Human cell mutants with very low mitochondrial DNA copy number (rho d).
Mutants of human Namalwa cells are described, denoted rho d (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, FV03 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 rho + cells. Quantitative PCR tests showed first, that the parent Namalwa rho + cells contain about 1800 molecules of mtDNA and second, that cells of each of FV02, FV03 and FV05 contain between 100- and 1000-fold less mtDNA than rho + cells. Subclones of each of FV02, FV03 and FV05 all contained detectable mtDNA, thus eliminating the possibility that these isolates are mixtures of rho o (devoid of mtDNA) and rho + cells. Each of the rho d cell lines FV02, FV03 and FV05 had a more rapid growth rate than the corresponding rho o cells but less than that of the parent Namalwa rho + cells. The experimentally induced rho d cells described here may represent a useful model for human diseases in which severe depletion of cellular mtDNA levels in tissues is encountered.
Vaillant
F
F
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Nagley
P
P
D002455:Cell Division; D002460:Cell Line; D003580:Cytochromes / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000378:metabolism; D015894:Genome, Human; D006651:Histocytochemistry; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D005810:Multigene Family; D009154:Mutation; D010101:Oxygen Consumption / Q000235:genetics; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D013053:Spectrophotometry
D003580:Cytochromes; D004272:DNA, Mitochondrial; D011773:Pyruvates; D019289:Pyruvic Acid