CONSTRUCTION AND PHYSICAL MAPPING OF PLASMIDS CONTAINING THE META GENE OF ESCHERICHIA-COLI K-12

Plasmids containing the metA gene (which codes for homoserine transsuccinylase of the methionine biosynthetic pathway) of E. coli K-12 were constructed in vitro using plasmid pBR322 as the cloning vehicle and .lambda.metA transducing phage as the source of metA DNA. EcoRI digests of pBR322 and .lambda.metA20 were jointed by ligase and plasmids carrying the metA gene were selected after transformation in a metA deletion strain. Recombinant DNA molecules contained 1 pBR322 fragment and 1 .lambda.metA20 fragment of 12.2 kb [kilobases] which was present in either of 2 possible orientations. Plasmids constructed by BamH1 digestion of .lambda.metA2 contained a single bacterial DNA fragment of 5.8 kb inserted in the tet gene. Insertion of the metA fragment led to loss of resistance to tetracycline in one orientation and partial resistance in the opposite orientation.