390 RICHARD SCHWEET AND JOHN BISHOP 



electrophorotic studios by the authors prox'ide good evidence that new 

 enzyme synthesis has occurred in this system. 



3. Function of Synthetic Polynucleotides in Cell-Free Systems 



The DNase-treatcd, iirciucuhatcd cell-free system from E. coli, 

 described earlier, was stimulated by various types of RNA (Nircnberg 

 and Matthaei, 1961). However, it was not clear that this RNA served 

 an infonnational function, e.g., directed the synthesis of specific proteins 

 by specifying the sequence of amino acids. Using poly-U as the stimu- 

 lating polynucleotide, however, only phenylalanine was incorporated. 

 Evidence was provided that the product was polyphenylalanine. In this 

 artificial system, therefore, the polynucleotide was sj)ecifying which 

 amino acid was incorporated. In a subsequent publication (Nirenberg 

 et al., 1962), it was shown that this system had many of the properties 

 of usual incorporating systems. For example, phenylalanyl-RNA was 

 an intermediate. Other synthetic polynucleotides were then tested in both 

 Nirenberg's and Ochoa's laboratories. The results of these studies have 

 been extremely fruitful, in that only certain polynucleotides stimulated 

 the incorporation of particular amino acids. Using certain assumptions 

 noted below, it was possible to assign particular nucleotide triplets to 

 particular amino acids and thus provide the solution to the code. 

 The findings have been published in some detail; references to these 

 are found in the most recent publications (Basilio et al., 1962; Matthaei 

 et al, 1962). 



The primary basis for assignment of code letters rests on the finding 

 that poly-U stimulates only phenylalanine incorporation. In fact, in- 

 corporation of leucine and isoleucine were also stimulated, but to a 

 much smaller extent. When polymers containing two or more nucleo- 

 tides were used, they were assumed to contain random sequences. For 

 example, the U:C polymer containing 5 times as much U as C was 

 considered to contain 5 times as many UUU triplets as UUC, UCU, or 

 CUU triplets. The use of a triplet is indicated as a minimum number 

 for each coding unit by the finding that incorporation of some amino 

 acids was stimulated only by polymers containing three different nucleo- 

 tides. The results of Crick et al. (1961) (see Chapter VI) also indicate 

 a triplet code. The term U2C includes all the possible arrangements of 

 these three letters; thus the sequence is not known for any code letter. 

 The letters in the code word are then decided by comparing the ratio 

 of phenylalanine stimulation to the amino acid in question. For example, 

 using the case given above, the only amino acids (besides phenylalanine) 

 whose incorporation was stimulated by poly-UC were leucine, proline, 

 and serine (Speyer et al., 1962). The ratios were 5, 13 and 4, respec- 



