MUTAGENESIS 105 



cate that the alkylation of guanine actually gives the main mutagenic 

 effect. 



If this is true, more and more guanine should be removed from DNA 

 when we incubate the treated DNA, let's say, at 37°. Therefore, we 

 treated phages for different times with ethylethane sulfonate and deter- 

 mined their survival after subsequent incubation for different times 

 at 37° C. AVhcn we plated the phages 12 or 24 hours after the treat- 

 ment, we found indeed that the phage titer had gone down appreciably, 

 and the more the longer the original treatment lasted. 



Lederberg: What do you visualize happens during this incubation 

 period? 



Freese: What I visualize is that the ethyl groups get first attached 

 to guanine. 



Lederberg: That happens during the treatment? 



Freese: Yes, that happens during the treatment, which is short. 

 Then, when the DNA duplicates right away, when you plate right 

 away, some ethylated guanine gets removed with a certain probability, 

 either during DNA duplication or before. I don't know why we get such 

 a strong inactivation right away, but when we incubate the free phages 

 at 37°, more and more of the guanines get removed from the DNA, 

 and therefore more phages are killed. 



Auerbach: What about mutations? 



Freese: The frequency of r and mottled plaques increases by about 

 a factor 2 after 24 hours of incubation. 



Auerbach: If I may commit the mistake against which I always 

 warn and extrapolate from phage to higher organisms, I should like to 

 point to a very similar phenomenon in Neurospora or bacteria that 

 have been treated with certain mutagens. When Dr. K0lmark and I 

 treated Neurospora conidia with diepoxy butane (42), we found quite 

 by accident that if one treats the spores, washes them thoroughly, 

 and then just lets them stand, there is a veiy considerable increase in 

 mutation frequency up to 10 hours or more. There was no concomitant 

 decrease in viability, but I don't think that killing in these experiments 

 has anything to do with mutation. Szybalski (71) found the same for 

 bacteria that had been treated with TEM. What we found difficult 

 to understand in our experiments was that this delayed effect was in- 

 terrupted by plating, although plating was done on minimal medium 

 without supplements where there should be no growth theoretically, 

 just as in the water in which storing had taken place. However, in 

 practice there always is some leakage and this perhaps stops the 

 delayed effect of diepoxybutane. 



