while others are >94% similar to the reference human mitochondrial DNA (mtDNA), and thus must be recent. Among the latter, two NUMTs, encompassing the COI gene, carry a set of transitions characteristic of the extant African-specific L macrohaplogroup mtDNAs and are more homologous to human mtDNA than to chimp. Screening for one of these NUMTs revealed its presence in all human samples tested, confirming that the African macrohaplogroup L mtDNAs were present in the earliest modern humans and thus were the first human mtDNAs. An analysis of flanking sequences of the NUMTs revealed that 59% were within 150 bp of repetitive elements, with 26% being within 15 bp of and 33% being within 15-150 bp of repetitive elements. Only 14% were integrated into a repetitive element. This association of NUMTs with repetitive elements is highly nonrandom (p<0.001). These data suggest that the vicinity of transposable elements influences the ongoing integration of mtDNA sequences and their subsequent duplication within the nDNA. Finally, NUMTs appear to preferentially integrate into DNA with different GC content than the surrounding chromosomal band. Our results suggest that chromosomal structure might influence integration of NUMTs.

Africa↗