266 VINCENT G. ALLFREY 



RNA are plotted against the time of incubation in the presence of [i-^^C]- 

 leucine. It is clear that the addition of chloramphenicol effectively stops 

 amino acid uptake into protein (as first pointed out by Breitman and 

 Webster [33]); despite the presence of the antibiotic, leucine enters the 

 ribonucleic acid fraction at a very high initial rate. 



Similar results have recently been obtained with puromycin [29]. 

 Yarmolinsky and de la Haba have called attention to the fact that the 

 structure of puromycin closely resembles that of the proposed amino 

 acyl-RNA complex [30]. Considering its structure, the effectiveness of 

 puromycin as an inhibitor of nuclear protein synthesis can be interpreted 

 as due to a close competition between the antibiotic and the natural amino 

 acyl-RNA complex. This would then be strong presumptive evidence that 

 most of the incorporation of amino acids into nuclear proteins proceeds 

 through an amino acyl-RNA intermediate. 



In the experiments described above, the formation of leucyl-RNA took 

 place in the intact thymus nucleus. An alternative method allows the 

 preparation of the amino-acid-carrier RNA complex in sub-nuclear 

 fractions. This procedure employs the nuclear activating enzymes (the 

 nuclear pH 5 fraction), ATP, RNA, and the radioactive amino acid [29, 



34]- 



It is of interest that much of the "transfer" or "carrier" RNA in the 



thymus nucleus occurs in association with the nuclear ribosome fraction 



and can be centrifuged down together with the ribosomes in the spinco. 



(This differs from the usual observation that in cytoplasmic extracts, the 



"carrier" RNA occurs in a relatively low-molecular weight, soluble form.) 



The pH 5 fraction as usually prepared from neutral extracts of thymus 



nuclei contains a few per cent of RNA which readily accepts activated 



amino acids. The addition of more nuclear RNA (prepared by the phenol 



method) results in a higher level of amino acid transfer. 



In this connection, it should be mentioned that not all nuclear RNA 

 fractions can function as leucine acceptors in the transfer reaction. For 

 example, there is good evidence that the high molecular weight RNA of 

 the nucleolus does not take part in this transfer process [19, 29]. (This 

 RNA also fails to go into solution during the usual phenol extraction pro- 

 cedure.) Similar tests for amino acid transfer to DNA in the nucleus have 

 led to negative results [19]. 



The transfer reaction in sub-nuclear fractions provides a convenient 

 method for the preparation of leucyl-RNA. That the I'^C-uptake observed 

 under these conditions actually represents a true incorporation of amino 

 acid into RNA has been shown in several ways: (i) all of the radioactivity 

 can be removed from the labelled pH 5 fraction by treatment with ribonu- 

 clease; (2) the presence of traces of ribonuclease during the reaction 

 effectively prevents any uptake of isotope. (This is of interest because 



