258 HEINZ SCHUSTER 



volume of 5% sodium dodecyl sulfate (Duponol C), and is held at 50 ± 1°C. for 5 

 minutes. Then the solution is rapidly cooled to 20°C. and brought to 33% saturation 

 by the addition of saturated ammonium sulfate. After a few minutes, the protein 

 precipitate is separated by centrifugation and the supernatant is held in a refrigera- 

 tor overnight to permit the macromolecular nucleic acid to precipitate. The precipi- 

 tate is then centrifuged, redissolved in water and reprecipitated with two volumes of 

 cold ethanol with the addition of a few drops of 3 M acetate buffer at pH 5 to facilitate 

 flocculation. The alcohol precipitation is repeated and the final aqueous solution is 

 freed from aggregated material and any contaminating virus by ultracentrifugation. 



The infectivity of the most active preparation amounted to 0.1-0.3% of 

 the theoretical value, and is, therefore, of the same order of magnitude as 

 in the case of RNA prepared by the phenol method. 



(3) Infectivity Due to the Ribonucleic Acid Component. The proof that the 

 infectivity obtained after the two extraction methods described is actually 

 due to RNA and not to intact virus particles still present in the prepara- 

 tions, can be obtained in a series of tests. The effect of ribonuclease upon 

 the preparations is the simplest test, since ribonuclease destroys free RNA 

 in a very short time, whereas the activity of intact virus is not influenced. 

 The opposite effect can be obtained with virus antisera which rapidly neu- 

 tralize intact virus particles without influencing the infectivity of RNA. 

 The marked difference in the sensitivity of virus and virus RNA solutions 

 to heat treatment can also be applied as a test. TMV is relatively heat 

 stable, whereas RNA solutions loose their activity, at least after several 

 days. Finally, the marked difference in the sedimentation constants for 

 virus and virus RNA can be employed as a further control. All these tests, 

 together, have been used to show that the activity of TMV-RNA prepara- 

 tions obtained by either the phenol or the detergent method is due to the 

 nucleic acid alone. On the other hand, well-known analytical tests must be 

 employed to determine the degree of purity of the RNA preparations. 



(4) Protein Content of Infectious Ribonucleic Acid. It is most important 

 to know the protein content of preparations of infectious RNA in order to 

 exclude the possibility that the infectivity of the preparation is due to the 

 presence of intact or only partially deproteinized virus particles. 



(i) Protein content of phenol RNA. The protein content of RNA extracted 

 with phenol is less than 2% when measured with the Sakaguchi reaction, 

 and less than 0.4% when measured with a modified biuret reaction. 61 In 

 order to detect smaller quantities of protein, an RNA preparation was 

 hydrolyzed with 1 M alkali at 20°C. The nucleotides arising in this hy- 

 drolysis were removed by dialysis and the nondialyzable fraction was sub- 

 jected to acid hydrolysis and tested for the presence of amino acids by paper 

 chromatography. Aspartic acid, glutamic acid, glycine, and serine could be 

 demonstrated in an amount corresponding to approximately 40 jug. of a 



61 R. Markham and J. D. Smith, Biochem. J. 46, 509 (1950). 



