Cloning the trpR gene.

Abstract

In Escherichia coli, the structural gene for purine nucleoside phosphorylase, deoD, is subject to insertional inactivation by prophage lambda. From one such secondary site lambda lysogen, strain SP265, one may isolate deletions that remove all or part of the trpR gene and other genes in the deo-thr sector of the E. coli chromosome. Specialized transducing phages harboring serB+ and trpR+ were liberated following induction of SP265. All such phages were N-defective, bio-type pseudolysogens whose DNA persisted in the form of plasmids. A collection of transducing phages, differing in their complement of bacterial DNA, was used to locate cleavage sites for BamHI, SalI, and PvuI within the deoD-trpR region of the E. coli genome. The trpR gene lies within a specific 950 base pair BamHI-PvuI segment. A 1250 base pair BamHI fragment carrying a functional trpR gene was cloned into the amplifiable plasmid pBR322. A single SalI site in this fragment was shown to lie within the TrpR gene. In two situations where increased gene dosage might generate elevated amounts of Trp repressor (N-defective trpR+ pseudolysogens and strains harboring pBR322 trpR+ plasmids) neither tryptophan auxotrophy, enhanced sensitivity to DL-5-methyl-tryptophan, nor super repression of the tryptophan biosynthetic enzymes was observed.