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.
Bodnar
A G
AG
Department of Neurological Science, Royal Free Hospital School of Medicine, London, England.
Cooper
J M
JM
Holt
I J
IJ
Leonard
J V
JV
Schapira
A H
AH
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