Cloning of E. coli pnp gene from an episome

Starting with an F' episome harboring a transposon inserted in the pnp gene (Portier 1980), we were able to identify an EcoRI restriction fragment carrying the pnp and argG genes. This fragment, from both wild-type and mutant episomes, was cloned ni pACYC184. The presence of argG on the fragment allowed positive selection of the desired clones in an auxotrophic strain (argG). A restriction map was established and a fragment of 3 megadaltons subcloned in the plasmid vector pBR322. The pnp gene corresponds to about 50% of this subcloned segment and was roughly located by deletion mapping. The direction of transcription and locations of the promotor and gene extremities were determined by analyzing proteins synthesized in "maxi-cells". In addition, the gene coding for a 10,000 dalton protein was found to reside adjacent to the beginning of the pnp structural gene. Strains carrying plasmids which express the pnp overproduce polynucleotide phosphorylase.