Functional and molecular mitochondrial abnormalities associated with a C --> T transition at position 3256 of the human mitochondrial genome. The effects of a pathogenic mitochondrial tRNA point mutation in organelle translation and RNA processing.
We have previously identified a mitochondrial DNA polymorphism (a C --> T transition at position 3256, within the mitochondrial tRNALeu(UUR) gene in a patient with a multisystem disorder. Although there were several indicators suggesting a pathogenetic role for this mtDNA polymorphism, its heteroplasmic nature made functional and molecular studies difficult to interpret. We have now fused enucleated fibroblasts from the patient with a mtDNA-less cell line to generate transmitochondrial cybrids harboring different proportions of mutated and wild-type mtDNA. Individual clones harboring essentially 100% wild-type or > 99% mutated mtDNAs were characterized and studied for respiratory capacity, respiratory chain enzymes activity, mitochondrial protein synthesis, and RNA steady-state levels and processing. Our results showed that cell lines containing exclusively mutated mtDNAs respire poorly, overproduce lactic acid, and have significantly impaired activity of respiratory complexes I and IV. Molecular studies showed that mutant clones have a decrease in steady-state levels of mitochondrial tRNALeu(UUR), and a partial impairment of mitochondrial protein synthesis and steady-state levels, suggesting that these molecular abnormalities are involved in the pathogenetic mechanism of the mtDNA 3256 mutation.
Hao
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Department of Neurology, University of Miami, Florida 33136, USA.
Moraes
C T
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D015152:Blotting, Northern; D004272:DNA, Mitochondrial / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D017240:Mitochondrial Myopathies / Q000235:genetics*; D010085:Oxidative Phosphorylation; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics*; D000077278:RNA, Mitochondrial; D012356:RNA, Transfer, Leu / Q000235:genetics*
D004272:DNA, Mitochondrial; D000077278:RNA, Mitochondrial; D012356:RNA, Transfer, Leu; D012313:RNA