RIBONUCLEIC ACIDS 



snake venom. These products are largely 5' nucleotides, but 

 equimolar amounts of purine nucleosides and pyrimidine 

 nucleoside 3 ',5 '-and -2 ',5 '-diphosphates (18-20) were also iso- 

 lated. Although equal amounts of such substances would be 

 expected from the two ends of a simple 3 ',5' (or 2 ',5') RNA 

 chain, the large quantities of these would require chain lengths 

 of only seven to ten nucleotides. However, any branches 

 originating in triply esterified ribose and proceeding via the 

 3 ',5' (or 2 ',5') linkage could yield these same products. The 

 difficulty in excluding the possibility of nuclease contamination 

 in the diesterase preparations, which are essentially whole venom 

 minus the 5' monoesterase activity, has undermined the validity 

 of this argument, as any nuclease present could reduce a long, 

 unbranched chain to short chains which could in turn give rise 

 to the products observed. Plausibility is attached to this latter 

 speculation because the cytidine diphosphate as isolated was 

 shown to be a mixture of 2 ',5' and 3 ',5' isomers, which may have 

 been formed from cytidine 2' :3 ',5 '-diphosphate, the latter 

 substance arising in turn from nuclease action on a longer chain. 

 Until this source of the diphosphates (and purine nucleosides) 

 can be eliminated from consideration, the diesterase evidence 

 for branching through ribose must be considered equivocal. 



Branching originating from triply esterified phosphate has 

 been considered ever since titration experiments became suffi- 

 ciently accurate to indicate an excess of secondary phosphate 

 groups (that is, singly esterified phosphates having a second 

 ionizable hydrogen with a pK. value of about 6) over end groups. 

 With increasing attention being paid to the prevention of hydro- 

 lytic degradation during the isolation of RNA samples, the 

 amounts of secondary phosphate observed have fallen to less 

 than 1 0% of the total phosphate present, but are seemingly still 

 present. The additional observation has been made that the 

 secondary phosphate ionizations are found at the expense of 

 primary, the phosphorus content of the sample being used as the 

 measure of primary ionizations to be expected (57). Since each 

 triply esterified phosphate represents a loss of one primary 



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