INDUCED MUTATIONS IN E. COLI 



267 



themselves not inactivate the mutator substance, 

 might nevertheless render it ineffective by altering 

 the cell surface of the bacteria so as to deny access 

 of the mutator substance to the interior of the cell. 

 Such a reagent may be chloroform, which Dr. Beale 

 has found ineffective as a mutagen despite its lethal 

 effect. 



Thus, the induction of mutations by chemicals 

 which do not themselves directly interact with the 

 gene would be a function of their lethality, the de- 

 gree of interaction between the chemical reagent 

 and the liberated mutator substance, and the degree 

 to which the permeability of the cell wall is altered. 

 In this manner we may account for differences in 

 "mutagenicity" of compounds with identical killing 

 rates. 



This hypothesis is susceptible of experimental 

 test in several ways. I shall suggest two such experi- 

 ments. One would be to test (a) the effect of lethal 

 reagents which do not induce mutations (particu- 

 larly methyl green) on the mutator substances of 

 Boivin and Avery before addition to the culture 

 substrates to determine whether inactivation does in 

 fact occur, and (b) the effect of adding the chemical 

 (particularly chloroform) to the appropriate cul- 

 ture before adding Boivin's or Avery's substance to 

 determine whether a reagent which does not itself 

 inactivate the mutator substance but nevertheless 

 fails to produce mutations, prevents the effect of 

 the mutator substance by its action in the otherwise 

 susceptible cells. Another would be to centrifuge 

 off the surviving bacteria after 99.9% had been 

 killed, wash them several times in water and then 

 resuspend them in a medium containing the chemi- 

 cal under investigation (let us say pyronin). Con- 

 trols would consist of bacteria left in the original 

 medium and bacteria resuspended in water. If the 

 bacteria resuspended in a fresh solution of the rea- 

 gent and those resuspended in water show a signifi- 

 cantly lower rate of continued mutation than those 

 permitted to remain in the original suspension (pro- 

 vided the killing rate remained near zero in all 

 samples), one could conclude that the mutagenic 

 factor was not the chemical reagent, but rather 

 some material which was present in the first super- 

 nate alone — this could only be derived from the 

 cultures themselves. If, on the other hand, muta- 

 tions continued to occur in all three test samples at 

 the same rate, the conclusion would again be that 

 the mutagenicity was probably not a direct func- 

 tion of the chemical (since one suspension is re- 

 suspended in water), but that the mutator sub- 

 stance was adsorbed to the washed bacteria or had a 

 significant lag time between its action on the bac- 

 teria and the emergence of the mutant. The third 

 result which might occur, namely that the sample 

 resuspended in a fresh solution of the chemical 

 showed a continued mutation rate equal to that of 

 the original suspension and greater than that of the 

 suspension in water, would disprove the hypothesis 



I have proposed above and demonstrate the capacity 

 of the chemicals themselves to produce mutations, 

 presumably by direct attack on the gene. 



I do not by any means, I repeat, wish to imply 

 that chemical mutations due to the direct action of 

 a chemical reagent on the gene do not occur. Quite 

 the contrary, there is good reason to believe that 

 such effects are possible. I wish only to interject a 

 word of caution in interpreting the induction of 

 mutations by a considerable number of reagents, 

 particularly when working with such material as 

 bacteria where the opportunity for interaction be- 

 tween large numbers of organisms exists. This pit- 

 fall may be avoided by suitable controls, as sug- 

 gested above, to distinguish between direct inter- 

 action with the gene and the indirect effect due to 

 autolysis. 



Witkin: It is, of course, possible that phage- 

 resistant mutants of strain B/r contain specific 

 mutator substances, similar to the transforming fac- 

 tors of Avery and Boivin. The addition of filtrates 

 of heat-killed cultures of B/r/1, however, does not 

 increase the number of resistant mutants in cultures 

 of B/r. Numerous other attempts to detect evidence 

 for transformation in this system have failed. This, 

 in addition to the fact that the number of induced 

 mutants obtained is independent of the background 

 number, over a range of one to fifty per 10 8 bac- 

 teria, seems to me to render Dr. Kurnick's hy- 

 pothesis unlikely. 



Bryson: In the event that we continue to find 

 numerous and unrelated chemicals that are able to 

 induce mutation, it may be of particular interpretive 

 value to study with care those substances that are 

 not mutagenic. It would also be desirable that more 

 simple chemical substances such as inorganic salts 

 be surveyed as mutagens, even as Drs. Greenstein, 

 Carter and Chalkley have surveyed them for effect 

 on enzymatic degradation of nucleic acid. One might 

 then bring to bear what is known of the effects of ions 

 on cells, and compare mutagenesis with what has 

 been established about permeability and relative 

 toxicity of various materials whose fate as reactants 

 in living systems has already been the subject of 

 extensive biochemical investigations. The problem of 

 toxicity itself presents a difficulty in the classification 

 of chemical mutagens since toxicity may act as a 

 limiting factor in exploring mutagenic potential. For 

 example, we have found that the induction of mu- 

 tations of phage resistance in E. colt by bis-beta- 

 chloroethylamine hydrochloride is most readily per- 

 formed on a strain of cells that has been through 

 twelve consecutive exposures to the inducing agent 

 with repeated selection of survivors for resistance to 

 the chemical. The number of zero point mutations 

 induced by nitrogen mustard in stock B 12 /M at a 

 survival level of 0.007% is 240 per 10 8 . It is always 

 possible that a chemical like methyl green that Dr. 

 Witkin has described as negatively mutagenic could 

 be made to induce mutations in a strain of cells 



130 



