278 



J. D. SMITH 



T 



F. 



T 

 Cy. 



T 



Ad. 



T 

 Gu. 



T T T 



E. Ur. D. 



T 

 C. 



T T 

 B.A. 



Direction of run 



Fig. 5. (a) Ultraviolet print at 260 mp and (b) autoradiograph of a short electro- 

 phoretic run of rat liver ribonucleotide fraction prepared by a modified Schmidt and 

 Thannhauser method. The substances showing in the autoradiograph contain P". 

 Cy, Ad, Gu, and Ur represent cytidylic, adenylic, guanylic, and uridylic acids, re- 

 spectively. (Reproduced from Davidson and Smellie.') 



compounds. [Cf. Smellie, Chapter 26.] P^^ \vas administered subcutaneously 

 to rats and after suitable intervals the animals sacrificed and their liver 

 tissue treated with liquid solvents and cold trichloracetic acid to remove 

 lipid and acid-soluble P. The tissue was then hydrolyzed in 0.3 A'' KOH 

 for 18 hr. at 37°, 60 % wt./wt. HCIO4 added to give pH 1, thus precipitating 

 the DNA and KCIO4 , the combined supernatant and washings adjusted to 

 pH 4 and run on paper electrophoresis at pH 3.5 in 0.02 M citrate buffer. 

 The nucleotides were located by their ultraviolet absorption and P^^.^q^. 

 taining compounds shown up on autoradiographs. The autoradiographs 

 (Figure 5) show the presence of six P^--containing compounds other than 

 adenylic, guanylic, cytidylic, and uridylic acids, of which one (A) absorbs 

 in the ultraviolet at 260 mfx. Component B is inorganic phosphate; the 

 other substances are unidentified organic phosporus compounds. As P^^ 

 (administered as inorganic phosphate) is initially incorporated into these 

 substances much more rapidly than into the ribonucleotides, contamination 

 of the latter by any of these substances leads to gross errors in the esti- 

 mation of P^2 incorporation into ribonucleic acid. By a long electrophoretic 

 run (18 hr., at 11 v. /cm.) cytidyhc, adenylic, and guanyhc acids may be 

 freed from other P^^.^ontaining substances and their true specific activ- 

 ities measured, but it is difficult to separate uridylic acid from components 

 D and E. These interfering substances can only be satisfactorily eliminated 

 by the prior isolation of PNA from the tissue. 



