HUMAN CELL MUTANTS WITH VERY-LOW MITOCHONDRIAL-DNA COPY NUMBER (RHO(D))

Mutants of human Namalwa cells; are described, denoted rho(d) (mtDNA-d epleted), which contain a very low mtDNA copy number, Three isolates, denoted FV02, FV03 and FV05, each selected on the basis of their pyruv ate-dependent phenotype, were shown to have severely reduced mitochond rial respiratory functions consistent with results of histochemical an d cytochrome analysis, Analyses of mtDNA by the polymerase chain react ion (PCR) and Southern blotting indicated that FV02, FV03 and FV05 eac h contained a full complement of mtDNA sequences, 16.6 kb in length, i n circular form but at a greatly reduced level compared with that of p arent rho(+) cells, Quantitative PCR tests showed first, that the pare nt Namalwa rho(+) cells contain about 1800 molecules of mtDNA and seco nd, 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, FV 03 and FV05 all contained detectable mtDNA, thus eliminating the possi bility that these isolates are mixtures of rho(0) (devoid of mtDNA) an d rho(+) cells, Each of the rho(d) cell lines FV02, FV03 and FV05 had a more rapid growth rate than the corresponding rho(0) cells but less than that of the parent Namalwa rho(+) cells, The experimentally induc ed 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.