28o 



NUCLEIC ACIDS AND GROWTH 



6 7 



hours 



In onion root-tips (Brachet, 1954a), ribonuclease (img/ml) produces a 90% 

 inhibition of amino-acid incorporation into proteins within 3 h. Fig. 5 illustrates 

 the residts obtained, with an autoradiography technique, in the case of phenyl- 

 alanine. The inhibition of the incorporation of the amino acids into the proteins is 

 faster and stronger than that of the penetration of the free amino acids; this 

 suggests that ribonuclease does not act primarily on cellular permeability. 

 Furthermore, ribonuclease strongly inhibits growth (Fig. 6) : the inhibition is 



almost complete within 3 h. and is usually 

 irreversible (Brachet, 1955b). Chemical 

 estimations of the protein content have 

 shown, as might be expected, that net 

 protein synthesis is completely inhibited 

 in the ribonuclease-treated onion roots. 

 Another interesting finding is shown in 

 Fig. 6: addition of yeast RNA to the 

 ribonuclease-treated roots partially re- 

 stores growth, at least for a few hours. 



RNA is only broken down to a limited 

 extent in the ribonuclease-treated roots; 

 but RNA metabolism is certainly deeply 

 affected : for instance, adenine incorpo- 

 ration in RNA is first stimulated, then inhi- 

 bited, and free nucleotides increase. It 

 seems that, in the treated roots, RNA 

 becomes abnormal in constitution and unable to fulfill its role in protein synthesis; 

 or that RNase combines with RNA to produce an inactive complex. 



The inhibition of protein synthesis in ribonuclease-treated roots is not due to an 

 indirect action of the enzyme on energy producing reactions, since the oxygen 

 consumption and the ATP content remain essentially normal after 3 h. treat- 

 ment. 



Still more interesting perhaps is the case of the amoeba A. proteus (Brachet, 

 1955c): low concentrations of ribonuclease (o.i mg/ml) are sufficient to produce 

 loss of locomotion followed by cytolysis after 2-3 h. If the amoebae are returned 

 to their normal medium after i h., they show poor motility and ultimately die, 

 even if they are well fed. But when the normal medium is fortified with yeast 

 RNA (1.4 mg/ml), complete recovery occurs in a given percentage of the amoeba 

 population (20-50% according to experimental conditions) : shape, locomotion 

 and ability to multiply if fed are almost normal in the amoebae which have re- 

 covered after the RNA treatment. 



Cytochemical observations and chemical micro-analyses have conclusively 

 shown that ribonuclease produces a marked drop (20-45%) ^^ the RNA content 

 of the amoebae, the nucleoli and the cytoplasm being both affected (Fig, 7) ; 

 simultaneously, free nucleotides increase; as in the onion root-tips, the oxygen 

 consumption and the ATP content undergo little, if any, change. The RNA con- 

 tent of the amoebae which have recovered from ribonuclease treatment in an RNA- 

 containing medium, practically returns to normal. 



Fig. 6. Inhibition of growth in onion 



roots by ribonuclease; partial recovery 



on addition of RNA. 



