THE PROBLEM OF NUCLEOTIDE SEQUENCE IN DEOXYRIBONUCLEIC ACIDS 73 



from the intact polymer; there is, moreover, a significant trend in the 

 composition of the products produced by the stepwise enzymic digestion. 

 No indications of any regularity in the release or the composition of 

 fragments were observed; no sub-units of recognizably recurrent structure 

 were seen : deoxyribonucleic acids apparently exhibit a largely arrhythmic 

 nucleotide sequence. 



APURINIC ACID 



This has proved a very useful compound; it is, in fact, the first inter- 

 mediate product in all procedures involving acid degradation [26]. When 

 a deoxyribonucleic acid is exposed to mildly acidic conditions (pH i-6 

 at 37^') all the purines are cleaved off gradually, leaving behind a polymer 

 representing the original polynucleotide, but composed of deoxyribo- 

 phosphate units, at the places of the previous purine nucleotides, and of 

 the pvrimidine nucleotides in unchanged ratio and at the same position 

 as in the native starting material. For instance, in the segment represented 

 in Fig. I, positions 3, 5-7, 9, i', 2', 4', 8', 10' would remain unchanged, 

 whereas in the remaining places the purines would now have made room 

 for the reactive aldehydo groups of the deoxy sugar. The presence of the 

 aldehydo group [27] and, therefore, of a free hydroxyl at 4' brings about a 

 remarkable labilization of the polymer: it is broken not only by alkali, 

 but even by buffers (pH 8-6) containing primary amino groups [28]. It 

 is likely that this susceptibility of the poly-sugar-phosphate backbone of 

 apurinic acid to amines is involved in the degradation reactions employing 

 diphenylamine under acidic conditions [29]. 



Studies on the arrangement of the pyrimidine nucleotides in apurinic 

 acid [28] were, in fact, the first that made possible an approach to the 

 problem of nucleotide sequence in deoxyribonucleic acid. The informa- 

 tion so obtained was limited, since it was only qualitative, but it showed 

 that a considerable portion of the pyrimidine nucleotides, and therefore 

 also of the purine nucleotides, was arranged in the form of tracts of several 

 nucleotides of one kind. 



Sequence studies through differential distribution analysis 



MECHANISM 



The important discovery that among the fragments produced by the 

 acid degradation of deoxyribonucleic acids there are found the 3', 5'- 

 diphosphates of deoxycytidine and thymidine is due to the work of Levene, 

 Thannhauser and their collaborators [30-33]. A more recent re-investiga- 

 tion [34] by improved techniques, which confirmed the occurrence of 

 these diphosphates, prompted a discussion of the possible bearing of this 



