Summary
The mitochondrial macromolecular synthesis and assembly processes of three mutants of the human cell line VA2-B, which are deficient to varying degrees in mitochondrial protein synthesis and resistant to chloramphenicol (CAP), have been analyzed. The mutant VA2/CAP23 was selected directly for resistance in vivo to CAP, while the mutants VA2/mtPS- 1 and 4 were selected as respiration deficient, and subsequently found to be resistant to CAP. The phenotypes of the three mutants are inherited cytoplasmically and thus the mutations are probably localized in mitochonrial DNA (mtDNA). The gross mtDNA sequence organization of the three mutants, as analyzed by digestion with several restriction enzymes, was found to be indistinguishable from that of VA2-B cells, and their relative mtDNA content comparable to or greater than that of the parental cell line. No difference was observed in the electrophoretic mobilities of mitochondrial rRNAs or mRNAs of the mutants analyzed on denaturing methylmercuric hydroxide-agarose gels as compared to the mobilities of the RNA species of the VA2-B cells. Similarly, no significant change, or only a moderate decrease in the rate of synthesis of mitochondrial 12s or 16s rRNAs was found in the mutants. By contrast, the incorporation of3H-uridine into mitochondrial ribosomal subunits was significantly reduced in the three mutants, and in each of them, this decrease paralleled the decrease in the level of mitochondrial protein synthesis. The complex phenotypes of the mutants analyzed here could be accounted for by assuming that, as previously reported for other CAP-resistant mouse and human cell variants, the 16s rRNA gene is the site of the mutation. Such a mutation would produce a CAP-resistant phenotype and at the same time affect the assembly of the large ribosomal subunit, with a resulting reduction in mitochondrial protein synthesis and a pleiotropic respiratory deficiency. However, other possible sites for the primary defect in the mutants analyzed here cannot be excluded.
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Communicated by E. Bautz
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Wiseman, A., Attardi, G. Cytoplasmically determined human cell mutants defective in mitochondrial ribosome assembly. Molec. Gen. Genet. 186, 364–371 (1982). https://doi.org/10.1007/BF00729455
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DOI: https://doi.org/10.1007/BF00729455