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Cloning the trpR gene

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Summary

In Escherichia coli, the structural gene for purine nucleoside phosphorylase, deoD, is subject to insertional inactivation by prophage λ. From one such secondary site λ 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-methyltryptophan, nor super repression of the tryptophan biosynthetic enzymes was observed.

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Communicated by G.A.O'Donovan

Journal Paper No. 7426 of the Purdue University Agricultural Experiment Station

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Roeder, W., Somerville, R.L. Cloning the trpR gene. Molec. gen. Genet. 176, 361–368 (1979). https://doi.org/10.1007/BF00333098

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