Transcriptional and translational expression of a chimeric bacterial-yeast plasmid in yeasts

Abstract

Chimeric plasmids composed of the bacterial plasmid pBR322, 2 micron yeast plasmid fragments and the 1.1 kb ura3+ fragment of yeast chromosomal DNA which codes for orotidine-5'-phosphate (OMP) decarboxylase were constructed and used to transform Escherichia coli and Saccharomyces cerevisiae recipient cells. The expression in yeast of one such plasmid was studied and compared to the expression of a chromosomally integrated bacterial plasmid. In the strain carrying the chimeric plasmid the level of OMP decarboxylase activity is about 25 times that found in either the wild-type strain or in the strain carrying the chromosomally integrated plasmid. The ampicillin gene of pBR322 is expressed in yeast. Labeling kinetics of RNA and measurements of the polyadenylated fractions showed that RNA hybridizing to the pBR322 plasmid was polyadenylated to the same extent as RNA hybridizing to the ura3+ gene. Half-lives of 10 and 20 min were estimated for the ura3+ and pBR322 transcripts respectively.