248 ERNST FREESE 



this property is genetically controlled and can itself nnitate. Thus, 

 "spontaneous" mutations can Ix^ conti'olled genetically and by the 

 environment. 



Some of these agents inside the cell act on duplicating DNA, others 

 on resting DNA. The freciuency of nmtations induced by the attack of 

 resting DNA increases with the time of exposure and may become large 

 if resting nucleic acid is stored for a long time, as in seeds, j)ollen 

 (Cartledge and Blakeslee, 1934), viruses, etc. Also in resting bacteria an 

 increase in the frequency of mutations has been observed (Ryan, 1959). 

 Such bacteria do not show any major turnover of DNA, but it is not 

 clear whether or not a minoi- DNA turnover occurs (Ryan et al., 1961). 



In general it is difficult to decide which mutagenic mechanism is 

 mainly responsible for the spontaneous mutations of a given organism. 

 This decision is relatively easy only in special cases in which specific 

 environmental conditions or a genetic factor greatly influence the rate. 

 I'sually one can only exclude certain mechanisms as unlikely in one of 

 two ways. On the one hand, one can compare the genetic extent and the 

 frequency distribution of spontaneous forward mutations with the extent 

 and frequency distribution of mutations induced by agents of known 

 chemical properties. This has been done for the rll mutants of phage 

 T4 and very different mutability spectra have been found for spontane- 

 ous mutations and those induced by 5-bromouracil (Benzer and Freese, 

 1958), i)roflavin (Brenner et al., 1958), 2-amino|mrine (Freese, 1959a), 

 and other mutagens (Benzer, 1961). One can conclude that the major 

 spontaneous mutagenic mechanism in phage must be different from that 

 caused by any one of the agents used for mutation induction. On the 

 other hand, one can induce the reverse mutation of spontaneous and 

 induced mutations by various mutagens and thus determine the proper- 

 ties of the base pair changes involved (see Section V). 



IV. The Extent of Point Mutations 



As shown in Section III, most mutagens induce in nucleic acids the 

 change of a single base or base pair. This does not necessarily imply that 

 such a simple change can show up as a phenotypically observable muta- 

 tion. Conceivably, several base pairs may have to be altered before such 

 a mutation would l)e produced. I shall now cite several experimental 

 results which indicate that actually a single base pair change is responsi- 

 ble for most mutations observed in phage T4. 



A. GENETIC MAPPING OF MUTATIONS 



The classical approach to an analysis of the extent of mutations has 

 been to cross as many independent mutants as possible with each other 



