434 H. FRAENKEL-CONRAT 



methods for the degradation of particles toward the end of isolatuig either or 

 both components in pure and preferably native form. The last objective, that 

 of separating both native protein and nucleic acid in good yield from the 

 same batch of degraded virus, has actually not yet been realized. 



A method for the separation of native (i.e., active) RNA which has proven 

 very useful is by means of phenol. This method originally proposed by Morgan 

 and Partridge (1941) w^as adopted by Gierer and Schramm (1956) for the 

 purpose of the splitting of TMV and isolation of its nucleic acid. It has since 

 proven applicable also to other viruses. The method resembles the older 

 Sevag (Sevag et al., 1938) procedure in using an interface separation of insol- 

 uble, denatured protein from w^ater-soluble sodium nucleate, but it substi- 

 tutes for chloroform the more water-soluble and more efficient denaturant, 

 phenol. After several cycles of shaking and centrifugation of the pH 7 phos- 

 phate buffer-phenol mixture at 0°C., the aqueous phase is freed from dis- 

 solved phenol by ether extraction, and from ether by a stream of Ng. 



The alternate procedure developed in our laboratory makes use of the de- 

 tergent, sodium dodecyl sulfate (SDS) (1 %) for the splitting of the virus 

 (Sreenivasnya and Pirie, 1938) and of ammonium sulfate for the separation 

 of nucleic acid and denatured protein (Fraenkel-Conrat and Singer, 1954, 

 1957). The most consistent results have been obtained when 0.002 M EDTA 

 (ethylenediamme tetraacetic acid or Versene) was used as a buffer at an 

 initial pH of 7.8, and the solution was heated to 50°C. until the viral opales- 

 cence had disappeared (1-10 mmutes for different strams of TMV). Splittmg 

 occurs also in a few minutes at pH 3.5 and room temperature, and yields 

 comparable nucleic acid preparations (Fraenkel-Conrat, 1957a). For the iso- 

 lation of the nucleic acid, the protein is first precipitated by the addition of 

 one-half volume of saturated ammonium sulfate. The nucleic acid precipi- 

 tates from the supernatant upon cooling, and is reprecipitated twice from 

 water by the addition of 2 volumes of ethanol, containing 1-2 drops of 3 M 

 acetate sodium. The preparation is finally subjected to ultracentrifugation, 

 in 2-ml. tubes, discarding the more viscous bottom drop (less than 10 %). 



A third procedure which has yielded active nucleic acid is based on the 

 original preparative method of Cohen and Stanley (1942). It consists in 

 heat-denaturmg and precipitatmg the protein in 0.1 M sodium chloride. No 

 detailed description of this method as adapted for the purpose of isolating 

 active nucleic acid has as yet appeared. It seems that the first two methods 

 give preparations which are similarly infective and that the last, and other 

 methods which have not been surveyed, give preparations which are at 

 least one order of magnitude less uifectious. 



In the particular case of the TYMV, treatment with 30 % ethanol in 

 presence of salts at room temperature was fomid sufficient to degrade the 

 virus and yield pure nucleic acid (Markham el at., 1948; Knight, 1957a). 



