34. THE RIBONUCLEIC ACIDS OF VIRUSES 277 



can be reduced or prevented by the presence of uracil in the culture, but 

 not by cytosine or thymine. Thiouracil is incorporated into the RNA of 

 TMV as was shown, using S ;fS -labeled thiouracil. 110 ' 11L> ' ,1:i Alkaline hy- 

 drolysis or hydrolysis with ribonuclease are best suited for demonstrating 

 the incorporation of thiouracil into RNA. After alkaline hydrolysis of 

 RNA, the mononucleotides were separated by chromatography and it was 

 found that all the radioactivity moved with the Rj expected for pyrimidine 

 mononucleotides. Separation by paper electrophoresis shows that all the 

 activity had about the mobility to be expected for thiouridylic acid. The 

 clearest results were obtained by degrading the RNA with ribonuclease 

 followed by a chromatographic and elect rophoretic separation of the prod- 

 ucts of hydrolysis. Then the radioactivity was found in several components: 

 thiouridine, thiouridylic acid, thiouridine-3', 5'-diphosphate, and thiouracil- 

 containing di- and polynucleotides. Thus, thiouracil seems to occupy cer- 

 tain positions in the nucleic acid which, presumably, are normally occupied 

 by uracil. 



The incorporation of thiouracil seems to occur chiefly at the end of the 

 polynucleotide chain since some 25% of the total incorporated S 35 is lo- 

 cated in thiouridine-3', 5'-diphosphate. How r ever, the presence of such a 

 large quantity of thiouridine-3 ',5 '-diphosphate excludes the possibility 

 that this is found only in a terminal position in a 2 X 10 6 molecular weight 

 RXA molecule. Approximately 3.5% of the uracil in RNA is replaced by 

 thiouracil, which means that there are about 50 thiouracil molecules per 

 RNA. Therefore, about 12 thiouridine-3', 5'-diphosphate molecules are 

 found per RNA molecule. If this should come only from a terminal position, 

 the polynucleotide can be only 500 nucleotides long, contrary to the con- 

 ception that the chain is some 6000 nucleotides long. 



Virus containing thiouracil in its RNA has approximately the same 

 infectivity per unit weight of virus as normal TMV. 114 



5-F I uoro uracil. Fluorouracil also inhibits the multiplication of TMV in 

 excised discs from tobacco leaves. Thereby, the yield of TMV is reduced to 

 some 50% of a control yield. 115 Fluorouracil can replace about y% of the 

 uracil of TMV-RNA according to base analyses on RNA after hydrolysis 

 with perchloric acid. If RNA is hydrolyzed with snake venom diesterase or 

 by alkaline digestion, it is possible to separate the 5'- or 2'- and 3 '-mono- 

 phosphates of fluorouridine from the corresponding uridine monophosphates 

 by paper electrophoresis. Nucleoside diphosphates could not be demon- 

 strated. 



112 R. E. F. Matthews, Biochim. et Biophys. Acta 19, 559 (1956). 



1,3 H. G. Mandel, R. Markham, and R. E. F. Matthews, Biochim. et Biophys. Acta 



24, 205 (1957). 

 114 R. Jeener, Biochim. et Biophys. Acta 23, 351 (1957). 

 116 M. P. Gordon and M. Staehelin, J. Am. Chem. Soc. 80, 2340 (1958). 



