31. SYNTHESIS OF POLYNUCLEOTIDES 127 



of a primer, then it is possible that the 5'-phosphomonoester group was 

 either present in the primer itself or it arises by the action of a nuclease pres- 

 ent in the enzyme preparation used. (The nuclease would be of the type that 

 is present in guinea pig liver nuclei. 42, 46 ) Alternatively, if a chain is synthe- 

 sized de novo then the pyrophosphate group of the terminal nucleoside-5'- 

 diphosphate must be hydrolyzed by some enzymic means. 



So far the preparation of five different types of polymers containing only 

 one kind of nucleoside residue (homopolymers) has been reported. 32, 33, 35, 47 

 These are polyadenylic acid, poly uridy lie acid, poly cy tidy lie acid, poly- 

 inosinic acid, and poly-(ribofuranosylthymine-phosphate). 47 The notable 

 omission is that of polyguanylic acid. Neither the Azotobacter enzyme 32 nor 

 the E. coli enzyme 33 is able to polymerize guanosine-5 '-diphosphate, al- 

 though guanosine nucleotide is readily incorporated into a polymer 32, 44 

 when the diphosphate is present among other nucleoside diphosphates. Of 

 the polymers containing more than one kind of nucleotide residue (hetero- 

 polymers), adenylic-uridylic acid polymers as well as polymers containing 

 all the four common ribonucleotides 32, 44 have been prepared. Detailed deg- 

 radative evidence has been presented 42, 43, 48, 48a to show that the products 

 obtained by using mixtures of nucleoside-5'-diphosphates contain chains 

 in which the different nucleotides are interlinked with each other. 



Polymers synthesized by polynucleotide phosphorylase are large, even 

 though molecular weight determinations by different methods have given 

 varying results. Thus, while the physical methods indicate molecular 

 weights of 30,000 to over a million, 34, 44, 48 determinations of average chain 

 length by end group assays gave lower values (an average chain length of 

 30 for mixed polymers of adenylic, guanylic, uridylic, and cytidylic acids, 

 120 for an adenylic-uridylic acid polymer, and 230 for polyadenylic 

 acid). 44, 48 Certainly, there is great latitude in the size as well as the type of 

 polymers that can be prepared enzymically and the scope has been enlarged 

 greatly by the studies of Heppel and co-workers as described below. 



c. Primers for Polynucleotide Phosphorylase 



(1) High Molecular Weight Polynucleotides as Primers. As mentioned 

 above, using a highly purified Azotobacter enzyme preparation, Mii and 

 Ochoa 40 demonstrated a lag phase in the reaction. The lag phase could be 

 overcome by certain high molecular weight polynucleotides. However, a 

 number of interesting but intricate relationships were noted between the 



" L. A. Heppel, P. J. Ortiz, and S. Ochoa, Science 123, 415 (1956). 



« B. E. Griffin, A. R. Todd, and A. Rich, Proc. Natl. Acad. Sci. U. S. 44, 1123 (1958). 



48 S. Ochoa and L. A. Heppel, in "The Chemical Basis of Heredity" (W. D. McElroy 



and B. Glass, eds.), p. 615. Johns Hopkins Press, Baltimore, 1957. 

 48a P. J. Ortiz and S. Ochoa, J. Biol. Chem. 234, 1208 (1959). 



