62 MUTATIONS 



hours, the concentration of the limiting substrate gets so low that the 

 bacteria give up the ghost and stop growing. The concentration of 

 limiting growth factor then builds up to a high enough level to start 

 the bacteria growing again. Thus the bacteria grow only part of the 

 time. For example, at an apparent 30-hour generation time, the 

 bacteria are growing only half the time, so the mutation rate per hour 

 is one half. 



Auerbach: Well, wouldn't Ryan's experiments agree with what 

 Dr. Freese said? — because he got discontinuous mutation frequencies, 

 I think it was a factor of 40 or so, between mutations occurring in 

 the stationary phase of bacteria and mutations occurring in the "log" 

 phase (31). 



Neel: Could somebody describe Ryan's experiment for us who don't 

 know it? 



Auerbach: As far as I remember, he did this work exclusively with 

 histidine reversions. He compared spontaneous mutation frequencies 

 in growing and resting bacteria, and he checked on residual growth in 

 various ways, for instance by the use of chloramphenicol. There were 

 mutations in resting bacteria, but as I said they were very much less 

 frequent than during the log phase. He used penicillin to check on 

 growth, as to whether or not there was growth, and he did get fewer 

 mutations, but he got an occurrence of mutations in that stage. 



Freese: But he also had shown that the DNA duplicated. 



Auerbach: He just showed that there was an increase in DNA 

 which might be DNA precursors. I don't think he showed that the 

 chromosome duplicated. 



Goldstein: May I add an experiment to this which bears on the same 

 question? In our studies on amino acid starvation in E. coli auxotrophs, 

 one can abruptly stop protein and RNA synthesis by the withdrawal 

 of the required amino acid and maintain this condition of starvation 

 for a period of one and a half to three hours. Three hours in this 

 organism corresponds to approximately four to five generations time. 



In the first half hour of this amino acid deprivation, there is about 

 a 30 per cent increase in the DNA, but then it remains stable at this 

 level for the rest of the starvation period. These cells are all physi- 

 ologically competent, because if they are refed with the missing amino 

 acid at the end of three hours, then protein, RNA, and DNA synthesis 

 immediately resume without lag, and at the original rate. 



We did do a study in this system on the frequency of streptomycin 

 resistance during the period of starvation, in which we could easily 

 have picked up the anticipated increase in the number of mutants if 



