A physiological study of functional expression in Escherichia coli of the cloned yeast imidazoleglycerolphosphate dehydratase gene

Abstract

Functional expression in Escherichia coli of bacteriophage λ and plasmid hybrids containing the Saccharomyces cerevisiae (yeast) gene for imidazoleglycerolphosphate (IGP) dehydratase ( his3) has been characterized in growing E. coli cells lacking the bacterial IGP dehydratase activity and during lytic infection of these cells. his3 expression of an integrated bacteriophage λhis3 hybrid requires transcriptional initiation from a “promoter” in the yeast DNA. A deletion mutant lacking this promoter, but containing the his3 structural gene has been isolated. Fusion of a DNA segment containing this promoter to a segment containing the intact structural gene for tetracycline resistance ( tet r ) but lacking an intact promoter results in expression of the tet r gene. Using four physiological criteria, the level of his3 expression in growing E. coli cells is affected by gene dosage. By these criteria, cells containing multiple copies of the his3 gene produce nearly wild-type E. coli activity levels of yeast IGP dehydratase. There is no evidence for regulation of his3 expression at the gene level as a function of histidine starvation. Expression of the his3 gene during lytic infection by bacteriophage λ hybrids has been assessed by the ability of such hybrids to grow in histidine-starved E. coli cells lacking IGP dehydratase. Phage containing a functional his3 gene grow with a single burst of five under these conditions and also form “plaques without lawns” on the starved cells. Lytic expression depends on transcription from the λ promoter P L; the level depends on the distance from P L to the his3 gene. The results indicate that expression in E. coli of a eukaryotic gene obeys prokaryotic rules of gene expression and that it can occur at a significantly high level. This suggests that the basic gene recognition signals of eukaryotes and prokaryotes may not be so different.