74 ERWIN CHARGAFF 



finding on the problem of nucleic acid structure (p. 366 of ref. [7]). It was 

 clear that for these fragments to serve as a tool in research on nucleotide 

 sequence the mechanism of their release had to be better understood. It 

 was also indispensable to be able to study them under rigorously controlled 

 conditions and on a quantitative basis. 



The kinetics of the liberation of the pyrimidine nucleoside diphos- 

 phates were first studied, in collaboration with Dr. Shapiro, on a series of 

 models, namely, the several deoxyribodinucleotides all having cytidylic 

 acid as one of their components [35]. These investigations served as the 

 basis of a well-controlled, difi'erential hydrolysis procedure which was 

 usually performed in three stages [21, 36]. The diagnostically most 

 valuable results are obtained in Stage I (o-i M H2SO4, 30 min., 100°), 

 when the release of pyrimidine nucleoside diphosphates can be taken to 

 reflect directly the abundance of these units as solitary pyrimidine nucleo- 

 tides in the polynucleotide chain. In the present survey I shall limit myself 

 to the findings based on this stage of the difi^erential hydrolysis procedure. 

 The results yielded in Stages II and III, which involve longer periods of 

 hydrolysis, are mainly indicative of the secondary cleavage of bunched 

 pyrimidine sequences; they are of great value as further means of differen- 

 tiation between analytically indistinguishable nucleic acids of different 

 cellular origin. This will be exemplified in the following section. 



In its simplest form the cleavage of a polynucleotide chain and the 

 formation of the diphosphates of the pyrimidine nucleosides (Py) may be 

 regarded as a series of ^ elimination reactions [21]. 



-O OH 



\/ 

 P 



o o 



CHO 



I 

 CH, 



CH 



CHOH 

 CH, 



O OH 



O 



o.. 



After the liberation of the purines the first elimination presumably occurs 

 at the broken line A. Since no extraneous sugar fragment is found in ester 

 linkage with the resulting nucleoside diphosphate, a subsequent hydrolysis 

 or elimination must take place at the other flanking sugar (broken line C). 

 It is likely that this cleavage follows a ^ aldehyde elimination (broken 

 line B), which has left a double bond between the second and third carbon 

 atom of the sugar. Similar considerations apply to the release of bunched 

 pyrimidine sequences. A more than tentative formulation of the reaction 



