Cloning of the dut (deoxyuridine triphosphatase) gene of Escherichia coli

Gene
A F TaylorB Weiss

Abstract

Through the molecular cloning of DNA, cells were obtained that could produce a 300-fold increased level of deoxyuridine triphosphatase (dUTPase). First, lambda pyrE-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 lambda pyrE-dut phage, 45-fold (10-fold for orotidylate pyrophosphorylase); a dut-ColE1 type plasmid, 15-fold; and a thermoinducible dut-lambda-ColE1 chimera, 14-fold before induction and 300-fold after induction.

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Citations

Jan 1, 1984·Molecular & General Genetics : MGG·A MarmenoutW Fiers
Mar 4, 2005·Biological & Pharmaceutical Bulletin·Mika HoriHiroyuki Kamiya
Jun 20, 2002·Preparative Biochemistry & Biotechnology·Rebecca PerssonPer Olof Nyman
Jul 1, 1983·Applied and Environmental Microbiology·S G RogersM J Brackin
Nov 1, 1980·Gene·S G Rogers, B Weiss
Aug 1, 1990·Journal of Bacteriology·I R Tsaneva, B Weiss
Sep 1, 2008·EcoSal Plus·Kaj Frank JensenMartin WillemoËs
Sep 8, 1998·Microbiology and Molecular Biology Reviews : MMBR·M K Berlyn
Mar 1, 1988·Journal of Bacteriology·H H el-HajjB Weiss

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