Evidence for transcriptional regulation of orotidine-5'-phosphate decarboxylase in yeast by hybridization of mRNA to the yeast structural gene cloned in Escherichia coli

Abstract

From a large population of strains of Escherichia coli carrying shear fragments of yeast (Saccharomyces cerevisiae) DNA attached by in vitro recombination to the plasmid vector pMB9, two hybrid plasmids were selected that relieve the pyrimidine requirement of nonreverting pyrF mutants of E. coli. An 1100-base-pair DNA fragment common to the two complementing plasmids was recloned into another plasmid vector, pBR322; these new hybrids retained the ability to specify orotidine-5'-phosphate decarboxylase (orotidine-5'-phosphate carboxy-lyase, EC 4.1.1.23) synthesis in E. coli. Evidence is presented that this common fragment is yeast DNA and thus apparently carried the structural information for yeast orotidine-5'-phosphate decarboxylase, the product of yeast gene ura3. A hybrid plasmid containing the 1100-base-pair fragment was used to measure levels of putative ura3 mRNA from yeast cultures labeled with [3H]adenine, ura3 mRNA was unstable with an apparent half-life of 10.5 min. Under different circumstances previously shown to alter the level of orotidine-5'-phosphate decarboxylase in yeast, a coordinate variation in proportion of labeled RNA complementary to the hybrid plasmid was found. These data support the hypothesis that regulation of the ura3 gene in yeast is at the level of transcription.