Characterization of the Escherichia coli thyA gene and its amplified thymidylate synthetase product.

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Abstract
The 7.8-kilobase HindII insert in phage .lambda.NM589thyA was confirmed as originating from E. coli by hybridization analysis and was shown to encode the thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase EC 2.1.1.45) of E. coli K-12 by using biochemical, structural and immunologic criteria. The 7.8-kilobase insert was reduced in size to a quasi-random population of DNA subfragments by partial digestion with the 4-base-pair recognition enzymes Alu I and Hae III. A clone containing a 1.1-1.2-kilobase fragment that encompassed the gene was obtained from this mixture by selecting for Thy+ recombinants. Fusion of this DNA fragment to the phage .lambda. .pi.L promoter in plasmid pKC30 revealed the direction of transcription of the thyA gene, and, in a phage .lambda. lysogen containing a thermolabile repressor, intracellular synthetase levels were increased .apprx. 700-fold. The enzyme was purified to homogeneity from this source by affinity chromatography and some of its properties are described.