Fig. 17. 



appears actually to be on the fibres. Therefore, 

 one of the first proteins synthesized in the early 

 development of the sea urchin and presumably 

 one of those for which the program is stored 

 in the egg prior to fertilization, is a protein 

 that has some function in the organization or 

 operation of the mitotic apparatus. 



I believe that my time is up and we will 

 therefore have to defer a discussion of other 

 products of early protein synthesis to another 

 occasion. 



POLLARD: Thank you very much. Are 

 there any questions for Dr. Gross? 



CHALKLEY: Do the ribosomes from the 

 mature egg support protein synthesis under in 

 vitro conditions? 



GROSS: There has been some argument 

 about whether unfertilized ribosomes are com- 

 petent to support protein synthesis. An alterna- 

 tive explanation to the maternal messenger 

 story might be that there is a lesion in the 

 ribosomes of unfertilized eggs which is healed 

 on fertilization. That is indeed a part, at least, 

 of the point of view of Monroy and his collabor- 

 ators (12). Nemer (13), on the other hand, has 

 presented what was, I believe, reasonably good 

 evidence that ribosomes from unfertilized eggs 

 work well. In his experiments, they operate 

 with poly-U and with other synthetic poly- 

 nucleotides. Monroy explains that the ribosomes 



from fertilized eggs respond well to natural 

 messages, while the ribosomes from unfer- 

 tilized eggs do not. The point of their recent 

 paper is that unfertilized ribosomes which 

 respond very poorly to natural messengers 

 in vitro can be made to respond normally by 

 a brief treatment with trypsin. They are sug- 

 gesting that the unfertilized ribosomes are 

 blocked, perhaps with a protein, and that one 

 of the first events of early development is the 

 removal of that block, possibly by proteolysis. 

 It should be pointed out, however, that the same 

 group of investigators have shown that in this 

 material endogenous mRNA levels are about the 

 same in unfertilized eggs and blastulae. 



DEERING: Do you know what happens to the 

 RNA situation when you artificially activate an 

 egg? 



GROSS: If you do this successfully, you 

 turn on both protein and RNA synthesis in the 

 normal way, since one gets a normal haploid 

 embryo. 



MAURER: What about nuclease activity? 

 Could it be that the stability of your messenger 

 is due to a low level of ribonuclease? 



GROSS: It could, but it is certainly not so. 

 These eggs have extremely high levels of 

 nuclease, so that the problems of handling the 

 RNA are very complicated, indeed. 



MAURER: Can you inhibit by bentonite? 



GROSS: Yes. You can inhibit the nuclease 

 activities sufficiently to make what look like 

 respectable RNA preparations, but this does 

 require rather heroic efforts. There is only 

 one way I know of dealing with the high levels 

 of nuclease when such activity must be stopped 

 entirely. We learned of the trick when working 

 with polyribosomes. This is to add either large 

 amounts of enucleate HeLa cells, that is to say, 

 HeLa cell cytoplasm, or large amounts of yeast 

 RNA. In both cases, what one is doing is provid- 

 ing the endogenous nucleases with a large excess 

 of substrate in the hope that the substrate in 

 which one is interested will remain, to a large 

 extent, untouched. 



CHALKLEY: Wouldn't this raise a very 

 interesting point, then? First, you have a very 

 stable RNA in the cell and a lot of nuclease 

 present and, presumably, not able to attack and 

 disrupt it; later the problem arises that it can 

 attack it. One might think of compartmentation 

 playing a role. 



GROSS: Yes, I believe it would be a neces- 

 sary conclusion. If the nuclease is really there. 



14 



