GROSS: It's a maximum of 5% in the sea 

 urchin. 



POLLARD: It is also interesting that this 

 increase in sRNA seems to be in response to a 

 need, because breaking down the yolk protein 

 gives a large supply of amino acids, and this 

 needs transfer RNA in order to be used, also. 



GROSS: Yes, and that suggests, of course, 

 that putting the CCA on has something to do 

 with protein synthesis. 



POLLARD: Well, a much higher concen- 

 tration of tRNA will be necessary anyway, and 

 whether the CCA addition is an essential part 

 of that process or not I don't know. The con- 

 centration of transfer RNA has to be raised in 

 order to actually get the amino acids in the 

 proper location on the template. Actually, it's 

 almost certain in a cell which is fairly big like 

 this that the numbers have to be raised very 

 considerably unless there is compartmentation, 

 and protein synthesis occurs only a small 

 regions. Still, I don't think that answers your 

 question. You want to know why the sudden 

 burst in the CCA part of it occurs. 



GROSS: Yes, there is no question that there 

 is a rapid net synthesis of sRNA when the 

 protein synthesis rates rise for a second time 

 at gastrulation. There begins a rapid internal 

 synthesis of sRNA at that point and that is 

 quite reasonable. However, why the entire 

 CCA triplet should be knocked off and put 

 back on, I don't know. At least I know of no 

 evidence indicating that such an event is neces- 

 sary in order for the sRNA to transfer and 

 activate amino acid. 



DEERING: Does anyone know whether this 

 end group is actually present during the earlier 

 stages or is it just added later? 



GROSS: There is some uncertainty about 

 this, but some evidence that has recently become 

 available indicates that functional RNA is present 

 in the unfertilized egg, that is, sRNA with its 

 CCA triplet intact. 



PAPACONSTANTINOU: Did your base 

 ratio for that RNA give a GC content of 78%? 



GROSS: No, but that is just the base com- 

 position of the new RNA, determined from 

 hydrolysis of the bulk. It gives information 

 only about what bases are being incorporated. 

 It is possible that they are being added to 

 populations of sRNA molecules that don't have 

 any CCA on them at all. 



PAPACONSTANTINOU: Then how could 

 you test those for aP32 base ratio of 78%? 



GROSS: Think of a piece of RNA in the 

 presence of medium containing radio-phosphate. 



Then add to it pC, pC and pA, all of them 

 radioactive. These are added to base X on every 

 molecule. Now, stop the reaction, purify the 

 material, and hydrolyze it. One residue comes 

 off with no phosphate because the chain runs 

 in the wrong direction. The next comes off with 

 the phosphate and then X comes off with the 

 radioactive phosphate. Nothing has been labeled 

 at the next location. Radioactive XP can be any 

 one of the four bases. Let's assume for the 

 moment randomly so. That is what the base 

 composition implies. Measuring the radioac- 

 tivity of the phosphate, the composition of the 

 material I have labeled in the way shown here, 

 will be determined at 75% C and the rest 

 distributed among A, U and G. 



PAPACONSTANTINOU: I still can't see 

 how you can explain a 70% GC unless you have 

 an active CC turnover. That's the only way 

 you can explain it. You have evidence that 

 there is CCA present in the early embryo 

 because of the 78% GC. However, suppose you 

 started out with all the sRNA having no CCA 

 on it. Would you still get this pattern? 



GROSS: Yes. 



As to the RNA of the unfertilized egg, there 

 is strong but indirect evidence that the tem- 

 plates carrying the information for most or all 

 of the protein synthesis that occurs during the 

 period of cleavage are already present in the 

 unfertilized egg. If that's so, then one is dealing 

 with RNA templates that are storable under 

 conditions of non-use and are very stable when 

 they do begin to be used. In the presence of 

 actinomycin, in doses sufficient to shut off new 

 RNA synthesis, the primitive pattern of protein 

 synthesis persists for a long time. It is a matter 

 of some interest, therefore, to attempt to dem- 

 onstrate directly that such a maternal mes- 

 senger fraction exists, and second, to isolate it. 

 It would be useful to isolate it because whatever 

 approach works for the isolation would surely 

 tell us something about the state of this material 

 in the cell, and that, in turn, might tell us 

 something about the control of its translation. 



Nothing has been done as yet about isola- 

 ting this material in bulk. A number of steps 

 have been taken, however, to demonstrate its 

 existence more directly. One approach is to 

 make the RNA of an unfertilized egg radioactive 

 during oogenesis in order to show that among 

 the radioactive species there are some that are 

 not ribosomal or transfer RNA. This has been 

 done with most success in the amphibian (e.g., 

 9), and I'll leave it for Dr. Kohne to discuss. 

 It has been possible to do the same sort of 



