25G HEINZ SCHUSTER 



6. Preparation of Biologically Active Ribonucleic Acid 



Since it has not been possible to test the biological activity of RNA iso- 

 lated from plant or animal tissues, no definite criteria exist, as yet, as to 

 the relationship of the isolated nucleic acid to that found within the cell. 

 The recent discovery of a well-defined biological activity of at least one 

 type of cellular RNA, the so-called "soluble RNA" 50 and the possibility 

 of isolating this RNA, has led to one approach to the solution of this 

 problem. 



The situation is much simpler when one turns to virus RNA since the 

 biological activity of such RNA, the production of certain diseases with 

 well-defined symptoms, is well known. If the biological activity is due 

 solely to the RNA component, one must seek suitable methods to isolate 

 such RNA so that the biological activity remains intact. 



Earlier procedures for the isolation of RNA from nucleoproteins, such as 

 treatment with alkali, hot 10% NaCl, 51 guanidine-HCl 52 (see Chapter 11 

 for a detailed description) do not meet the requirements for isolating a 

 "natural" RNA molecule. Even special techniques formerly employed for 

 isolating TMV-RNA, such as heating the virus in NaCl at neutral pH 53, 54 

 or splitting the nucleoprotein with Sr(N0 3 )2 , 55 are not useful owing to the 

 sensitivity of RNA. The splitting of TMV with 67 % acetic acid has been 

 employed to obtain native virus protein, but the RNA obtained, thereby, 

 has a very low biological activity. 56 TMV has also been split with amino 

 alcohols, 57 but the applicability of this procedure is not known. 



Two methods will be described in some detail since they meet the require- 

 ments described above. The phenol method was developed by Zillig et a/. 58 

 for the splitting of TMV nucleoprotein. It has proved a most useful method 

 for the preparation of RNA, not only from viruses, but also from plant 

 and animal tissues. The second method, employing detergents, had been 

 used for splitting nucleoproteins for some time. Fraenkel-Conrat 4 employed 

 this technique to isolate an infectious RNA. The phenol method seems to 

 be superior to the detergent method with regard to ease of use and general 

 applicability. 



50 M. B. Hoagland, P. C. Zamecnik and M. L. Stephenson, Biochim. et Biophys. 

 Acta 24, 215 (1957). 



51 J. N. Davidson and C. Waymouth, Biochem. J. 38, 375 (1944). 

 62 E. Volkin and C. E. Carter, J. Am. Chem. Soc. 72, 1516 (1951). 



53 S. S. Cohen and W. M. Stanley, J. Biol. Chem. 144, 589 (1942). 



54 C. A. Knight, J. Biol. Chem. 197, 241 (1952). 

 66 N. W. Pirie, Biochem. J. 56, 83 (1954). 



56 H. Fraenkel-Conrat, Virology 4, 1 (1957). 



57 P. Newmark and R. W. Myers, Federation Proc. 16, 226 (1957). 



58 H. Schuster, G. Schramm, and W. Zillig, Z. Naturforsch. lib, 339 (1956). 



