then either the RNA or the nuclease is seques- 

 tered. 



CHALKLEY: Then the point I'm aiming at 

 is that the RNA is not in some mysterious way 

 stabilized. 



GROSS: It's not easy to distinguish at this 

 point between the two proposals. 



HYMER: I would like to comment on this 

 point. Dr. E. L. Kuff and I demonstrated the 

 presence of an endonuclease within nuclei iso- 

 lated from murine plasma cell tumors. This 

 enzyme preferentially attacked rapidly labeled 

 high molecular weight RNA, and its activity 

 could be completely inhibited by the addition of 

 cytoplasmic soluble fraction. 



GROSS: Well, in any case, the whole prob- 

 lem of stability and instability in messages is 

 both interesting and difficult, and it is by no 

 means restricted to embryos. On the basis of 

 a large body of accumulating evidence, one can 

 now safely conclude that stable and unstable 

 messages coexist in the cells of higher orga- 

 nisms. 



UNKNOWN DISCUSSANT: You mention that 

 you are able to hybridize the nucleic acid from 

 the unfertilized egg. What percentage of hybrid- 



ization were you getting and what technique were 

 you using? 



GROSS: Our technique was a modification 

 of the Nygaard-Hall method, essentially the one 

 described by McConkey and Hopkins in the 

 Proceedings of the National Academy of Science 

 about a year ago (14). The method gives low 

 values of hybridization. In fact, McConkey and 

 Hopkins got a value for the size of the ribosomal 

 fraction that is obviously much too low. Their 

 method has the one virtue that it reduces so- 

 called mistaken identity hybrids to the lowest 

 values that I know without the use of ribo- 

 nuclease. We use this method, therefore, be- 

 cause our low specific activities and large 

 amounts of ribosomal RNA demanded it. With 

 it, we get something like 1-1/2% hybridization. 

 That is 1-1/2% of the total counts in a prepara- 

 tion of the type for which we saw gradients 

 earlier, hybridized under the conditions of 

 saturation routinely employed. By using a 5 to 

 15-fold excess of unlabeled RNA, we can reduce 

 the counts by only a very small amount - 8 or 

 10% of the original number. From that reduction, 

 we got the estimate of the fraction of the genome 

 occupied by the ribosomal cistrons. 



15 



