31. SYNTHESIS OF POLYNUCLEOTIDES 129 



were effectively incorporated into chains of polyadenylic and polynridylic 

 acids and poly-(ribofuranosylthymine-phosphate). 



The oligonucleotides initiated polynucleotide chain formation by utiliza- 

 tion of their 3'-hydroxyl groups even with enzyme preparations which did 

 not show primer requirement. In experiments with preparations of highly 

 purified Azotobacter enzyme, the oligonucleotides were effectively incorpor- 

 ated even when their concentration far exceeded that required for maximal 

 rate of synthesis. 



Using the oligonucleotides as primers, and especially by adjusting the 

 primer to nucleoside diphosphate ratio, the stepwise addition of nucleoside- 

 5'-phosphoryl units to the 3'-hydroxyl ends of preformed oligonucleotide 

 chains could be convincingly demonstrated and the products obtained were, 

 in general, of much lower chain length than those obtained in the absence of 

 primers. 



By a mechanism as yet obscure, small oligonucleotides bearing 3'-phos- 

 phomonoester end groups and thus containing no free 3'-hydroxyl groups, 

 also served to abolish the lag period in the polymer synthesis, although 

 they were not incorporated into the polymers formed. [It may be noted 

 that working with the enzyme from M. lysodeikticus, Hendley and Beers 49 

 have recorded the inhibition of polynucleotide synthesis by certain mixtures 

 of larger oligonucleotides bearing 3'-phosphomonoester end groups (cf. 

 Beers 50 )]. 



d. Phosphorolysis of Polynucleotides Catalyzed by Polynucleotide Phosphoryl- 

 ase 



(1) Phosphorolysis of High Molecular Weight Polymers and Ribonucleic 

 Acids. Studies of the phosphorolysis of enzymically synthesized polynu- 

 cleotides and a variety of ribonucleic acids have been reported. 32 ' 33 ' 51, 52,53 

 Phosphorolysis of enzymically synthesized polynucleotides containing one 

 kind of nucleotide is facile. 32 ' 33 ' 51 In contrast, mixing of polyadenylic acid 

 and polyuridylic acid confers great resistance to phosphorolysis. 51 This is 

 obviously due to the interaction to form multistranded chains. 54 " 56 ' 56a The 

 mixed synthetic polymer of the four nucleotides as well as highly polymer- 

 ized yeast, leaf, and rat liver ribonucleic acids are also phosphorolyzed 



49 D. D. Hendley and R. F. Beers, Jr., Federation Proc. 18, 245 (1959). 



60 R. F. Beers, Jr., Nature 183, 1335 (1959). 



61 S. Ochoa, Arch. Biochem. Biophys. 69, 119 (1957). 



52 D. D. Hendley and R. F. Beers, Jr., Federation Proc. 17, 240 (1958). 



53 P. Lengyel and S. Ochoa, Biochim. et Biophys. Acta 28, 200 (1958). 

 64 R. C. Warner, J. Biol. Chem. 229, 711 (1957). 



56 G. Felsenfeld and A. Rich, Biochim. et Biophys. Acta 26, 457 (1957). 



56 L. A. Heppel, M. F. Singer, and R. J. Hilmoe, Ann. N. F. Acad. Sci. 81, 635 (1959). 



56a M. Grunberg-Manago, J. Mol. Biol., 1, 240 (1960). 



