T4 phage-coded dihydrofolate reductase. Subunit composition and cloning of its structural gene.

The structures of phage-coded dihydrofolate reductases are of interest because of 1) possible relationship to plasmid-coded dihydrofolate reductases; 2) unusual regulation of enzyme synthesis; and 3) multiple roles and intermolecular interactions involving the protein. To prepare for primary structural studies, we have cloned the T4 frd gene, which codes for dihydrofolate reductase, and we have determined for redetermined some physical properties of the enzyme. The native enzyme has a molecular weight of about 44,500, as determined by sedimentation velocity and gel filtration, and a subunit molecular weight of about 23,000, as determined by aminopterin titration and denaturing gel electrophoresis. We conclude that the enzyme is a dimer, with each subunit containing one methotrexate-binding site. A 1.1-kilobase pair fragment from a HindIII restriction digest of cytosine-substituted T4 DNA was cloned into pBR322, and recombinants were identified by trimethoprim resistance. Cells carrying this recombinant plasmid produce both the host cell and phage-coded dihydrofolate reductases.