The trimethoprim-resistant dihydrofolate reductase associated with the R plasmid R388 was isolated from strains that overproduce the enzyme. It was purified to apparent homogeneity by affinity chromatography and two consecutive gel filtration steps under native and denaturing conditions. The purified enzyme is composed of four identical subunits with molecular weights of 8300. A 1100 bp long DNA segment which confers resistance to trimethoprim was sequenced. The structural gene was identified on the plasmid DNA by comparing the amino acid composition of the deduced proteins with that of the purified enzyme. The gene is 234 bp long and codes for 78 amino acids. No homology can be found between the deduced amino acid sequence of the R388 dihydrofolate reductase and those of other prokaryotic or eukaryotic dihydrofolate reductases. However, it differs in only 17 positions from the enzyme associated with the trimethoprimresistance plasmid R67.

DNA fragments of the R factor R388 which renders E. coli resistant to trimethoprim by inducing a trimethoprim resistant dihydrofolate reductase (Amyes and Smith, 1974) were inserted into plasmids and screened for the expression of the trimethoprim resistance gene. By means of a two step deletion procedure a 1770 bp EcoRI/BamH1 fragment was isolated which conferred drug resistance and which was found to induce the synthesis of the same dihydrofolate reductase as the parental R factor. Gene dosage experiments indicated that the induction was due to the presence of a dihydrofolate reductase structural gene on the 1770 bp fragment. The gene could be assigned to a segment which was less than 1200 bp long. The 1770 bp fragment and a recombinant plasmid consisting of pSF2124 and part of R388 were mapped with several restriction nucleases. The R factor induced enzyme was partially purified from a strain carrying a multicopy recombinant plasmid into which the 1770 bp fragment was inserted and which induced high levels of dihydrofolate reductase. The enzyme was found to be stable at 100 degrees. Some aspects of the synthesis of dihydrofolate reductase are discussed.

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