Expression of a prokaryotic gene in yeast: isolation and characterization of mutants with increased expression

Summary The Escherichia coli Tn9 derived chloramphenicol resistance gene (cam ^r) is functionally expressed in the yeast Saccharomyces cerevisiae. This gene was introduced into yeast cells as part of a hybrid yeast/E. coli shuttle plasmid. A number of plasmid associated yeast mutants overproducing the cam ^r gene product, chloramphenicol acetyltransferase (acetyl-CoA: chloramphenicol 3-0-acetyltransferase, E.C. 2.3.1.28) were isolated. One of the plasmid mutants was analyzed in some detail. Even though this mutant showed a 1,000 fold overproduction of chloramphenicol acetyltransferase in the yeast host the level of RNA complementary to the cam ^r gene was not increased. A deletion of 127 base pairs in the region immediately upstream from the 5 end of the cam ^r gene appeared to be responsible for the up phenotype of this mutant. This mutation affected the expression of the cam ^r gene in E. coli in a down fashion, in contrast to its effect in yeast.