VIII. PROTEIN SYNTHESIS AND GENE ACTION 387 



of cell-free synthesis of /^-galacto.'^idase by DNase was reported by 

 Kameyama and Novelli (1960). Complete inhibition was obtained and, 

 in fact, the activity of the initial jS-galactosidase present was decreased. 

 A thorough investigation of the DNase inhibition (Matthaei and 

 Nirenberg, 1961) showed that decreases in protein synthesis was noted 

 only after 10-15 minutes of incorporation. The amounts of RNase and 

 trypsin contamination of the DNase preparation were estimated and 

 these amounts shown not to inhibit protein synthesis. The products of 

 DNase digestion did not account for the inhibition. Thus, a require- 

 ment for DNA for protein synthesis was demonstrated. The effect of 

 DNA was probably not direct, since complete inhibition (particularly 

 in the initial phases) was not attained. This was attributed to the 

 presence of endogenous messenger RNA which was utilized in the early 

 phases of amino acid incorporation and then destroyed, leading to a 

 complete dependence on DNA at later stages. 



Restoration of activity for protein synthesis by RNA (Nirenberg and 

 Matthaei, 1961) as expected was the next step forward. For these studies, 

 the authors used the crude E. coli supernatant containing ribosomes, 

 which had been preincubated with DNase and energy. The use of the 

 energy-generating system resulted in incorporation of C^--amino acids 

 which presumably led to loss of endogenous messenger RNA. This latter 

 point has not been demonstrated, but, in any event, the treatment pro- 

 vided a system which showed almost no amino acid incorporation in 

 the absence of added messenger RNA. Increases in amino acid incorpo- 

 ration by addition of E. coli and yeast ribosomal RNA and by TMV- 

 RNA were observed. The most striking increase was that given by 

 TMV-RNA, which increased incorporation from 42 to 872 counts per 

 minute/mg of ribosomal protein. The RNA-stimulated incorporation was 

 inhibited by puromycin, required energy, and in general showed the 

 properties of the usual amino acid-incorporating systems. Excess soluble 

 RNA was present and did not account for the observed stimulations. 

 Finally, in this paper, stimulation of phenylalanine incorporation by 

 polyuridylic acid was observed (Nirenberg and Matthaei, 1961). These 

 results represent the most significant advance in the understanding of 

 information transfer in protein synthesis in recent years and many new 

 fields are open to investigation as a result. However, the RNA types 

 which gave increased incorporation did not resemble messenger RNA, 

 as defined earlier, and in fact these results did not prove that DNA 

 destruction was involved in the loss of activity. 



Tissieres and Hopkins (1961) were able to remove DNA from an 

 E. coli system by precipitation with magnesium chloride. By adding back 

 E. coli DNA ])lus spermidine, some stimulation of amino acid incorpo- 



