V. MOLECULAR MECHANISM OF MUTATIONS 249 



and to decide whether or not recombinants can arise between them. The 

 frequency of single and double recombinants could be used to determine 

 the sequence and relative distance of the various mutations. 



As already outlined in Section I,C, the most refined genetic map is 

 that of the rll region of phage T4 which Benzer (1961) has evolved 

 using a new technique of deletion mapping. This experimental system is 

 sensitive enough to detect any recombinant that occurs. Within the 

 genetic region of the rll functional property at least 304 different 

 mutational sites have been found and according to the Poisson distribu- 

 tion at least 120 more sites have not yet been detected (Benzer, 1961). 

 Actually even more sites must exist, because so far certain mutagenic 

 agents of a restricted specificity have been used mainly. Since the length 

 of the rll region is at most 10* and probably of the order of 10^ nucleo- 

 tide pairs, each mutational site can correspond at most to a few nucleo- 

 tide pairs and recombination can occur between any two such sites. Most 

 spontaneous and practically all chemically induced mutations so far 

 analyzed involve only one of these sites. The induced mutations include 

 those induced by 5-bromouracil (Benzer and Freese, 1958), proflavin 

 (Brenner et al., 1958), 2-aminopurine (Freese, 1959a), nitrous acid 

 (Freese, 1959c; Benzer, 1961), ethyl methanesulfonate (Benzer, 1961), 

 low pH (Freese, 1959c), and hydroxylamine (Freese et al., 1961a). 

 Only about 5% of the spontaneous mutations extend over several of the 

 presently known mutagenic sites. 



B. SPONTANEOUS AND INDUCED REVERSE MUTATIONS 



If most mutations really correspond to the change of a single nucleo- 

 tide pair in DNA they should occasionally mutate back to the original 

 genotype, i.e., the original sequence of nucleotide pairs. Furthermore, in 

 some cases this reversion should be inducible by some of the mutagenic 

 agents. In contrast, a mutation which extends over several nucleo- 

 tide pairs might find it difficult to revert all these nucleotide pairs 

 simultaneously. 



Actually, practically all T4-r7/ mutants induced by the aforemen- 

 tioned chemicals can revert to the original phenotype spontaneously 

 and most of them can be induced to revert by some of the agents. Also 

 all but 10% of the spontaneous mutants can revert spontaneously. The 

 question is only whether these reverse mutations to the original pheno- 

 type arose by a genuine back mutation at the mutant site or rather by 

 some other ("suppressor") mutation at a different (or even the same) 

 genetic site. 



Suppressor mutations will be treated in more detail later and it may 

 suffice here to say that, for most spontaneous and induced mutants which 



