We describe methods that allow isolation, identification, and counting of mitochondrial mutants that are resistant to antibiotics (antR ) or respiratory deficient (rho− ). (1) Analysis of diploid and meiotic progenies generated in crosses between mutants and tester strains allows distinguishing nuclear from mitochondrial mutants, for either antibiotic resistance or respiratory deficiency. (2) The mutation rate of mitochondrial deoxyribonucleic acid (mtDNA) can be estimated from the average frequency ofantR mutants produced in a large number of independent clones. (3) The frequency of retention of mtDNA fragments inrho− genomes accumulating in nuclear respiratory-deficient mutants can be determined by a genetic test based on the ability of theserho− genomes to restore cellular growth on glycerol in crosses with selected mutants bearing punctual mutations in their mtDNA (mit− ).
