278 HEINZ SCHUSTER 



Infectivity was tested again by local lesion count and the infectivity per 

 milligram of virus was not different for virus containing fluorouracil and 

 normal virus. 



e. Reaction with Nitrous Acid and Production of Mutants 



(1) The "Sensitive" Size of the Ribonucleic Acid Molecule. The oxidative 

 deamination of NH 2 groups with HNO2 is well known in the field of protein 

 chemistry (Van Slyke). The reaction may also be carried out on the amino 

 groups of purines and pyrimidines regardless of the nature of the com- 

 pounds (free bases, nucleosides, nucleotides, or polynucleotides). Only the 

 reaction rate differs for these different compounds. Under identical condi- 

 tions, nitrous acid reacts more rapidly with amino acids than with adenine, 

 guanine, or cytosine; 116 the slowest reaction is obtained with nucleotides in 

 a polynucleotide chain. 117 ' 118 



TMV-RNA is inactivated by HNO2 according to first-order kinetics in 

 the presence of an excess of HNO2 at 20°C, in the pH range 4-4. 5. 102 In 

 the absence of HNO2 , the RNA is stable over a long period of time in this 

 pH range. The inactivation is not accompanied by a degradation of the 

 polynucleotide chain. The only observable alteration in the bases is de- 

 amination, and it is presumably this reaction that leads to the inactivation. 

 The reaction products of adenine, guanine, and cytosine with HNO2 are 

 hypoxanthine, xanthine, and uracil, respectively. Thus, deamination of the 

 purine bases leads to the presence of "unnatural" bases in RNA and de- 

 amination of cytosine produces an alteration of the cytosine/uracil ratio. 



The HNO2 inactivation of RNA occurs in the order of minutes (Fig. 8). 

 The per cent of deaminated nucleotides arising in such short time periods 

 is much too low to allow a direct measurement of them. It is only possible 

 to detect the reaction products after the RNA has been exposed to HNO2 

 for hours. This is accomplished by chromatography of a hydrolyzate of 

 RNA. The increase in hypoxanthine, xanthine, and uracil is linear in time 

 under conditions of constant pH and constant nitrite concentration. 



If an average of 1 nucleotide out of N nucleotides must be deaminated 

 before inactivation occurs, then the decrease is : 



It = Le~ Nat (5) 



where It = infectivity at time /, Io = infectivity at time t = 0, and a = 

 moles of nucleotides transformed per minute. N may be determined using 

 the experimentally found decrease in infectivity along with the measured 



116 D. D. Van Slyke, J. Biol. Chem. 9, 185 (1911). 



117 H. Bredereck, M. Kothnig, and G. Lehmann, Ber. deut. Chem. Ges. 71, 2613 (1938). 



118 W. E. Fletcher, J. M. Gulland, D. O. Jordan, and H. E. Dibben, J. Chem. Soc. p. 30, 

 (1944). 



