38 RIBONUCLEIC ACIDS AND PROTEIN SYNTHESIS 



of a homogenate of roots. The significance of this soluble RNA 

 will be discussed later (Brachet and Six, 1959). 



In conclusion, there is no doubt that ribonuclease can penetrate 

 into onion roots, where it inhibits protein synthesis. Its mode of 

 action is probably a dual one : it breaks down the soluble RNA and 

 it apparently forms a complex with the RNA of the microsomes 

 and the nucleoli. Evidence for such a complex formation is found 

 in the fact that onion root-tip cells, as might be expected from the 

 direct RNA estimations, do not lose their basophilia unless they 

 have been acted upon by ribonuclease for more than 3 hours. But, 

 if the ribonuclease-treated roots are fixed by freeze-substitution, 

 instead of the usual Zenker acetic treatment, cytoplasmic baso- 

 philia disappears from the outer layer after only 1 hour; nuclear 

 basophilia is destroyed in almost all cells. After a 3-h ribonuclease 

 treatment, basophilia (except for chromatin) practically disappears 

 in the roots fixed by freeze substitution; on the other hand, staining 

 remains unimpaired in the Zenker-fixed roots, which cannot be 

 distinguished from the control roots. Obviously ribonuclease, es- 

 pecially in the nucleoli, forms a complex with RNA. This complex 

 remains intact in the roots fixed by freeze substitution, while it is 

 broken down by an energetic fixative such as Zenker. 



More interesting perhaps is the case of the amoeba, Amoeba pro- 

 teus (Brachet, 1955a, 1956b). Very low concentrations of ribonu- 

 clease (0.05-0.01 mg/ml) are sufficient to produce loss of locomo- 

 tion, followed by cytolysis after 2-3 h. If the amoebae are returned 

 to a normal medium after H h, they ultimately die, even if they are 

 provided with food. But complete recovery of a given percentage 

 (20-50 %) of the amoebae population is obtained if the ribonuclease- 

 treated amoebae are placed in the same medium supplemented with 

 yeast RNA (1.4 mg/ml). The shape, locomotion and abihty to 

 multiply (if properly fed) are almost normal in the amoebae which 

 have recovered in this way. 



Cytochemical observations indicate that, in contrast to onion 

 root-tip cells, ribonuclease-treated amoebae lose to a considerable 

 extent their affinity for basic dyes (Figs. 15, 16, 17, p. 35 and p. 36). 

 Both the cytoplasm and the nucleoli show decreased basophilia, 



