Virus Action and Replication 1 39 



70. Nirenberg, M., and Matthaei, J. H.: The dependence of cell-free 

 protein synthesis in E. coli upon naturally occurring or synthetic 

 polyriljonucleotides. Proc. Nat. Acad. Sci. USA, 47. ■ \ 588 -\ 602, 1961. 



DISCUSSION OF CHAPTER VI 



Saul Kit (Houston, Texas): I sincerely congratulate Dr. Darnell 

 on his very interesting and informative paper. He may have 

 already settled this point, but it is one that is extremely important 

 to us. It has been postulated that the messenger-RNA is destroyed 

 at the time the proteins are synthesized and that, as a result of this, 

 there is a need for continual synthesis of messenger-RNA. Dr. 

 Darnell's system would seem to be an excellent one for testing 

 this possibility, since after synthesis of the polio proteins in the 

 reconstructed ribosome system, one could re-extract the polio RNA 

 and test for infectivity. I wonder whether he has already done this. 



James E. Darnell, Jr. (Cambridge, Massachusetts): It turns 

 out not to be necessary to try to extract the polio RNA and to 

 look for any infectivity. Rather, what happens is this: The Niren- 

 berg system is made from E. coli, and E. coli ribosomes contain quite a 

 large amount of ribonuclease. It has been shown in Watson's 

 laboratory (J. D. Watson, personal communication) that natural 

 messenger-RNA from E. coli, which can be attached in vitro to the 

 ribosomes, is broken down completely during protein synthesis. 

 This is the case with polio also. However, we cannot say definitely 

 that this is linked to protein synthesis because the breakdown goes 

 on if the system is incubated in the absence of an ATP generating" 

 system. One possible way to get around this is to use ribosomes 

 from cells which do not contain ribonuclease, such as Bacillus 

 megaterium. An additional point of interest in this general area 

 is, of course, whether messenger-RNA from animal cells is handled 

 difTerently from bacterial messenger-RNx'\. 



Kit: Are the E. coli ribosomes not stabilized sufficiently by the 

 amount of magnesium used in your medium so that there is no 

 release of ribonuclease activity? 



Darnell: Not completely. Incubation of polio RNA in the 

 synthesizing system without an energy source, and, therefore, with 

 no resulting protein synthesis, still results in degradation of the 

 viral RNA. 



