Summary
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 in 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 “maxicells”. 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.
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Portier, C., Migot, C. & Grunberg-Manago, M. Cloning of E. coli pnp gene from an episome. Molec. Gen. Genet. 183, 298–305 (1981). https://doi.org/10.1007/BF00270632
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DOI: https://doi.org/10.1007/BF00270632