34. THE RIBONUCLEIC ACIDS OF VIRUSES 273 



under conditions (6 hours, 20°C, pH 5) where all singly esterified phos- 

 phoric acid is quantitatively degraded from a commercial preparation of 

 yeast RNA. 76 Phosphate, which must be set free by phosphomonoesterase 

 action, could not be detected in the case of TMV-RNA. If the TMV-RNA 

 molecule with a molecular weight of 2 X 10 6 is composed of 6000 covalent 

 bonded nucleotides, then the expected amount of phosphates (1 mole 

 phosphate per 6000 moles nucleotides for RNA of either type (I) or type 

 (II), see Fig. 4) must lie well below the limits of sensitivity for phosphate 

 determination. 



TMV-RNA was also treated with polynucleotide phosphorylase from Azotobacter 

 vinelandii 9 * and in this case a slow rate of inactivation was found. 76 This enzyme 

 catalyzes the reversible reaction: 



n X-R-PP ^ (X-R-P)„ + nP (4) 



where R stands for ribose, PP for pyrophosphate, P for orthophosphate and X for 

 one or more of the following bases: adenine, guanine, uracil, or cytosine. It is, there- 

 fore, capable of degrading polynucleotides by phosphorolysis. The products of phos- 

 phorolysis, nucleoside-5'-diphosphates of all four bases, were chromatographically 

 determined after a 6-hour incubation of TMV-RNA with the enzyme. 96 While syn- 

 thetic polynucleotides containing only one kind of nucleotide, such as polyadenylic 

 or polyuridylic acid, are rapidly phosphorolyzed, yeast and bacterial RNA, as well 

 as synthetic polymers containing more than one kind of nucleotide, or the polymer 

 aggregate formed by mixing solutions of polyadenylic and polyuridylic acid are 

 phosphorolyzed slowly. On the contrary, TMV-RNA is phosphorolyzed at an inter- 

 mediate rate. Ochoa concluded that the slow phosphorolysis of polynucleotides can 

 be related to the fact that they consist largely of multi-stranded rather than single- 

 stranded chains. 



By using a homogeneous TMV-RNA, primer free phosphorylase, and employing 

 radioactive phosphorus, it might be possible to determine the terminal nucleotide in 

 the TMV-RNA and, also, to determine how many nucleotides must be split from the 

 terminal position before the biological activity is destroyed. 



(3) Action of Chemical Agents. Reaction of infectious RNA with aldehydes. 

 Formaldehyde. Treatment of TMV with formaldehyde leads to inactivation 

 of the virus. 96 Since this type of inactivation does not bring about a sig- 

 nificant loss or alteration of the serological properties of the virus, it is 

 generally used for the preparation of vaccines, for example, with polio- 

 virus. Since RNA is the infectious component of TMV, the inactivation by 

 formaldehyde must be due to its reaction with RNA, though the aldehyde 

 also reacts with viral protein. Formaldehyde inactivation of TMV obeys 

 first-order kinetics, at least in the early part of the reaction. The reaction 



94 M. Grunberg-Manago, P. J. Ortiz, and S. Ochoa, Biochim. et Biophys. Acta 20, 269 

 (1956). 



95 S. Ochoa, Arch. Biochem. Biophys. 69, 119 (1957). 



96 W. M. Stanley, Science 83, 626 (1936). 



