348 R. B. ROBERTS, H. .1. HHITTP:N. and B. J. MCCARTHY 



whose synthesis is directed by DNA, then self-replication would be 

 ruled out. 



H. CELL-FREE SYNTHESIS OF RIBOSOMES 



Several of the cell-free systems for protein synthesis have been 

 reported to show a sensitivity to DNase and to require ribonucleoside 

 triphosi)hates for optimal synthesis (Kameyama and Novelli, 1960; 

 Matthaei and Nirenberg, 1961; Tissieres et al., 1960). Presumably in all 

 these systems there is a DNA-dei)endent synthesis of RNA which allows 

 a more prolonged period of protein synthesis. 



The incorporation of labeled nucleotides into RNA has been observed 

 both in purified enzyme preparations and in some of the cell-free systems 

 capable of protein synthesis. The purified enzyme preparations yield 

 RNA of random sequence (Grunberg-Manago et al., 1955), copies of a 

 DNA primer (Hurwitz et al, 1960; Weiss and Nakamoto, 1961), or 

 copies of an RNA primer (Reddi, 1961), depending on which enzyme is 

 present. As these preparations are not designed to allow protein synthesis, 

 no ribosomes are formed. 



In most of the experiments using cell-free systems capable of protein 

 synthesis the RNA concurrently synthesized has not been characterized. 

 In one experiment P^--labeled nucleotides were added to the cell free 

 system of Nirenberg and Matthaei. The products (analyzed by chroma- 

 tography on DEAE) showed RNA eluting at 0.5 M NaCl and at 0.6 M 

 NaCl. These concentrations elute S-RNA and eosomes, respectively (see 

 Section III,A). The peak at 0.6 M NaCl, but not the peak at 0.5 M NaCl, 

 was absent when the system had been treated with DNase. As free RNA 

 elutes at a much higher NaCl concentration, this system seems to be 

 capable of the synthesis of the first ribosome precursor. The composition 

 of the eosome-like RNA showed the typical 50-50 proportions of D-RNA 

 and R-RNA. Thus both D-RNA and R-RNA, but not S-RNA, seem to 

 be synthesized only when DNA is present. 



Since these systems have been optimized on the basis of their 

 capacity to synthesize protein it seems likely that ways to improve their 

 capacity for ribosome synthesis could be found. Synthesis of complete 

 ribosomes in cell free systems is a likely development in the future. 



I. STATE OF RIBOSOMES IN LIVING CELLS 



It is by no means established that the ribosomes observed in the 

 analytical centrifuge or the electron microscope exist as definite entities 

 of the same form in the living cell. For several years indications have 

 accumulated that ribosomes undergo certain irreversible changes at the 

 instant of breaking the cells. 



