A System to Study Human Mitochondrial Genes: Application to Chloramphenicol Resistance

Abstract

A set of procedures has been developed that permits the genetic analysis of mtDNA variants in cultured human cells. With this system, mutants resistant to chloramphenicol have been analyzed in detail. The chloramphenicol-resistant (cap^R) mutation was shown to be cytoplasmic by cybrid transfer and has been assigned to the mtDNA by linkage to mtDNA restriction-site polymorphisms. Using a pair of polymorphic proteins synthesized in the mitochondria, it was found that when chloramphenicol-resistant and chloramphenicol-sensitive (cap^S) mtDNAs were mixed within cells, the cap^R allele permitted the expression of the cap^S mtDNA in the presence of chloramphenicol. This implies that the mitochondria within a cell metabolically cooperate and that chloramphenicol resistance is dominant. Cloning and sequencing of the 3′ end of the large mitochondrial rRNA gene from one mouse and two human cap^R cell lines revealed that all had single-base changes in a highly conserved region of this gene. Thus, chloramphenicol resistance is the result of an altered 16S rRNA gene, which renders the ribosome insensitive to the drug.

INTRODUCTION
Human mtDNA is now known through sequence analysis to code for a 12S rRNA and a 16S rRNA, 22 tRNAs, three cytochrome oxidase subunits, one ATPase subunit, cytochrome b, and eight or nine polypeptides of unknown function (Ojala et al. 1980; Anderson et al. 1981). The genome is efficiently transcribed into structural RNAs and mRNAs (Battey and Clayton 1980; Ojala et al. 1980) and the mRNAs are translated into polypeptides on mitochondrial cap^S ribosomes.

Although molecular analysis has contributed much to our...