Cloning arg3, the gene for ornithine carbamoyltransferase from Saccharomyces cerevisiae: expression in Escherichia coli requires secondary mutations; production of plasmid beta-lactamase in yeast

Abstract

The yeast arg3 gene, coding for ornithine carbamoyltransferase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3), has been cloned on a hybrid pBR322-2-micrometers plasmid. The cloned gene gives a normal regulatory response in yeast. It is not expressed at 35 degrees C when a mutation preventing mRNA export from the nucleus at this temperature is included in the genetic make-up of the carrier strain. In Escherichia coli, no functional expression can be observed from the native yeast arg3 gene. The study of a mutant plasmid (M1) producing low levels of yeast carbamoyltransferase in E. coli has permitted the localization and orientation of arg3 on the plasmid. The mutation involved is a deletion that alters the regulatory response of arg3 in yeast. The plasmid bla gene produces detectable amounts of beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) in yeast: the data provide an estimate of the beta-lactamase activity associated with one exemplar of the plasmid expressing arg3 (0.6 units).