Isolation and characterization of DNA fragments containing the dihydrofolate-reductase gene of coliphage T4.

DNA of a mutant of the bacteriophage T4, which contains cytosine instead of glucosylated hydroxymethylcytosine, was shown to direct the synthesis of enzymatically active dihydrofolate reductase in a coupled in vitro transcription-translation system. The DNA-directed synthesis of the enzyme was used to localize the dihydrofolate-reductase gene frd on a 2300 bp long restriction-nuclease-generated DNA fragment. Fine structure mapping showed that the gene is encoded on a segment of less than 1850 bp but more than 700 bp length. The enzyme, which is synthesized in vitro from the DNA fragment, has a molecular weight of 18 500 to 19 500. A restriction map was constructed which extends about 10 kb to both sides of the reductase gene and which covers the T4 genome between the genes 55 and 63. The two genes which flank the frd gene, genes 32 and td (thymidylate synthetase), were mapped in detail. A correlation between the physical and genetic maps was established.