Defects in mitochondrial DNA (mtDNA) are associated with mitochondrial encephalomyopathies (McKusick 251900). The mutations include deletions but also duplications and point mutations. In individuals with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), a common A-to-G substitution at position 3243 in the leucine (UUR) tRNA gene has been elucidated (Goto et al 1990). Furthermore, this mutant mtDNA always coexists with the wild-type mtDNA, and the proportion of the mutated mitochondrial genome is different in each tissue of one individual.This mutation creates an ApaI restriction site, and we can then easily make the molecular diagnosis for this syndrome. Blood samples are also available for molecular genetic analysis (Hammans et al 1991), but a new problem arises: do the individuals with the mutation always develop MELAS or not? It is important to answer this question whether the point mutation affects the functional mitochondrial abnormality. A defect of complex I has been detected in muscles from patients with MELAS. However, activity of this enzyme does not always decrease in fibroblasts from a MELAS patient despite their containing the mutated mitochondrial genome. We therefore studied how the mutant mtDNA coexists with wild-type mtDNA in one cell and affects functional abnormality. MATERIALS A N D M E T H O D SSkin fibroblasts from patients with MELAS were grown in Eagle's minimum essential medium (MEM) supplemented with 10% (v/v) fetal bovine serum, 0.15 % (w/v) sodium bicarbonate, 50#g/ml streptomycin and 100 U/ml penicillin. When cells were 60% confluent in a 25 crn z flask, the medium was replaced with 8 ml of opti-MEMI (Gibco). A 20 #g sample of SV40 DNA lacking its replication origin were diluted in 50 #1 of water. SV40 DNA and lipofectin solutions (50 #1 of each) were gently mixed and incubated for 15 rain at room temperature. The SV40 DNA-lipofectin complex was dropped into the cells in the opti MEM. After incubation for 12-16 h under 5% carbon dioxide at 37°C, the cells were harvested with phosphate-buffered saline containing 0.3% trypsin. About 3000 cells were distributed into each a welt of 96-well culture plate filled with 0.8 ml of the culture medium. The cells were taken out 797

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