238 Discussion 



component of the gene, if indeed the gene has got a protein component. 

 In that case, you would have an abnormal protein which was in fact 

 a part of the gene ; you would then have a system which could be, up to a 

 point, capable of repeating itself, but gradually it would revert to nor- 

 mal as the stimulus which evoked the production of the abnormal protein 

 disappeared. I should like to know what is thought about this problem of 

 the relation between the protein and the DNA in the gene. 



Hotchkiss: As far as I know, DNA has never exerted its function in a 

 cell that did not contain protein! We don't know any of the steps in the 

 process of gene action, but one of them might well be a forming of a 

 complementary protein-like structure ; but we have not the evidence to 

 give an answer. 



Eagle: As a fairly recent convert to the thesis that most resistant 

 variants which we see in cultures arise as a result of mutation and selec- 

 tion rather than physiological adaptation, I confess that I am still 

 puzzled with respect to terminology. I am referring specifically to so- 

 called first-, second- and third-step mutations. If one takes a culture not 

 previously exposed to antibiotics, then, varying with the organism and 

 with the antibiotic, the number of survivors in an agar plate may 

 fall off steeply as the concentration of antibiotic is increased, or may 

 fall very slowly. In either case, if a surviving colony is subcultured, and 

 the distribution of resistance redetermined, then as Dr. Gyorffy has just 

 reported, some have essentially the same spectrum of resistance as the 

 parent population. Usually, however, the average resistance of sample 

 clones growing out at a given concentration of antibiotic tends to be 

 related to the concentration to which it had been exposed. A single clone 

 may therefore give rise to colonies which differ widely in their resistance 

 to antibiotic ; and the gradations are almost imperceptibly fine. Are all of 

 these organisms first-step mutants varying widely in resistance, as these 

 results and those of Dr. Hughes would imply; or are there first-, second-, 

 third-, fourth- and even fifth-step mutants within a single clone? 



Cavalli-Sforza: It refers to the sequence in which you have selected. 



Eagle: I think we have to be quite clear on this point. I had assumed 

 that a first-step mutant represented the first mutational step toward 

 increased resistance, and it has been so described. If we now redefine the 

 first-step mutant as that isolated on the first attempt at selection, such a 

 mutant could be a second- or third-step mutant in terms of what actually 

 transpired. 



Dr. Hughes has reported some observations which indicate that 

 extremely fine gradations of resistance may occur within a single clone. 

 Operationally, as Prof. Cavalli-Sforza would define them, these are all 

 first-step mutants; in fact, they could be second-, third, or fourth-step 

 mutants. The terms should be used with caution, and perhaps avoided. 



Cavalli-Sforza: In relation to the first-step variations observed by Dr. 

 Hughes, I don't know that they are mutants. 



Eagle: The differences we have observed are certainly stable. Whether 

 Dr. Hughes' extremely fine steps are similarly stable apparently remains 

 to be determined ; but at least for streptomycin, penicillin and chloram- 

 phenicol, these small differences are real and stable. Are they all to be 



