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 cha racteristic 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 th e activities of all the respiratory-chain enzymes were severely decrea sed in mitochondria isolated from these fibroblasts. In order to eluci date the underlying molecular defect, cell fusions were performed betw een enucleated fibroblasts from this patient and a human-derived rho0 cell line (rho0A549.B2). The resulting cybrids were plated in medium l acking pyruvate and uridine, to select for the restoration of respirat ory-chain function. Complementation was observed between the nuclear g enome of the rh0A549.B2 cells and the mtDNA of the patient's cells, re storing mtDNA levels and respiratory-chain function in the cybrid cell s. These results indicate that mtDNA depletion in our patient is under the control of the nuclear genome.