Molecular and General Genetics MGG

, Volume 176, Issue 3, pp 361–368

Cloning the trpR gene

Authors

  • William Roeder
    • Department of BiochemistryPurdue University
  • Ronald L. Somerville
    • Department of BiochemistryPurdue University
Article

DOI: 10.1007/BF00333098

Cite this article as:
Roeder, W. & Somerville, R.L. Molec. gen. Genet. (1979) 176: 361. doi:10.1007/BF00333098

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.

Copyright information

© Springer-Verlag 1979