282 ELLIOT VOLKIN 



B. THE USE OF POLYNUCLEOTIDES IN ESTABLISHING THE CODE 



From the laboratories of Nirenberg (Matthaei and Nirenberg, 1961 ; 

 Nirenberg and Matthaei, 1961; Martin et al., 1962; Nirenberg et al., 

 1962) and Ochoa (Lengyel et al, 1961, 1962; Speyer et al., 1962) have 

 come the most startling applications of the microbial ribosome-siiper- 

 natant amino acid incoi-porating systems. Nirenberg and ]\Iattliaei 

 (1961) discovered that the addition of heat-stable RNA from different 

 sources increased the incorporation of C^^-labeled amino acids by the 

 ribosome-supernatant system. It is of interest that these authors note 

 that "the correlation between the amount of incorporation and the 

 amount of added RNA suggested stoichiometric rather than catalytic 

 activity of the template RNA" (Nirenberg and Matthaei, 1961). In the 

 process of testing a variety of RNA's, synthetic polynucleotides synthe- 

 sized by polynucleotide phosphorylase were tested for stimulatoiy ac- 

 tivity. It was found that the addition of polyuridylic acid resulted in a 

 rather massive synthesis of poly-L-phenylalanine, and the polymeriza- 

 tion of only this amino acid was specifically directed (Nirenberg and 

 Matthaei, 1961). In a subsequent thorough investigation of this reaction, 

 Nirenberg et al. (1962) determined that the phenylalanine was linked to 

 soluble RNA as a true intermediate in the process. Polyuridylic acid 

 associated in a double- or triple-stranded configuration is ineffective in 

 amino acid stimulation, suggesting that active RNA is a single-stranded 

 template (Nirenberg et al., 1962). In any case, the observations with 

 polyuridylic acid have been fully corroborated by Lengyel et al. (1961), 

 indicating that uridylic acid residues serve as the information centers 

 for phenylalanine insertion into protein. 



The laboratories of Nirenberg and Ochoa have fully exploited the 

 advantages of such synthetic polynucleotides as synthetic "messengers," 

 whereby copolymers enriched in uridylic acid and containing known 

 proportions of one or more of the other nucleotides have been tested for 

 stimulatory activity with virtually the entire spectrum of natural amino 

 acids. In this fashion, relatively long sequences of uridylic acid in the 

 chain serve as templates for the production of polyphenylalanine chains, 

 which in turn serve as "handles" (Lengyel et al., 1961) for the attach- 

 ment of other amino acids whose linking is directed by the specific neigh- 

 boring base sequences. Thus, one may correlate the observed incorpora- 

 tion of a particular amino acid with the calculated frequency ratio of 

 the uridylic sequences to the other nucleotide sequences in the polymer. 

 Assuming a minimum coding ratio of three nucleotide sequences for the 

 direction of a specific amino acid insertion into protein, it has been 

 possible thus far to establish nucleotide coding letters for a larger num- 



