Abstract
mtDNA with a point mutation in the tRNA(Ile) gene at nucleotide position 4269 found in a patient with fatal cardiomyopathy and mtDNA with a point mutation in the tRNA(Arg) gene at 10410 found in a patient with Alpers disease were transferred cytoplasmically to rho zero HeLa cells (HeLa cells lacking mtDNA) to determine whether these novel mtDNA mutations in the tRNA genes are responsible for the defects in mitochondrial respiration function observed in these diseases. Cybrid clones (clones of rho zero HeLa cells with mtDNA from the patients) were isolated, and respiratory function and morphology of the mitochondria of the cybrid clones containing wild-type mtDNA and mutant mtDNA predominantly were compared. The results showed that accumulation of mutant mtDNA at 4269 alone without defects in the nuclear genome was sufficient to produce a disease phenotype, while mutant mtDNA at 10410 was not related to pathogenesis and reflected one of the rare polymorphic sites of human mtDNA. Moreover, we found that mitochondria in living cells were significantly swollen only when they contained predominantly the pathogenic mutant mtDNA, suggesting that the functional abnormality of mitochondria induced by pathogenic mtDNA mutations in tRNA genes is always associated with their swollen structure.
Highlights
Tant mtDNA, suggesting that the functional abnormal- Besides these clinically distinct syndromesof mitochondrial itiy of mitochondria induced by pathogenic mtDNA mue-ncephalomyopathies, many kinds of mtDNA mutations, partations in tRNA genes is always associated with their ticularly in tRNA genes [13,14,15,16,17,18], have been identified in assoswollen structure
Functionaland morphological abnormalities of muscle and brain mitochondria, and they have beednivided into threedistinct subgroups,chronic progressive external ophthalmoplegia (CPEO),' mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and myoclonus epilepsy associated with ragged red fibers(MERRF) [1].Recently, some of these changes were latefor und t o be those a t rare polymorphic sites in the human population, even when they occurred at sites that are highly conserved in different species [26, 27]
By intercellular transferof CM- andAD-derived mtDNA t o clonal p" HeLa cells, we showed that accumulation of CM-derived mutant mtDNA with tRNA1Ie4269 alone was sufficient t o produce the disease phenotype without the involvement of possible defects in the nuclear genome, while AD-derived mutant mtDNA with tRNAArg10410 was not related to pathogenesis of disease; i.e. it reflected a rare polymorphic site of human mtDNA
Summary
Hyde, 0.05 M phosphate buffer, pH 7.4, for 15 min and stainedfor COX [33]with slight modifications [34]. Isolation of Cybrid Clones-Intercellular mtDNA transfer was carried mize phototoxicity. Out as described previously [9]by fusion of enucleated fibroblasts with Northern Blot Analysiosf Mitochondrial tRNAs-Total cellular RNA p' HeLa cells and cybrid clones wereisolatedinselectionmedium. HeLa cells, and fusion was carried out in the presence of 50% (w/v) buffer and 7 M urea. The fusion mixture a nylon membrane (Hybond N', Amersham Corp.) by a vacuum blotting was cultivated in selection medium withoutglucose Unfused parental p' used as hybridization probes for detecting tRNAL""'" and tRNA"", HeLa cells were removed by culture inDM170 medium, since thecyould respectively.
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