Human cell mutants with very low mitochondrial DNA copy number ( d)

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Abstract
Mutants of human Namalwa cells are described, denoted ρd (mtDNA-depleted), which contain a very low mtDNA copy number. Three isolates, denoted FV02, FV03 and FV05, each selected on the basis of their pyruvate-dependent phenotype, were shown to have severely reduced mitochondrial respiratory functions consistent with results of histochemical and cytochrome analysis. Analyses of mtDNA by the polymerase chain reaction (PCR) and Southern blotting indicated that FV02, Fy03 and FV05 each contained a full complement of mtDNA sequences, 16.6 kb in length, in circular form but at a greatly reduced level compared with that of parent ρ+ cells. Quantitative PCR tests showed first, that the parent Namalwa ρ+ cells contain about 1800 molecules of mtDNA and second, that cells of each of FVO2, FVO3 and FVO5 contain between 100- and 1000-fold less mtDNA than ρ+ cells. Subclones of each of FV02, FV03 and FV05 all contained detectable mtDNA, thus eliminating the possibility that these isolates are mixtures of ρ0 (devoid of mtDNA) and ρ+ cells. Each of the ρd cell lines FV02, FV03 and FV05 had a more rapid growth rate than the corresponding ρ0 cells but less than that of the parent Namalwa ρ+ cells. The experimentally induced ρd cells described here may represent a useful model for human diseases in which severe depletion of cellular mtDNA levels in tissues is encountered.

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