434 D. M. BROWN AND A. R. TODD 



cleotidic linkages in the presence of alkaline reagents and the difficulty of 

 assessing the completeness of methylation.^^sa 



In discussing structure XXIII, Brown and Todd*^- suggested that the rapid 

 production of pyrimidine mononucleotides during ribonuclease digestion 

 might be explained by postulating that branches were short and frequent — 

 perhaps consisting of only one nucleoside unit in some cases — and consisted 

 of pyrimidine nucleotide residues. Fission at the C2' — O — P bond at the 

 branch-point would yield pyrimidine nucleotides (via cyclic phosphates) 

 readily. Rather similar suggestions based, however, on branched structures 

 of undefined type, were also advanced by Carter and Cohn^^ and Magasanik 

 and Chargaff.27 ^pj^g more recent developments in our knowledge of ribo- 

 nuclease action make it clear that it is not necessary to postulate branches 

 as the source of the pyrimidine mononucleotides, but it has made it evi- 

 dent that in any branching of the type shown in XXIII the first residue in 

 the branch must be a pyrimidine nucleotide i^^ if it were not, branched oligo- 

 nucleotides would be formed. 



Cohn and Volkin^^^ have brought forward further evidence bearing on the 

 problem of branching from studies using snake venom diesterase, and have 

 discussed their results in terms of structure XXIII. Following the work of 

 Gulland and Jackson^^ with snake venoms which contain diesterases and 

 5 '-nucleotidases, they were able to confirm that, acting on ribonucleic acids, 

 large amounts of inorganic phosphate are liberated. In addition, however, 

 they showed that cytidine diphosphate and uridine diphosphate were pro- 

 duced simultaneously in an amount corresponding to about 30% of the 

 pyrimidine content of the nucleic acid. That the inorganic phosphate origi- 

 nated mainly in nucleoside-5 '-phosphates was shown by experiments using 

 venom which had been freed of 5 '-nucleotidase by the method of Hurst, 

 Little, and Butler. ^^ This purified preparation acting on ribonucleic acids 

 from calf-liver, thymus, and yeast, liberated very little inorganic phos- 

 phate but yielded large amounts of all four nucleoside-5 '-phosphates, the 

 pyrimidine nucleoside diphosphates, some nucleosides (mainly purine), 

 and about 10 % of pyrimidine h nucleotides. The diphosphates were shown 

 by enzymic degradation to be mixtures of cytidine-2' ,5'- and cytidine 3',5'- 

 diphosphate (XXI Va and b; R = cytosine residue) and the corresponding 

 uridine diphosphates (XXI Va and b; R = uracil residue), structurally 



123a More recent work (D. M. Brown, D. I. Magrath, and A. R. Todd, J. Chem. Soc. 

 1954, 1442) has shown that during the methylation of uridylic acid b, phosphoryl 

 migration occurs; the method is therefore unlikely to afford reliable evidence 

 when applied to polynucleotides since internucleotidic bond fission must on this 

 evidence be expected to accompany methylation. 



12^ E. Volkin and W. E. Cohn, /. Biol. Chem., 203, 319 (1953). 



