ISOLATION AND CHARACTERIZATION OF CHLORAMPHENICOL RESISTANT MITOCHONDRIAL MUTANTS IN SACCHAROMYCES CEREVISIAE.
Chloramphenicol is a broad spectrum antibiotic, cheaply and easily available. It works by inhibiting protein synthesis in prokaryotes such as E. coli. Its main mode of action is via its binding to the large ribosomal subunit of the bacterial ribosome and inhibiting peptidyl transferase activity, thereby terminating protein chain elongation (Moazed and Noller, 1987). Saccharomyces cerevisiae, commonly known as baker’s yeast, is a model eukaryotic organism. It can be easily grown in the laboratory and is ideal for testing resistance against microbial drugs. This is because the structure of the mitochondrial genome is similar to that of prokaryotic microbes and thus allows for successful comparison. The mitochondrial genome of S. cerevisiae is a molecule of approximately 75 kb and codes for the two rRNA components of the mitochondrial ribosomes. Similar to E. coli, S. cerevisiae when exposed to chloramphenicol shows inhibited mitochondrial protein synthesis (Clark-Walker, 1967). Resistance to chloramphenicol can arise in yeast via point mutations in the mitochondrial genome. These mutations affect the structure of the large ribosomal subunit in such a way that the antibiotic can no longer bind to it and thus inhibit protein synthesis. A known site of resistance to chloramphenicol in that region of the mitochondrial genome is called cap1. In this study spontaneous mutants of S. cerevisiae that showed resistance to chloramphenicol were generated from multiple strains. These mutants were then tested for their recombination frequencies with one another and with known cap1 testers. Allelic and non-allelic mutants were determined, and some of those were selected for mitochondrial DNA isolation and sequencing. The purpose of this study was to not only confirm the published sequence of the cap1 mutation but also to characterize and sequence new mutants that display additional sites of mutation. It is hoped that with a greater understanding of how chloramphenicol binds to the mitochondrial ribosome in yeast, new strategies for the development of synthetic antibiotics can be developed.