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Two hydroxylamine-induced mutants of bacteriophage T4 defective in modification of host valyl-tRNA synthetase have been isolated by assay of crude extracts for the activity that is characteristic of the wild-type virus. The mutations define a single gene that is situated between the rI and e genes on the T4 genetic map. This new gene is designated vs for valyl-tRNA synthetase. One of the mutations may be of the missense type since it results in the production of a valyl-tRNA synthetase activity that has unusual ureainactivation properties. The other appears to be an amber mutation since the viral enzyme can only be found after infection of cells that are permissive for amber mutations. No differences in growth properties were found between wild type and amber mutant strains on the nonpermissive host. We conclude that the bacteriophage T4 valyl-tRNA synthetase is not essential for viability under prevailing laboratory conditions.

The dihydrofolate (FH 2 ) reductase specified by the T4 frd gene (previously called the wh gene) is apparently nonessential for phage growth on Escherichia coli because the bacterial FH 2 reductase can partially substitute for the phage enzyme. To study the effect in vivo of folate analogs which specifically inhibit the T4 FH 2 reductase, the enzyme must be made essential for phage production. Partial inhibition of the E. coli FH 2 reductase with the folate analog trimethoprim strongly inhibits phage production by a T4 frd mutant and slightly inhibits wild-type phage production. Adding an additional T4-specific folate analog, a chlorophenyl triazine, strongly inhibits wild-type phage production without preventing the uninfected E. coli cells from multiplying. We have isolated spontaneous mutants of T4 which are capable of producing progeny in the presence of chlorophenyl triazine and another folate analog pyrimethamine. These mutants are designated folate analog resistant ( far ) and have been separated into two general classes. Class I mutants induce T4-specific FH 2 reductases which are less sensitive to the action of the folate analogs than the normal FH 2 reductase. Class II mutants induce an unaltered FH 2 reductase but contain mutations in a variety of T4 genes. Some class II mutants induce more phage FH 2 reductase activity than wild-type T4 and appear to be regulatory mutants.

Mutagenized T4 was screened for hydroxyurea sensitivity on mutant strain Escherichia coli OK305, which was growing slowly on a synthetic medium. Most of the hydroxyurea-sensitive mutants of T4 isolated in this manner are different from those previously described by Warner et al. (1970) and Hercules et al. (1971), and several may be in new cistrons. Most of them produce fewer progeny per infected cell than wild-type T4, even in the absence of hydroxyurea. The first seven mutants studied map at seven distinct sites and affect at least five different cistrons which are scattered throughout the major early region of the T4 genetic map (between gene 38 and rI). At least one of these mutants is defective in the ability to degrade E. coli DNA.

Hydroxyurea (HU) has been used successfully (Warner el al. , 1970; Hercules et al. , 1971) to select bacteriophage T4 mutants which are deficient in degrading Escherichia coli DNA after infection. Using a different set of plating conditions, HU has also been used to isolate another set of T4 mutants ( hus ) ( Goscin and Hall, 1972 ). All the backcrossed hus mutants degrade bacterial DNA normally. The original hus 13 contained another mutation N. The double mutant hus 13-N degrades E. coli DNA slowly in the absence of phage DNA synthesis, leaving DNA fragments with a molecular weight midway between the sizes of fragments left by mutants defective in genes 46 and 47 and by endonuclease II ( den A) mutants at similar times after infection. Endonuclease IV activity is normal in hus 13-N infected cells. The HU sensitivity of three mutants, hus 3 (which is deficient in exonuclease A), hus 7 and hus 23 is partially due to inefficient utilization of host-derived deoxyribonucleotides as substrates for phage DNA synthesis. This is related to the DNA delay phenotype of these 3 mutants. The mutants hus 7 and hus 23 also appear to package phage DNA into phage particles inefficiently. The backcrossed hus 13 is apparently HU sensitive due to a defect in packaging DNA into phage particles. The HU sensitivity of two other mutants ( hus 19 and hus 20) remains unexplained as these mutants degrade bacterial DNA normally, synthesize phage DNA normally using host-derived deoxyribonucleotides, and package the DNA into phage particles efficiently.

T4 induces dCMP deaminase activity in Escherichia coli B. Mutants ( cd ) of T4 unable to induce this activity have been isolated at a high frequency by hydroxylamine treatment. No selection procedure was needed other than an enzymatic assay of extracts of phage-infected cells. The isolation of these mutants indicates that at least one T4 cistron controls the production of dCMP deaminase activity. Studies of one cd mutant suggest that either it makes no dCMP deaminase or else one that is structurally altered. The cd + function does not appear to be essential for T4 growth in E. coli B nor does it significantly affect the production of the other early enzymes dTMP synthetase and dCMP hydrooxymethylase.

Genetic mapping of a T4 mutant ( cd ) deficient in the ability to induce dCMP deaminase in Escherichia coli B has shown that it is very closely linked to a mutant ( td ) deficient in the ability to induce dTMP synthetase. The finding that these genes controlling sequential biosynthetic reactions are very closely linked and probably adjacent to each other suggests that each of these genes may be part of a larger unit of genetic organization, possibly a unit of coordinate expression. A new type of T4 mutant ( wh ) has been found. The three genes appear to be contiguous and in the order cd, td, wh . Like the cd and td functions, the wh function does not appear to be essential for growth of T4 in E. coli B. Preliminary analysis of one wh mutant suggests a defect in the ability to induce synthesis of dihydrofolate reductase.