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Plating techniques which eliminate T4 plaque formation on Escherichia coli by folate analogue inhibition of dihydrofolate (FH 2 ) reductase (EC 1.5.1.3) allowed the isolation of folate analogue-resistant ( far ) mutants of T4. One class of far mutants overproduces the phage-induced FH 2 reductase. Deoxycytidylate deaminase (EC 3.5.4.12), thymidine kinase (EC 2.7.1.21), and deoxycytidine triphosphatase (EC 3.6.1.12) are also overproduced by 20 min after infection at 37 C. The overproduction of FH 2 reductase by these far mutants is not affected by the absence of DNA synthesis. Other types of mutations that affect the synthesis of early enzymes cause overproduction in the absence of DNA synthesis of some of the above enzymes but not of FH 2 reductase. Therefore, overproducing far mutants apparently have mutations in previously undescribed genes controlling the expression of the T4 genome. Three of four mutants under study map near gene 56, and one maps near gene 52. All of these mutants show delays in DNA synthesis, phage production, and lysis and appear to show decreased levels of RNA synthesis based on the cumulative incorporation of uridine.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.

New mutants of T4 have been isolated by using a strain of Escherichia coli lacking thymidine kinase activity. These T4 mutants, designated tk , are able to grow on this E. coli strain under light on plates containing 5-bromodeoxyuridine and were all found to be unable to induce thymidine kinase (ATP: thymidine 5′-phosphotransferase, EC 2.7.1.21). All of these tk mutants fall into one complementation group which maps just to the right of rI on the standard T4 genetic map, far from most other genes coding for enzymes involved in pyrimidine metabolism. The tk mutants grow as well as wild-type T4, indicating that thymidine kinase is a non-essential enzyme.