The first mitochondrial mutation isolated in cell cultures of higher organisms was obtained in mouse cell lines treated with increasing concentrations of chloramphenicol for 2.5 months (Bunn et al., Proc. Natl. Acad. Sci. USA 71:1681, 1974). In animals, chloramphenicol is an inhibitor of mitochondrial protein synthesis. A long selection period is necessary because of resident populations of multiple mitochondria per cell. In plants, attempts to obtain mutants resistant to inhibitors of organellar protein synthesis are complicated by the presence of two organellar genomes, those of the plastids and the mitochondria, sensitive to a similar range of drugs. Since most of the products of the plastid genome are components of the photosynthetic apparatus (or of the protein synthesizing apparatus required to make components of the photosynthetic apparatus), plastid gene expression may not be necessary for the heterotrophic growth of cultured cells. It should therefore be possible to select directly for mitochondrial resistance in cell cultures. In a first effort to obtain chloramphenicol-resistant, mitochondrial variants in corn, we have treated BMS cells with the drug for several months. BMS wild-type cultures are completely inhibited and eventually killed by 20 µg/ml chloramphenicol, although some slow population doubling (Td = 144h) occurs at 10 µg/ml. Selection was begun at the latter concentration, and after 5 months we obtained a line of cells able to grow slowly (Td = 72h) in 20 µg/ml drug.
Analysis of the mitochondria of the resistant cell line indicates that:
1. Their protein synthesizing apparatus is functional. Protein synthesis by mitochondria isolated from the resistant cell line is, however, as sensitive to chloramphenicol as protein synthesis by mitochondria from the sensitive cells; the dependence of inhibition on chloramphenicol concentration is similar for mitochondria from the two types of cell. Thus resistance is apparently not due to an alteration to the mitochondrial protein synthesizing apparatus.
2. The steady-state levels of mitochondrial gene products (relative to nuclear gene products) are lower in mitochondria of the resistant cells grown in the presence of chloramphenicol than in wild-type cells grown in nonselective conditions. Thus chloramphenicol affects gene expression in the resistant cells. These preliminary results suggest that chloramphenicol has harmful effects on the resistant cells, but they are altered in a way that increases their tolerance of these harmful effects. Alternatively, the drug may be partially excluded, so that the resistant cells are affected similarly to sensitive cells grown at lower concentrations.
J. Stadler and E. Hack
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