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EVELYN M. WITKIN 



plates at 48 hours, starting with a sample of the same 

 size. Thus, for each chemical it is possible to show 

 that new mutants do actually appear. 



Dr. Hotchkiss suggests that the induced mutants 

 may differ from the spontaneous mutants in re- 

 sistance to the chemical used to induce them. In 

 the case of desoxycholate, where there is a linear 

 relation between the number of induced mutations 

 and the time of exposure to the chemical, in spite 

 of the fact that the rate of killing changes markedly 

 during the period of exposure, it is difficult to see 

 how selection can be involved. If the increase in 

 number of mutants were due in large measure to 

 the selective survival of a few mutants induced early 

 in the treatment, surely the rate of increase in the 

 proportion of mutants with time could not be total- 

 ly independent of the survival curve. 



Dr. Hotchkiss suggests that the mutant colonies 

 actually obtained after treatment with the chemical 

 should be compared with the nonmutant stock with 

 respect to resistance to the chemical. It seems to 

 me that this would not constitute a critical test 

 of the hypothesis Dr. Hotchkiss has raised. First 

 of all, a certain amount of the chemical is carried 

 over onto the plates, and then mutant colonies de- 

 velop in the presence of a very low concentration 

 of the chemical, negligible in terms of toxic or 

 mutagenic activity, but possbily sufficient to permit 

 selection during the growth of the colony of spon- 

 taneous variants better able to withstand the action 

 of the substance. Thus, to find that the mutant 

 colonies obtained after exposure to the chemical are 

 more resistant to its toxic action than the untreated 

 nonmutant stock, is not a fair test of the situation 

 before the development of the colony on the plate. 

 Should the mutant colonies prove no more resistant 

 to the action of the chemical than the untreated 

 non-mutant stock, the possibility of a temporary 

 resistance, as Dr. Hotchkiss has suggested, can be 

 raised. I cannot at present see any way of testing 

 this possibility critically, since it is based on the 

 state of a recently induced mutant, during treatment 

 with the chemical, and a transient resistance to the 

 chemical can be postulated. One possible way to 

 avoid considerations of this kind might be to start 

 out by developing a strain which is maximally re- 

 sistant to the chemical to begin with, and use this 

 strain throughout. 



The best evidence that new mutations are in fact 

 appearing throughout the exposure, to my mind, 

 will be based upon comparison of survival curves 

 and curves of induced mutations as a function of 

 exposure time. If, as has been found for desoxy- 

 cholate, the proportion of mutants increases at a 

 constant rate, regardless of changes in killing rate, 

 the possibility that recently induced mutants sur- 

 vive selectively will become quite remote. 



Kxjrnick: Dr. Witkin has expressed some sur- 

 prise at the fact that 3 out of 4 tested chemicals 

 proved mutagenic in her experiments and suggested 



that it will be found that mutagenicity is a com- 

 mon property of chemical reagents. This appears 

 to be a not unlikely prognostication. While not 

 wishing to deny the possibility of direct chemical ac- 

 tion to induce mutations, I should like to present 

 an alternative hypothesis here. 



Dr. Witkin searches only for a specific mutation- 

 resistance to Tl phage — not random mutations. She 

 observes that this mutation is present spontaneously 

 in her controls in 5 to 10 per 10 8 bacilli. It is not im- 

 probable that this mutant contains a specific muta- 

 genic substance similar to that isolated by Avery for 

 pneumococcic strains and by Boivin for a colon 

 bacillus strain. Dr. Witkin has observed that the 

 mutation effect of a given chemical corresponds 

 more nearly to its killing effect than to any other 

 factor, such as concentration of the chemical or 

 the time of exposure. It is obvious that if, as she 

 has shown, the mutant and the original strains 

 are equally sensitive to the lethal effect of the drugs 

 used, the higher the percentage of lethality, the 

 greater will be the chance of destruction of some of 

 the 5 to 10 spontaneous mutants per 10 8 bacteria. 

 The autolysis of the killed mutant (or penetration 

 through its wall whose permeability is apt to differ 

 from the living cell) will free the specific mutator 

 substance (for Tl phage resistance) into the 

 medium where it may cause the specific, directed 

 mutation of other organisms. The nearer the killing 

 rate approaches 100%, the higher will be the con- 

 centration of this mutator substance, and so the 

 higher the mutation rate. 



Dr. Witkin has observed that after about 99.9% 

 of the bacteria have been killed, the killing rate 

 approaches zero, but the production of mutations 

 continues. She suggests that this may mean that the 

 lethal and mutagenic properties of her chemicals 

 are unrelated, and that the 0.1% of surviving cells, 

 while no longer susceptible to the lethal effect are 

 still susceptible to the mutagenic effect of the drug. 

 If the hypothesis presented herein were correct, we 

 could readily account for the continued occurrence 

 of mutations in the surviving bacteria by the fact 

 that they are continuously exposed to the liberated 

 mutator of the killed spontaneous mutants and so 

 continue to undergo directed mutation. 



Of the 4 chemicals studied by Dr. Witkin, only 

 methyl green was inert as a "mutagen," although 

 its lethality in the concentrations used was compar- 

 able to that of the others. Now if, in fact, as is 

 indicated by Boivin and Avery, desoxyribonucleic 

 acid is an essential component (perhaps the only 

 component, but this argument is not germaine to 

 this discussion), a chemical which combined spe- 

 cifically with this nucleic acid might inactivate 

 the mutator substance. Methyl green is such a com- 

 pound (as opposed to pyronin, for example). Thus, 

 we may account for the lethality of this compound 

 without the expected production of mutations. 



On the other hand, some chemicals, which might 



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