NUCLEAR PROTEIN SYNTHESIS 267 



amino acid uptake in the intact nucleus cannot be blocked by adding RNA- 

 ase to the incubation medium. This result fits in with other observations 

 that added RNAase does not attack the ribosomal RNA as long as it remains 

 within the nucleus (see below).) (3) The RNA can be prepared by the 

 phenol isolation method and show^n to contain bound ^^C-amino acid; 

 (4) all the counts are removed by alkaline digestion of the RNA or by 

 treatment with hot 5^0 trichloroacetic acid to remove nucleic acids; the 

 protein residue after these treatments is not radioactive. 



PROPERTIES OF THE AMINO ACYL-RNA COMPLEX 



The chemical nature of the leucyl-RNA complex has been studied 

 using the in vitro system of labelling the RNA associated with the nuclear 

 pH 5 fraction. The product of the reaction (nuclear leucyl-RNA) has 

 many properties which show its similarity to the amino acyl-RNA com- 

 plexes studied in cytoplasmic systems. It is non-dialyzable and stable in 

 dilute acids (e.g. 2 per cent HCIO4). On the other hand, a brief exposure 

 to dilute alkali (0-005 '^ NaOH) removes all the radioactivity from leucyl- 

 RNA, and subsequent chromatography on paper separates the counts 

 as free P^C] -leucine. 



A short ribonuclease digestion of the nuclear leucyl-RNA complex 

 (adding 10 /xg. of RNAase for 15 min. at 37°) separates the radioactivity 

 in an acid-soluble form. It has been shown that the action of ribonuclease 

 releases the amino acid bound to a nucleoside [29, 34]. By following the 

 procedure described by Zachau et al. [35], we have isolated the amino 

 acyl-nucleoside by ionophoresis on filter paper. At pH 3-2 the leucyl- 

 nucleoside diff^ers in its mobility from any of the free nucleosides in the 

 RNA digest and it can be located on the paper as a radioactive, u.v.- 

 absorbing spot. Alternatively, the leucyl-nucleoside separates nicely in 

 the paper chromatographic system described by Preiss et al. [36]. In both 

 cases treatment of the radioactive, u. v. -absorbing spot with dilute alkali 

 releases the radioactivity as free ^^C-leucine and leaves the nucleoside. 

 Both components are readily separable by chromatography or electro- 

 phoresis [34]. 



In order to get sufficient material for identification of the nucleoside, a 

 large-scale preparation of leucyl-RNA was carried out, using the pH 5 

 fraction from over 500 g. of isolated nuclei. The product was digested wath 

 ribonuclease, and the amino acyl-nucleoside separated by ionophoresis. 

 Alkaline treatment then released the nucleoside, which, in its mobility, 

 Rf, and ultraviolet absorption spectrum appears to be adenosine [34]. 



It follows that the receptor group in nuclear "carrier" RNA is a 

 terminal adenylic acid, as was found earlier in the s-RNA of cytoplasmic 

 systems [35]. 



