100 MUTATIONS 



in the sense of the German biophysicist Jordan; that is, it produces 

 its effects by discrete, more or less random events in the genetic 

 material, similar to X-rays. This was proved by dose-effect curves. 

 For X-rays, it can be shown that individual "hits" are responsible 

 for mutations and chromosome breaks, because with increasing dose 

 the frequency of chomosome rearrangements, for instance transloca- 

 tions, increases approximately as the square of the frequency of 

 lethals. This is so because translocations require two independently 

 produced breaks. Dose-effect curves for chemical mutagens seem quite 

 unreliable to me because they obviously must be distorted by factors 

 such as penetration, diffusion, etc. But what one can do in an organism 

 like Drosophila is to plot one effect against the other. When this was 

 done (56) for mustard gas it was found that translocation frequency in- 

 creased almost exactly as the square of lethal frequency. This indi- 

 cates that two independent events, one of which is sufficient to produce 

 a lethal, are required for producing a translocation. By this criterium, 

 mustard gas has been shown to act as "hit-poison." 



How exactly alkylating agents act cannot be derived from work 

 with higher organisms. Recently, Loveless (47) from the Chester- 

 Beatty Institute in London reported that one of them — ethylmethane 

 sulfonate — produces mutations in phages in vitro. Ethyl sulfate had the 

 same effect. This was quite a breakthrough because until recently it 

 had not seemed possible to make phages mutate by treating them in 

 vitro. Then Krieg in Oak Ridge managed to do this with ultraviolet 

 radiation, and this was followed by Loveless' success with chemicals. 



Green in Oak Ridge confirmed Loveless' results, and he further 

 established that the mutations are not produced by ethylmethane 

 sulfonate that is carried passively into the bacterium and acts there 

 during phage formation. He showed this by infecting bacteria with a 

 mixture of treated and untreated phage, and all the mutations occurred 

 in the treated one. This, as far as I know, is the only evidence for an 

 alkylating agent acting directly on DNA. Dr. Freese said that he has 

 an explanation for this effect. 



Freese: I think it is important to point out that ethylmethane sul- 

 fonate is a monofunctional ethylating agent; only the ethyl group can 

 react whereas the methyl group is firmly attached to the sulfate. We 

 have worked with a similar agent, ethylethane sulfonate, in which 

 the ethyl group is again the active one. This has been done mainly by 

 Mr. Bautz in our laboratory, and we have found the following (6) : 



First, we made a theoretical consideration; one knows, from experi- 

 ments by Reiner and Zamenhof (60) and by Lawley (43), that diethyl 



