EXPRESSION OF A PROKARYOTIC GENE IN SACCHAROMYCES CEREVISIAE (CHLORAMPHENICOL)

Abstract

The antibiotic chloramphenicol (CM) inhibits the peptidyl transferase reaction of bacterial, mitochondrial and chloroplast ribosomes. Resistance to CM can be mediated by several different mechanisms; among these in the enzyme encoded by the cam('r) gene, chloramphenicol acetyl transferase (CAT), which inactivates CM. We have placed the transposon derived cam('r) gene present on pBR325 onto the plasmid vector pYT11LEU2. This vector is maintained as an autonomously replicating multicopy plasmid in both Escherichia coli and the yeast Saccharomyces cerevisiae. The wildtype cam('r) gene on pTY11Leu2 is functionally expressed in S. cerevisiae, albeit at a low level, and we have shown that yeast transformants harboring pYT11LEU2 acquire a Cam('R) phenotype (resistance to low levels of CM), and synthesize a protein with physical and kinetic properties similar to those of CAT synthesized in E. coli. Starting with S. cerevisiae transformants harboring pYT11LEU2, we have isolated a series of plasmid mutations which confer a phenotype, in S. cerevisiae, of resistance to very high levels of CM (Cam('SR) phenotype). We have examined three of these mutant plasmids in detail, and have shown that they encode a CAT with physical, immunological and kinetic properties identical to those of wildtype CAT. Yeast transformants harboring these mutant plasmids have 50 - 1000 fold higher levels of CAT activity and CAT protein, and higher levels of CAT mRNA activity then transformants containing pYT11LEU2. We have used DNA sequencing to determine the structure of the mutation in two of these plasmids, and used northern blotting to compare the cam('r) homologous transcripts synthesized in yeast from pYT11LEU2 and the mutant plasmids. A hypothesis is presented to explain the molecular basis by which these mutations act.