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A procedure for selective isolation of T4t mutants is described. At 120 min after infection ofEscherichia coli cells with a low multiplicity of T4 bacteriophage, the mixture was sedimented through a linear sucrose gradient, and infected cells that remained intact were collected as the fastest sedimenting fraction. Ten to 50% of the phage released by chloroform treatment of this fraction weret mutants. Collection of a high proportion oft mutants depended on efficient elimination of cells that would survive because of superinfection lysis inhibition. This was accomplished by early addition of anti-T4 serum and heat-killed cells to inactivate progeny wild-type phage released at the normal burst time. Of 85t mutants that were isolated and mapped, 23 new mutations were found, 14 of which are suppressible by anrII mutation and 9 of which are suppressible byrII or amber suppressors. Two hot-spot sites for spontaneous mutations were found; 14 mutants at one site, represented by a frameshift mutation, and 12 mutants at a second site were obtained from 39 spontaneous mutants independently isolated from different parental plaques. On our map of thet gene, the distance between the farthestt mutations is 6% recombination. A nonreverting triplet mutant, constructed to contain a frameshift mutation between two amber mutations, exhibited the samet mutant phenotype observed with revertiblet mutants.

Isolation and Mapping of t Gene Mutants of Bacteriophage T4D