274 VINCENT G. ALLFREY 



which suggests that the transport of amino acids into the nucleus is an 

 active process requiring the participation of ATP or some similar energy 

 source. For example, the addition of o-oo2 M cyanide or dinitrophenol 

 (both of which suppress nuclear ATP synthesis) causes some inhibition of 

 alanine transport. Similarly, the removal of nucleotides by selective extrac- 

 tion with acetate buffers [8] destroys the nuclear capacity for subsequent 

 amino acid accumulation [46]. 



Other experiments have made it clear that the primary reason for the 

 sodium dependence of amino acid incorporation into the proteins of intact 

 nuclei is simply that sodium ions are needed for the transport of amino 

 acids to the sites of synthesis. For example, we have found that thymus 

 nuclei will synthesize ^*C-labelled protein even in an all-potassium medium, 

 provided they are first exposed to a sodium-rich medium containing the 

 radioactive amino acid. (Other tests have shown that isolated nuclei cannot 

 retain sodium ions taken up during this preincubation.) Once transport 

 has occurred, the continued presence of sodium ions in the medium is not 

 necessary for the incorporation process [46]. 



Thus the sodium dependence of protein synthesis in the nucleus is due 

 to the operation of a specific transport mechanism. This sodium ion require- 

 ment becomes a useful test for establishing the intranuclear localization of 

 protein synthetic reactions, especially when one considers that many cyto- 

 plasmic systems display a specific requirement for potassium ions in amino 

 acid transport [48] and incorporation [15]. 



Other synthetic processes in isolated nuclei may or may not show a 

 sodium ion requirement. The range of the sodium ion requirement will 

 not be discussed here, except to mention that a good correlation has been 

 found between the ionic requirements for uptake and for transport of 

 several isotopic precursors of ribo- and deoxyribo-nucleic acids [46]. 



DNA EFFECTS ON ISOLATED NUCLEAR RIBOSOMES 



A useful test for the nuclear localization of a synthetic process is a 

 measure of its DNA dependency. The addition of DNAase to suspensions 

 of intact isolated nuclei leads to a cessation of nearly all synthetic activity 

 [4, 5, 13]. As mentioned before, this is largely due to the fact that DNA 

 plays a role in mediating nuclear ATP synthesis : removal of the DNA 

 therefore has an indirect effect on all nuclear processes that require ATP. 



However, some recent results suggest that DNA may play a more 

 specific role in controlling the synthesis of nuclear proteins. These results 

 were obtained in tracer experiments with isolated nuclear ribosomes. The 

 incorporation of amino acids into ribosomes outside the nucleus is not 

 affected by deoxyribonuclease [44] because the ATP needed for amino 

 acid activation is supplied by an exogenous ATP-generating system. It 



