Through the molecular cloning of DNA, cells were obtained that could produce a 300-fold increased level of deoxyuridine triphosphatase (dUTPase). First, λpyrE-dut phages were constructed from restriction endonuclease fragments. They contained a segment of Escherichia coli DNA that spanned the structural genes for dUTPase (dut) and orotidylate pyrophosphorylase (pyrE). The initial isolates demonstrated poor enzyme production and impaired growth. Improved enzyme yields were then obtained from large-plaque derivatives and from mutants with partial deletions of the cloned DNA. The deletion mutants were isolated after the induction of a recombinant prophage whose DNA was too large to be packaged. Finally, a 3.3-kb segment of DNA, containing the dut gene, was transferred to plasmid vectors. The recombinants and their levels of dUTPase overproduction (relative to that of wild type cells) were as follows: a thermoinducible λpyrE-dut phage, 45-fold (10-fold for orotidylate pyrophosphorylase); a dut-ColE1 type plasmid, 15-fold; and a thermoinducible dut-λ-ColE1 chimera, 14-fold before induction and 300-fold after induction.
Bibliographical noteFunding Information:
the caFable technical assistance of Brian J. White. This work was supported by a grant from the ~merican Cancer Society (NP-126) and from the National Cancer Institute (5 P01 CA16519).
- Recombinant DNA-E. coli pyrE gene
- dUTPase overproduction
- λ tranducing phages
- λ-plasmid vectors