Nuclear complementation restores mtDNA levels in cultured cells from a patient with mtDNA depletion.
We have studied cultured skin fibroblasts from a patient with a fatal mitochondrial disease manifesting soon after birth. These fibroblasts were found to grow only in the presence of pyruvate and uridine, a characteristic of cells lacking mtDNA (rho0 cells). Southern blot and PCR analyses confirmed that the patient's fibroblasts contained less than 2% of control levels of mtDNA. Biochemical analyses indicated that the activities of all the respiratory-chain enzymes were severely decreased in mitochondria isolated from these fibroblasts. In order to elucidate the underlying molecular defect, cell fusions were performed between enucleated fibroblasts from this patient and a human-derived rho0 cell line (rho0 A549.B2). The resulting cybrids were plated in medium lacking pyruvate and uridine, to select for the restoration of respiratory-chain function. Complementation was observed between the nuclear genome of the rho0 A549.B2 cells and the mtDNA of the patient's cells, restoring mtDNA levels and respiratory-chain function in the cybrid cells. These results indicate that mtDNA depletion in our patient is under the control of the nuclear genome.
Department of Neurological Science, Royal Free Hospital School of Medicine, London, England.
A G
AG
Bodnar
J M
JM
Cooper
I J
IJ
Holt
J V
JV
Leonard
A H
AH
Schapira
D002459:Cell Fusion; D002467:Cell Nucleus; D002869:Chromosome Aberrations / Q000235:genetics*; D002950:Citrate (si)-Synthase / Q000172:deficiency / Q000235:genetics; D030401:Cytochrome-c Oxidase Deficiency; D004272:DNA, Mitochondrial / Q000032:analysis / Q000235:genetics*; D042967:Electron Transport Complex I; D003576:Electron Transport Complex IV / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D006801:Humans; D006822:Hybrid Cells; D007231:Infant, Newborn; D008291:Malate Dehydrogenase / Q000172:deficiency / Q000235:genetics; D008297:Male; D008928:Mitochondria / Q000201:enzymology / Q000473:pathology*; D008931:Mitochondria, Muscle / Q000201:enzymology / Q000473:pathology; D017240:Mitochondrial Myopathies / Q000201:enzymology* / Q000235:genetics*; D009247:NADH, NADPH Oxidoreductases / Q000172:deficiency / Q000235:genetics; D015325:Pyruvate Dehydrogenase Complex Deficiency Disease / Q000235:genetics; D013384:Succinate Cytochrome c Oxidoreductase / Q000172:deficiency / Q000235:genetics
D004272:DNA, Mitochondrial; D013384:Succinate Cytochrome c Oxidoreductase; D008291:Malate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; D003576:Electron Transport Complex IV; D002950:Citrate (si)-Synthase; D042967:Electron Transport Complex I