may well be proteins other than histones in- 

 volved, but I was basing it on the results in 

 which we've come to the conclusion that the hor- 

 mone hasn't got as one of its functions to bind 

 histone, in any sort of binding we could detect. 

 So, one of the original reasons we developed 

 this idea was because we had to explain an 

 increase in template activity. So, we had to 

 postulate some sort of a dynamic equilibrium 

 and determine if we could interfere, not by 

 reaction with histone, but by reaction with 

 another protein. The suggestion here is that 

 the protein interacts with a hormone. 



The point I really wanted to make and really 

 wanted to stress is that we would like to approach 

 the problem in terms of a dynamic situation and 

 then see if we can disturb the equilibrium in 

 some way at some point; and this may give rise 

 to derepression. If you remove the inhibitor, 

 everything flows back and you set up the original 

 equilibrium. 



EPEL : I'd like to try to integrate your think- 

 ing with Dr. Wright's stimulating paper this 

 morning. In your evidence regarding the hor- 

 monal action you claim that 95% of the hormone 

 is in the cytoplasm - is that right? 



CHALKLEY: We've found large percent- 

 ages in the cytoplasm but not large percentages 

 bound in the cytoplasm. 



EPEL: Well, here is what I want to bring 

 out. In our discussion here, we know that these 

 hormones, such as estradiol or leutenizing hor- 

 mones, can fantastically affect intermediary 

 metabolism. These are very fast effects. Let's 

 take ACTH in the adrenal cortex; it will acti- 

 vate cyclic AMP formation and thence glycogen 

 phosphorylase. There are also a number of 

 papers showing direct cyclic AMP stimulation 

 of enzymes involved in steroid synthesis and 

 there is, also, activation of protein synthesis 

 via pre-existing messenger RNA. So, isn't it 

 conceivable that you have a system here in 

 which numerous changes are triggered as a 

 natural consequence of the hormonal action, 

 not through the hormone directly but through 

 the changing levels of various intermediates? 

 This, then could act on the nucleus? 



CHALKLEY: Yes, I would say that is en- 

 tirely possible. 



EPEL: I'm just trying to integrate these 

 two very interesting lines of reasoning. 



CHALKLEY: Well, some of these hor- 

 mones, of course, repress and activate pre- 

 existing enzymes. This is well catalogued; and 

 we know that they bind slightly into the cyto- 

 plasmic fraction. But on the other hand there is 



a body of evidence available which suggests that 

 an early biochemical effect of hormone adminis- 

 tration is increased RNA and protein synthesis. 

 It is entirely possible that the nuclear binding and 

 the increased RNA synthesis are related. 



EPEL: The hormone can be many steps re- 

 moved from its final action. It' s at least 3 or 4 

 steps back, for instance, in initiating cyclic 

 AMP formation. 



MAURER: Yes, but why do we always find 

 a lot of hormone in the nuclei? In the case of 

 ecdysone, for example, we find hormone about 

 20 minutes after injection into the larvae of 

 Calliphora erythrocephala in the nuclei; and the 

 earliest time it's been found is about 10 minutes 

 after injection. However, you are quite right; 

 it's still not ruled out that there isn't some kind 

 of reaction with the cytoplasm. 



CHALKLEY: There is one thing I possibly 

 didn't bring out fully enough. If we do this hor- 

 mone incorporation experiment, a great deal of 

 the hormone goes into the cytoplasm, but if you 

 look at the amount bound there is a much greater 

 degree of binding occurring in the nucleus. 



EPEL: Are these experiments in the range 

 of physiological concentration? Perhaps the 

 experiment to do would be to use an extremely 

 small amount of hormone, and then see if it 

 appears in the nucleus. 



MAURER: Actually this has been done with 

 physiological 10-^ M concentration. So, it' s con- 

 ceivable although not completely proved. 



GROSS: What were you saying about ecdy- 

 sone? When we studied this system, although it 

 is binding to the ribosomal particles, the amount 

 of binding in the nucleus is not substantial. 



CHALKLEY: I was recalling the results 

 with binding in the mitochondrial and ribosomal 

 fractions, because it has been shown that binding 

 occurs in these particular fractions. 



GROSS: Where does the hormone exert its 

 first recognizable characteristic effect? In this 

 case isn't the first characteristic response in 

 the genome? 



MAURER: I treated the insect larvae with 

 ecdysone and I found the highest activity in the 

 nuclei and not in the mitochondria. I found some 

 activity in the mitochondria and even in the 

 microsomes, but the highest activity after one 

 hours was in the nuclei. This was then followed 

 by a decreasing level; so it looks suspiciously 

 as though the hormone first migrates into the 

 nuclei and then by some process is depleted. 



SCHRAER: You keep using the word hor- 

 mone. Do you mean labeled hormone? 



CHALKLEY: Yes. 



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