Studies on Polynucleotides Synthesized by Polynucleotide Phosphorylase

The preparation of synthetic ribonucleic acid in which one of the nucleotide species is labeled with P32, makes possible studies on the distribution of the labeled nucleotide in the polynucleotide chain. Fig. 1 presents the structure of a polynucleotide in which labeled adenylic acid is randomly distributed. Hydrolysis of such a polymer with snake venom phosphodiesterase (Fig. 1A) will yield a mixture of nucleoside 5’-monophosphates of which only adenylic acid will be labeled and the specific radioactivity will be the same as that of the adenylic acid incorporated. On the other hand, hydrolysis with spleen phosphodiesterase (Fig. 1B) will yield nucleoside 3’-monophosphates all of which will be labeled and, as nonlabeled adenosine 3’-monophosphate is also released, its specific radioactivity will be lower than that of the adenosine 5’-monophosphate incorporated. Hydrolysis with alkali will give the same result except that the nucleotides will consist of the isomeric mixtures of nucleoside 2’and 3’-monophosphates. If the polymer contains Ps2-labeled uridine 5’monophosphate, or other labeled nucleotide, what has been said of adenylic acid will apply to this nucleotide. Two kinds of labeled ribonucleic acid have been used in the work reported in this paper. One (poly A*GUC)’ was prepared with approximately equimolar amounts of P32-labeled adenosine 5’-diphosphate (adenosine-P32-P32), and nonlabeled guanosine, uridine, and cytidine 5’-diphosphates. The other (poly AGU*C) was prepared in the same way but with PWabeled uridine 5’diphosphate (uridine-P32-P) and nonlabeled adenosine, guanosine, and cytidine 5’-diphosphates. Isolation of the products of hydrolysis of these polymers with spleen phosphodiesterase (or alkali) and determination of their amount and radioactivity has shown that adenylic and uridylic acids are extensively linked to each of the other three nucleotide species. The results suggest a random distribution of the labeled nucleotide units in the polynucleotide chains. They do not indicate, however, whether linking between all four nucleotide species occurs within a single