Discussion 21 



we can extract both protein and RNA by means of dilute citric acid. 

 If we use labelled nuclei isolated from an animal that has received radio- 

 active phosphorus beforehand, then the RNA which is extracted from 

 the isolated nuclei has a lower specific activity than the RNA which 

 remains. We do not yet know whether there is any difference in base 

 ratios, but there certainly is a difference in specific activity, and more- 

 over, both the easily extractable and the non-extractable RNA have 

 specific activities which differ from those of any of the RNA's of the 

 cell cytoplasmic fraction. We have done the same sort of thing with 

 nuclei which have been isolated in sucrose-CaClg media. Whether we 

 label the animals with radioactive phosphorus or radioactive carbon 

 in the form of i*C-formate, when the nuclei are treated with dilute 

 citric acid or dilute phosphate buffer, an RNA can be extracted which 

 has a different specific activity from that of the RNA which remains, 

 and a different specific activity from that of any of the cytoplasmic 

 fractions. So here we have soine fairly substantial evidence that the 

 RNA of the nucleus is heterogeneous. 



One might, of course, argue that the easily extractable RNA is simply 

 cytoplasmic RNA which is adhering to the nuclei. Against this is the 

 fact that nuclei prepared by the methods which we use are very clean 

 indeed according to microscopic examination. We have had them 

 examined microscopically by critics who were doing their best to find 

 flaws in our technique and they failed to do so, so that we do believe 

 that the RNA which is extracted is essentially nuclear RNA. 



The possibility of nuclear RNA being the precursor of cytoplasmic 

 RNA is extremely interesting, but as Prof. Brachet said, the evidence 

 for a direct move of RNA from nucleus to cytoplasm is not good at the 

 moment. 



Brachet: The localization of these two RNA's in the nuclei is not 

 known. If you study it by means of interference microscopy — I suppose 

 there is enough RNA which can be removed by the treatment with 

 citric acid — you might find out where it is located. There may be one 

 difficulty in this work on analysis of bases, i.e. it is difficult to get homo- 

 geneous RNA fractions, even from the tissues; so that it may be that 

 in the case of nuclei which have already been isolated, for instance by 

 extraction with citric acid, one may not get the true specific activity. 

 It may be a biochemical artifact to a certain extent. 



Butler : WTiat is the evidence for the presence of RNA in chromatin ? 



Brachet: As far as I know, there are only two pieces of evidence for 

 RNA in chromatin: one is that with cytochemical methods one finds 

 that there are differences in the staining ability of the nuclei from 

 different tissues. As a rule, the nuclei of tissues which do not synthesize 

 proteins stain almost green with methyl green-pyronin. In the case of 

 liver, pancreas, etc., there is a lilac or violet colour of the nuclei. If you 

 treat the sections with ribonuclease, you will find that the nuclei now 

 stain completely green. The red colour of the chromatin disappears as 

 well as the red colour of the cytoplasm and the nucleolus. Furthermore, 

 Mirsky and Ris, in their work on isolated chromosomes, obtained some 

 RNA in the threads. 



