298 HEINZ SCHUSTER 



in this case. Infected tissue culture fluid of tumor cells was centrifuged until most of 

 the virus was sedimented. The infectious RNA obtained by phenol extraction of the 

 supernate was of much higher titer than was to be expected if RNA were extracted 

 solely from virus. On the other hand, treatment of infected cell homogenates with 

 ribonuclease removed UV absorbing material without affecting the infectivity which 

 could afterwards be extracted by phenol. This indicates that the potentially infective 

 material must normally be present intracellularly in a form not identical with the 

 virus but resistant to the action of ribonuclease. The origin and cellular location of 

 this material and the part it plays in virus synthesis are, at present, completely un- 

 clear. 



It has also been possible to prepare infectious RNA by phenol extraction of pig 

 kidney tissue cultures and from the muscle and heart of suckling mice which had been 

 infected with foot and mouth disease virus. 171, 172 Infectivity of the RNA was tested 

 in pig kidney cell monolayer cultures as well as by intramuscular or intracerebral 

 injection in mice. The various tests designed to differentiate infectious RNA from 

 infectious virus were also employed here. There was a further differential test in 

 that infectious RNA had little or no infectivity when tested by the intraperitoneal 

 route, whereas virus is highly infectious when inoculated by this route. 



The phenol extract of mice brains infected with Semliki forest virus, a virus which 

 can also multiply in mosquitoes, yielded an RNA fraction which was also studied in 

 comparison with intact virus by the known differentiating procedures. 173 Virus and 

 RNA also reacted differently after treatment with sodium deoxycholate. This rea- 

 gent, which has been shown to inactivate many of the arthropod-borne viruses, 174 

 also inactivates the Semliki forest virus but does not affect the infectivity of RNA. 



4. Size of Infectious Ribonucleic Acids 



The determination of the molecular weight of infectious RNA from 

 animal viruses presents certain technical difficulties. The main difficulty 

 lies in the preparation of sufficient virus RNA in pure form. This difficulty 

 is present in the extraction of RNA from purified virus preparations, as 

 well as from infected cells. In the latter case, the entire cell RNA will be 

 extracted and this will interfere with any characterization of the virus RNA. 

 On the other hand, it is relatively easy to separate the total RNA from 

 cellular DNA by employing certain salts during the phenol extraction which 

 prevent the release of DNA from cell structures into the aqueous phase, 175 

 or by making use of the different solubilities of the two types of nucleic 

 acid in 1 M NaCl. RNA, itself, can also be fractionated with 1 M NaCl. 

 Only the high molecular weight RNA can be precipitated in 1 M NaCl, 176 ' 177 



171 F. Brown, R. F. Sellers, and D. L. Stewart, Nature 182, 535 (1958). 



172 M. Mussgay and K. Strohmaier, Zentr. Bakteriol., Parasitenk. Infektionskrankh. 

 u. Hyg. 173, 163 (1958). 



173 P. Y. Cheng, Nature 181, 1800 (1958). 



174 M. Theiler, Proc. Soc. Exptl. Biol. Med. 96, 380 (1957). 



175 K. S. Kirby, Biochem. J. 66, 495 (1957). 



176 F. F. Davis and F. W. Allen, /. Biol. Chem., 227, 907 (1957). 



177 J. S. Colter and R. A. Brown, Science 124, 1077 (1956). 



