and allowed time to go through the entire dif- 

 ferentiation process, in fact, well beyond when 

 it had ended in the untreated controls. We then 

 fixed all the samples and examined them to 

 determine the percentage of cells reaching a 

 recognizable stage. The curves on the right show 

 the normal pattern of papilla formation in the 

 control cultures and the release of the spores 

 at the end of the experiments. 



There was no inhibition of papilla forma- 

 tion by actinomycin at 17^ hr. That is to say, 

 there is essentially no effect on the papillae 

 if added just before the papillae actually ap- 

 pear, but if the actinomycin was added about 

 a half-hour earlier, 16-16/^ hr, then no papillae 

 formed and it gave 100% inhibition. The same 

 thing was true at a later point for spore for- 

 mation, the obvious "cleavage" of the protoplast 

 to form the individual spores. At 11% hr it was 

 100% effective; by 19 hr it was completely in- 

 effective. Essentially the same curve was ob- 

 tained when we used PFP. If you accept the 

 fact that these are well-synchronized cultures 

 and notice that the control patterns are super- 

 imposable, then the experimental curves for 

 actinomycin D and PFP are virtually super- 

 imposable also; puromycin is only indicated 

 here for spore formation because of the 

 100 /ug/ml concentration used it did not inhibit 

 papilla formation. It did result in abnormal- 

 looking papillae. They were long, multiple, and 

 somewhat twisted in contrast to the short, 

 single, and symmetrical papilla at the tip of the 

 normal plant grown under our conditions. We 

 do not know the reason for this effect. PFP, on 

 the other hand, completely mimics the behavior 

 of actinomycin. 



GROSS: Do you know that the puromycin is 

 doing what you want it to do? 



LOVETT: No, we don't. This is really the 

 only positive result that we have with puromycin, 

 and I am not going to say any more about it. 



CHALKLEY: In our work with tobacco cells, 

 it shows a similar lack of protein inhibition. 

 If the puromycin does get into the cells, then 

 one may assume that there is some degradation 

 of part of the molecule. 



LOVETT: I'm glad to hear that, because 

 we haven't yet tried other concentrations. We 

 were not sure that we were using an adequate 

 concentration and thought that it might not be 

 getting in fast enough. 



CHALKLEY: I think the problem is not 

 unsolvable. 



GROSS: I think the pea work is really much 

 more dramatic. 



LOVETT: It is interesting and also most un- 

 expected. 



B. WRIGHT: Is the time of effect of the 

 actinomycin accompanied by permeability 

 changes? 



LOVETT: It is so effective in inhibiting 

 uracil incorporation that I think this means 

 that it is getting into the cells. 



B. WRIGHT: Yes, but I mean the time at 

 which it first starts to inhibit. 



LOVETT: No, at least I choose to interpret 

 it as meaning something about when it is acting. 

 This is indirect evidence, to be sure. Before we 

 see obvious morphological changes it stops 

 everything. When it stops papilla formation, it 

 has obviously stopped growth. However, the point 

 is, if you add it later it no longer has any effect, 

 even on papilla formation. It has no effect on 

 papilla formation but will stop later develop- 

 ment. 



Returning to the fact that PFP acts like 

 actinomycin on development, you will note that, 

 as near as we can tell, it shuts down leucine 

 incorporation almost immediately. It is an 

 effective inhibitor. This, plus the fact that it 

 mimics the actinomycin effects almost iden- 

 tically in the morphological progression, leads 

 us to suggest that it may be acting in some 

 way other than simple incorporation, as has 

 been shown in E. coli (8), to form "nonsense" 

 protein. I have heard rumors of a similar 

 situation in another system. I think that in- 

 hibition by producing nonsense proteins would 

 take some time unless there was a critical 

 protein, and nothing could proceed unless it 

 was functional. This could be true, but I am 

 interpreting it somewhat differently. 



GROSS: Well, does it shut down RNA? 



LOVETT: We haven't done this yet, but I 

 think it shuts down protein synthesis. 



GROSS: It shuts down protein synthesis as 

 measured by leucine incorporation? 



LOVETT: We have only measured leucine 

 incorporation so far, but on the basis of the 

 fact that it is so effective on total development 

 1 think that it shuts down all protein synthesis. 



The inhibition results suggest that the 

 actinomycin effect is on RNA made half an hour 

 before the papillae are formed, and that the 

 necessary protein is probably also made at 

 nearly the same time. This could explain why 

 PFP mimics the effect of actinomycin. Though 

 we have much to do before we can really prove 

 it unequivocally, it is nicely consistent both in 

 this case and in the case of the spore formation 

 later on. To me, it suggests that we do not 



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