Cloning of Dihydrofolate Reductase Gene of Escherichia coli K12

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Abstract
Resistant strains for trimethoprim, a potent inhibitor of dihydrofolate reductase, were obtained by transforming the ligated products ofEscherichia coli K12 DNA and plasmid pBR 322 BamH I fragments. The strains carry a 13.6 kbp plasmid, pTP 1, which contains the trimethoprim- and ampicillin-resistance determinant genes. The trimethoprim-resistance determinant gene was estimated to consist of more than 500 nucleotides and less than 1,500 nucleotides and was restricted by EcoR I and Sal I. Trimethoprim-, ampicillin-, and tetracycline-resistant plasmids were made in the following way, and the resultant plasmids contained a unique EcoR I “insertional inactivation” site for trimethoprim resistance: the DNA sequences extraneous to the determinant gene of the trimethoprim resistance on BamH I fragment of pTP 1 were eliminated by digestion with a double-strand-specific exonuclease BAL 31, and the resultant fragments were ligated with pBR 322 which had been digested by EcoR I and a single-strand-specific nuclease S1. The strains carrying pTP 1 or trimethoprim-resistant plasmids produced about 10 times more dihydrofolate reductase than control strains. The enhancement of the enzyme production, which is due to an increase in the copy number of the enzyme gene, seems to be responsible for the trimethoprim resistance of the transformed cells.