Molecular cloning, correlation of genetic and restriction maps, and determination of the direction of transcription of gnd of Escherichia coli.

Abstract

Expression of the gene gnd of Escherichia coli, which encodes 6-phosphogluconate dehydrogenase, is regulated by growth rate. Using deoxyribonucleic acid from the specialized transducing phage lambda h80 dgnd his as the source of gnd, we cloned restriction fragments carrying the complete gene and portions of it on the plasmid vector pBR322. A hybrid plasmid carrying a 3.7-megadalton HindIII restriction fragment from the phage was prepared and found to be gnd+. Through restriction mapping of this fragment and subcloning segments of it, we prepared a gnd+ hybrid plasmid which carried only 1.85 megadaltons of E. coli deoxyribonucleic acid. A cleavage site for the restriction endonuclease PstI was located on the genetic map of gnd by cloning adjacent EcoRI-PstI restriction fragments and crossing the resulting hybrid plasmids with previously mapped gnd deletion and bacteriophage Mu insertion mutants. A maxicell experiment was used to determine the direction of transcription of gnd, to identify which EcoRI-PstI fragment contains the gnd promote, and to localize th beginning of the structural gene to a region about 850 +/- 150 base pairs from the PstI cleavage site. A fine-structure restriction map surrounding the PstI cleavage site was prepared for endonucleases KpnI, HincII, HaeIII, HpaII, and TaqI.