Discussion 163 



strain reverts eventually, but Hinshelwood has often stated that the 

 reversion can be very unpredictable. 



Yudkin : What we said was that it makes no difference if you had 12 or 80 

 subcultures, for example. Indeed we have got a culture which we used 

 in our transformation experiments, which was trained to a high level of 

 resistance in that way, and it still is resistant 5 years later. 



Dean: Of course there may be quite a variation in technique. 



Hughes: In your culture, which is not multiplying in the presence of 

 proflavine, how satisfied are you that there is not a turnover of cells ? 



Yudkin: When we did these experiments, we were rather impressed 

 by the work of Szybalski and Akiba. We took even more precautions 

 than they did, but until we go back now and look at the same sort of 

 things that Szybalski looked at, we cannot be absolutely certain. 



Fredericq: You get transformation with DNA extracted from a resis- 

 tant mutant. Did you try to transfer the resistance conferred by pro- 

 longed induction. 



Yudkin: No, that is one of the things we shall have to do. 



Fulton: How difficult is it to get DNA out of bacteria? Can you hope 

 for purity of the specimen without prolonged chemical operations ? 



Yudkin: We did not attempt to make entirely pure preparations. We 

 made preparations which had DNA activity. It involved lysing, extrac- 

 tion, precipitation of protein, then taking up in saline. We were content 

 at this stage, to show (a) that, prepared in this way, the extracts are 

 active if they come from resistant organisms — whether from a mutant 

 isolated by replica plating, or from a trained culture — and not w^hen 

 prepared in exactly the same way from a sensitive culture ; and (b) that 

 the activity is lost on treatment with DNAse. 



Pollock: You said that there was a cycle of 20 minutes in changes in 

 proflavine resistance, 30 minutes in the actual division time; yet later 

 on you seemed to imply that there was a relationship between the two. 

 Could you clarify that? 



Yudkin: I don't know the answer. This is an odd situation. It was 

 Dr. Hotchkiss who started this. He got changes in transformability 

 which were longer than the division cycles. We get changes in resistance 

 levels which are shorter. There are elaborate ways in which you can 

 possibly explain this by imagining that the cooling process interrupts at 

 more than one level and so on. But I feel there is no explanation at the 

 moment. I still feel that it is likely that these changes in cycles of 

 resistance are in some way tied up with cell division. 



Hotchkiss: We have the same problem. The two factors we tried to 

 correlate were the cyclical transformability, i.e. the percentage of cells 

 which respond to DNA; and the number of cells undergoing division, 

 exemplified by colony formation. The lack of correspondence of the 

 cycles might be because cell division is not the same as nuclear division, 

 so that the time at which nuclear processes are going on may well precede 

 the time at which cells become separate enough to form single colonies. 

 Furthermore, when we are measuring cell division, we are measuring a 

 property of all of the cells; what 1000 cells do in 5 minutes, and in the 

 succeeding 5 minutes, and so on. We found steps of division such as 



