WALDO E. COHN 



ionization and a gain of one secondary in its place, this has been 

 a favored explanation of the results. 



Additional evidence for this hypothesis was at first indicated 

 by the release of small amounts (less than 10%) of inorganic 

 phosphate from RNA samples by bone phosphatase (19,20). 

 Here again, as in the case of ribose branching just discussed, the 

 ever-present possibility of a nuclease contaminant in the bone 

 phosphatase leads to some reservations about accepting this 

 evidence. It has also been observed that alkaline hydrolysis of 

 RNA previously treated with bone phosphatase leads to the 

 formation of pyrimidine nucleosides in amounts equivalent to 

 the inorganic phosphate released. As in the case of ribose branch- 

 ing discussed, this is consistent with the mode of action of 

 ribonuclease, which causes fission of the phosphodiesters of 

 pyrimidine nucleosides, thus leading to their appearance as 

 unique products upon subsequent hydrolytic cleavage. There- 

 fore, the exclusion of nuclease action is necessary before the 

 enzymic evidence for either type of branching can be accepted. 

 Until then, the most that can be said is that if branching of either 

 type does occur, pyrimidine nucleotides are involved, not purine 

 nucleotides (18). 



In arriving at these conclusions, it should be understood that 

 none of the experiments which underlie them are of such 

 quantitative nature as to exclude small (i.e., up to 5%) amounts 

 of other types of material. The presumption of complete alkali 

 lability, the classic distinguishing characteristic of RNA, has 

 not been quantitatively verified and the persistence of some 

 polynucleotide material beyond a reasonable time limit may 

 indicate a small amount of an alkali-resistant linkage (60). 

 Difficulties in isolating, or even in testing for, all the sugar 

 components of a given preparation, and the necessity of referring 

 all analytical data to phosphorus rather than mass of material, 

 makes difficult the demonstration of a strict 1:1:1 ratio of bases 

 to ribose to phosphate or of the exclusion of all other materials 

 or of other sugars. However, important as these discrepancies 

 and unresolved questions may be, there can be little doubt at 



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