10 15 20 



FRACTION NUMBER 



Fig. 10. 



25 



(Fig. 1, Gross, Malkin and Hubbard, ]. Mol. Biol. 13, 463. 

 1965, reproduced with permission of Academic Press.) 



that the deviations from constancy of specific 

 activity do represent the first condition, that is, 

 the presence of a small amount of RNA of high 

 specific activity, not coincident with the ribo- 

 somal species. From the sizes of the specific 

 activity variations, one can make a crude esti- 

 mate of the amount of heterogeneous radio- 

 activity. There is no good theoretical way for 

 making such an estimate, but it is possible 

 to make simple models composed of Gaussian 

 error curves to represent the bulk species and 

 extra counts distributed in roughly the way one 

 might expect heterogeneous RNA to be dis- 

 tributed. You see in Fig. 10 that the order of 

 maximum deviation of specific activity from 

 unity is two (circles). Figure 10 shows a real 

 gradient of specific activity. The reason that 

 we drew the optical density curves (smooth 

 curve, solid) continuously is that this is how 

 they emerge from the Gilford recorder. We do, 

 however, in each case, select individual frac- 

 tions, measure their optical densities again, 

 and then count them so that the specific activity 

 as plotted results from the division of an ac- 

 tually measured optical density by an actually 

 measured count. 



With the model shown in Fig. 11, which is 



4-- 



--2.0 



J ooo°°: 



GO O o 



°°po, 



--0.5 



10 15 20 25 



FRACTION 



Fig. U. 



the closest one that we've been able to construct 

 to the experimental results, there are 15% 

 extra counts (large circles) distributed hetero- 

 geneously among the total in these preparations. 

 (Specific activity, triangles). 



There's only one final objection to this, 

 and it is another kind of technical error. There 

 might be absorption, or simply quenching, that 

 results from the presence of RNA in these 

 samples, and the amount of quenching could 

 therefore be directly proportional to the amount 

 of RNA. This has been checked, and it is not 

 so. The specific activity deviations are there- 

 fore real and, on the basis of the model, they 

 result from the presence of some 10 to 15% 

 extra radioactivity in these preparations, sedi- 

 menting out of coincidence with the ribosomal 

 and transfer RNA's. The suggestion is, there- 

 fore, that this is the messenger RNA in the 

 unfertilized egg. 



UNKNOWN DISCUSSANT: Let me ask you 

 a technical question. What label were you using 

 in these studies? 



GROSS: The first one, without data points 

 (Fig. 9), was labeled with P^-^; the second one 

 (Fig. 10) was labeled with uridine. 



UNKNOWN DISCUSSANT: And did you use 

 DNA digestion to eliminate any possibility of 

 DNA labeling? 



GROSS: Yes, DNase digestions are done 

 routinely. There are a number of alternative 

 possibilities for checking the conclusion that 

 this represents messenger RNA. One is to 

 examine the hybridizability of the radioactive 

 RNA with DNA. We've done this, and it is by 

 no means an easy thing to do because the 

 specific activities of these preparations are 



10 



