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276 Taylor et al.8. Quantitating the level of heteroplasmy in single cells (e.g., individual musclefibers) will confirm whether high levels of the mutation above the required thresholdfor disease expression precipitate an observable biochemical defect. In thiscase, single-fiber PCR analysis will reveal significantly higher levels of mutantmtDNA in COX-deficient fibers than in COX-positive fibers. The protocolsdescribing the isolation of template DNA from single cells are described in detailin the preceding chapter.AcknowledgmentsThe financial support of the Muscular Dystrophy Group of Great Britain,the Medical Research Council, and the Wellcome Trust is gratefullyacknowledged.References1. Chinnery, P. F. and Turnbull, D. M. (1997) Clinical features, investigation, andmanagement of patients with defects of mitochondrial DNA. J. Neurol. Neurosurg.Psychiatr. 63, 559–563.2. Shoffner, J. M. (1996) Maternal inheritance and the evaluation of oxidative phosphorylationdiseases. Lancet 348, 1283–1288.3. Schon, E. A., Bonilla, E., and DiMauro, S. (1997) Mitochondrial DNA mutationsand pathogenesis. J. Bioenerg. Biomembr. 29, 131–149.4. Taylor, R. W., Chinnery, P. F., Haldane, F., Morris, A. A. M., Bindoff, L. A., Wilson,J., and Turnbull, D. M. (1996) MELAS associated with a mutation in the valinetransfer RNA gene of mitochondrial DNA. Ann. Neurol. 40, 459–462.5. Chinnery, P. F., Johnson, M. A., Taylor, R. W., Lightowlers, R. N., and Turnbull, D.M. (1997) A novel mitochondrial tRNA phenylalanine mutation presenting withacute rhabdomyolysis. Ann. Neurol. 41, 408–410.6. Chinnery, P. F., Johnson, M. A., Taylor, R. W., Durward, W. F., and Turnbull, D. M.(1997) A novel mitochondrial tRNA isoleucine mutation causing chronic progressiveexternal ophthalmoplegia. Neurology 49, 1166–1168.7. Taylor, R. W., Chinnery, P. F., Bates, M. J. D., Jackson, M. J., Johnson, M. A.,Andrews, R. M., and Turnbull, D. M. (1998) A novel mitochondrial DNA pointmutation in the tRNA Ile gene: studies in a patient presenting with chronic progressiveexternal ophthalmoplegia and multiple sclerosis. Biochem. Biophys. Res.Commun. 243, 47–51.8. Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M. H., Coulson, A. R.,Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H.,Smith, A. J., Staden, R., and Young, I. G. (1981) Sequence and organisation of thehuman mitochondrial genome. Nature 290, 457–465.9. Lightowlers, R. N., Chinnery, P. F., Turnbull, D. M., and Howell, N. (1997)Mammalian mitochondrial genetics: heredity, heteroplasmy and disease. TrendsGenet. 13, 450–455.10. Tanaka, M., Hayakawa, M., and Ozawa, T. (1996) Automated sequencing ofMitochondrial DNA. Methods Enzymol. 264, 407–421.