Vol. 44, 1958 BIOCHEMISTRY: BENZER AND FREESE 113 



be beyond the limited resolving power of genetic techniques for the organism used. 



The absence of this limitation makes phage a suitable organism for our purposes. 

 There have been reports of induction of mutations in phage by ultraviolet light,* '^^ 

 nitrogen mustard, '' streptomycin, '^ and proflavine. ^^ A very provocative discovery 

 is that analogues of the normal bases may be built into DNA in place of the usual 

 ones and also raise the mutation rate. In particular, one such analogue, 5-bromoura- 

 cil, has been proven by Dunn and Smith'* to be incorporated into the DNA of 

 phage (in place of thymine), and Litman and Pardee'^ have shown that it greatly 

 increases the frequency with which phage mutants of various types arise. 



In the present work, this possibility of directly affecting the DNA structure is 

 combined with a genetic analysis of high resolving power, to make a fine-structure 

 study of mutagenesis. Our attention will be restricted to the rll region of the 

 genome of phage T4. The mutational alterations arising by 5-bromouracil induc- 

 tion are compared to, and shown to differ from, those which occur spontaneously. 



METHODS AND MATERIALS 



Strains: phage T4B; bacterium B {E. colt B) for the isolation of mutants and as 

 plating bacterium for the determination of phage titers; S (E. coli K12S) for the 

 preparation of phage stocks; K (E. coli K12S lysogenic for prophage lambda) as 

 the selective strain for genetic tests. 



Media: broth 1 per cent bacto-tryptone (Difco) plus 0.5 per cent NaCl; glucose- 

 salts medium;'^ sulfanilamide medium same as used by Litman and Pardee,'* except 

 for higher sulfanilamide concentration (2 mg/ml) and addition of 1 /xg/ml calcium 

 pantothenate, 1 Mg/ml pyridoxine, 1 Mg/ml thiamine, 1 Mg/ml uracil, and 20 Mg/ml l- 

 tryptophane (tryptophane required for adsorption of phage T4B to B in synthetic 

 medium). Plates contain broth plus 1.3 per cent agar (Difco) with a top layer of 

 broth plus 0.7 per cent agar. 



A sample of 5-bromouracil purified by ion-exchange column, was kindly supplied 

 by Dr. Rose Litman. 



Isolation of the Mutants. — Spontaneous mutants : Details on the isolation of spon- 

 taneously arising r mutants of phage T4, their properties, and the methods used in 

 mapping them genetically are given in earlier publications.^' " In brief, a stock of 

 standard ("wild") type phage T4 (derived from a single T4 particle) is plated on B. 

 Each phage particle produces a plaque containing around 10^ progeny. The 

 progeny include occasional r mxUtants, which can be found by picking the plaque 

 and replating its contents. In order to assure that each mutant arises by an inde- 

 pendent mutational event, no more than one r mutant is isolated from any one 

 plaque of the standard type. Each r mutant is replated (to free it from any contam- 

 inating particles of standard type), an isolated r-type plaque is picked, and a stock 

 of the mutant grown on bacteriums in broth. 



Induced mutants: These were isolated from the yield of bacteria infected and 

 allowed to burst in the presence of sulfanilamide and 5-bromouracil. A culture of B 

 was prepared by inoculation of 0.4 ml. of overnight culture (grown in glucose-salts 

 medium) into 20 ml. of sulfanilamide medium and aeration for 3.5 hours to reach a 

 cell concentration of 7 X 10* per ml. At this time, 1 mg. of 5-bromouracil and 4 X 

 10* particles of T4 standard-type phage'* were introduced simultaneously. Drops 



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