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



tablishes 6(3') as the linkage point for pyrimidine nucleoside residues, 

 studies with this enzyme can give no information about the corresponding 

 purine nucleoside residues. Evidence on this point comes from studies 

 using other nuclease preparations with different specificities, several of 

 which appear to exist. ^'"' Volkin and Cohn"** showed that spleen nuclease 

 prepared according to Maver and Greco"^ yields the h isomers of both 

 pyrimidine and purine mononucleotides when it acts upon ribonucleic acid. 

 Further purification of this enzyme preparation by Heppel and Hilmoe"' 

 gave a product which degrades ribonucleic acids and oligonucleotides, 

 giving high yields of mononucleotides which, in the case of adenylic and 

 guanylic acids, were shown to be the 6 (3') -isomers -j'^" since no evidence for 

 intermediate cyclic phosphates was observed, it was concluded that the 

 purine nucleotide residues were linked at the 6 (3') -position in the intact nu- 

 cleic acid. More definite evidence has been provided by Brown, Heppel, 

 and Hilmoe,*-^ who have shown that the same enzyme preparation, as well 

 as others from intestine, potato, and rye-grass, hydrolyze cytidine benzyl 

 phosphate b and adenosine benzyl phosphate b to the corresponding b 

 nucleotides while they have no action on esters of the a isomers. 



Regardless of the mechanism of spleen nuclease action it is clear that 

 these results, together with the above studies of ribonuclease hydrolysis 

 of nucleotide esters, establish with a high degree of certainty that the inter- 

 nucleotidic linkage in ribonucleic acids involves the 6(3') -position and not 

 the a(2')-position in both purine and pyrimidine nucleoside residues. As- 

 suming the validity of the constitutions proposed for the mononucleotides, 

 i.e., that the b isomers are the 3 '-phosphates, the ribonucleic acids must, on 

 the evidence presented, be considered to be polynucleotides in which the 

 individual nucleoside residues are joined one to the other by phosphodi- 

 ester linkages between the 3'- and 5 '-positions as indicated in structure 

 XVI. 



Although the oligonucleotides produced by ribonuclease action appear to 

 be unbranched, this fact cannot of itself be accepted as evidence that intact 

 ribonucleic acids are linear polynucleotides. To complete our discussion it 

 is necessary to consider the question of chain-branching. 



8. Chain-Branching in Ribonucleic Acids 



There has been much discussion in the past about branched-chain as 

 distinct from linear structures for the ribonucleic acids. This involved, in 



"8 M. E. Maver and A. E. Greco, J. Biol. Chem. 181, 861 (1949). 



"9 L. A. Heppel and R. J. Hilmoe, Federation Proc. 12, 217 (1953). 



'2" L. A. Heppel, R. Markham, and R. J. Hilmoe, Nature 171, 1152 (1953). 



'2' D. M. Brown, L. A. Heppel, and R. J. Hilmoe, J. Chem. Soc, 1954, 40. 



