Identification of the protein products of the rrnC, ilv, rho region of the Escherichia coli K-12 chromosome

Abstract

Two methods have been used to identify the protein products of the Escherichia coli K-12 ilv region at 84 min and the flanking rrnC (counterclockwise) and rho (clockwise) loci. First, a set of lambda dilv specialized transducing phages, including some phages that carry rho and others that carry part of rrnC, was used to infect UV irradiated cells. The proteins produced by the infecting lambda dilv phage were selectively labelled with radioactivity amino acids and identified by SDS gel electrophoresis and autoradiography. Second, restriction enzyme fragments were cloned from the lambda dilv phage into pBR322 and the plasmid specific gene products produced in maxicells were similarly identified by SDS gel electrophoresis and autoradiography. The proteins produced were correlated with specific genes and restriction enzyme fragments present in the lambda dilv phage and the pBR322 derivatives. Several ilv gene products that have previously been refractory to protein purification attempts have been identified for the first time by this technique. The presence of mutations at the ilvO site is shown to activate the cryptic ilvG gene and to result in the production of a 62,000 dalton protein. A 15,000 dalton protein of unknown function is synthesized from a DNA segment between ilv and rrnC. The rho gene was cloned from lambda dilv phage into pBR322 and shown to be dominant to a rho mutation on the host chromosome. The rho gene product and four additional proteins coded by genes near or between rho and ilv have been detected.