V. MOLECULAR MECHANISM OF MUTATIONS 251 



If mutation induction is a one-hit phenomenon, i.e., a single chemical 

 reaction can induce a mutation, the frequency of mutants per viable 

 l)hage must increase linearly with the number of treated bases, that 

 means with the number of lethal hits. If we call M the frequency of 

 mutants per viable phage we thus have: 



M = ilf + ixn 



where jx is a constant which depends only on the kind of mutation which 

 one examines. It measures the probability with which the mutational 

 site or sites are attacked by the chemical, per lethal reaction of the 

 phage. If, in contrast, more than one chemical reaction (hit) were neces- 

 sary to induce a mutation, M would increase with some power of ixn. 



Kinetic measurements of his kind have been done with nitrous acid 

 as mutagen for TMV (Mundry and Gierer, 1958) and phages T2 

 (Vielmetter and Wieder, 1959) and T4 (E. B. Freese and E. Freese, 

 1960), with ethyl ethanesulfonate (Bautz and Freese, 1960; E. B. 

 Freese, 1961), with hydroxylamine (Freese et al., 1961a,b), and with 

 low pH treatment (E. B. Freese, 1961). It was always found that the 

 frequency of forward mutations per visible phage (e.g., from standard 

 type to r type) increases linearly with the number of lethal hits. This 

 indicates that each of these mutations gets induced by the chemical 

 reaction with a single base of DNA. Not only the initial but also the 

 ultimate change in this mutated DNA apparently involves only one 

 nucleotide pair, since for the induction of reverse mutations again the 

 frequency of revertants per viable phage increases linearly, as can be 

 seen in Fig. 17 (p. 259). 



The various mutagens induce about 10 *-10~'' revertants per lethal 

 hit for highly reversion inducible mutants. This frequency is about 

 lO'-lO"* times smaller than that of the induction of rll forward muta- 

 tions. Since a T2 or T4 phage has about 2 X 10^ nucleotide pairs, these 

 figures are of the right order of magnitude if one assumes that the hit 

 of only one or any one of a few bases can induce a reverse mutation 

 while an rll forward mutation can be induced by the alteration of any 

 one of many bases within the rll region, and a lethal effect can be 

 produced by the alteration of any one of even more bases. In contrast, 

 if the alteration of more than one base were necessaiy to induce a 

 reverse mutation, a two or more hit curve should be observed, with a 

 gradually increasing slope, and the frequencies of reverse mutations per 

 lethal hit should be much less (e.g., of the order 10~^"). 



It should be noted that quite a number of mutants do not respond to 

 any in vitro mutagen known so far. Obviously, the above kinetic argu- 

 ments do not hold for them. 



