206 SUBCELLULAR PARTICLES 



cntial centrifugation in gradients, of lowering the whole cell concentration below 3 per 

 cent? Second, have you perhaps looked in nuclei for ribonucleoprotein particles similar 

 to those in the cytoplasm? 



Dr. Allfrey: The few whole cells which are present in preparations of thymus 

 nuclei do not constitute a problem in the experiments we have carried out so far. It 

 is possible to separate and remove these cells by centrifuging in 2.2 m sucrose solu- 

 tions. In such dense media, the nuclei will sediment and the cells go to the top of the 

 tube. Unfortunately, once the nuclei are exposed to such hypertonic media they lose 

 ability to incorporate radioactive amino acids or radioactive orotic acid. 



We do not know whether the nucleus isolated in sucrose-CaCU has in it ribonucleo- 

 protein particles similar to those seen in the cytoplasm. Electron microscopy of nuclei 

 isolated in calcium-containing media is disappointing from a morphological point of 

 view, because there seems to be a general precipitation or clumping of intranuclear 

 structures. 



Dr. Marshak: Would you discuss further the degree of contamination in your 

 preparations and its significance in the results you report. It seems to me that the cri- 

 terion of the effect of added DNA is one which may be so indirect as in possible 

 interpretations to be not reliable. In this connection I recall that the figure given for 

 contamination in published data is 10 per cent by volume. Cbuld not this mean more 

 than 10 per cent in terms of cRNA contamination? Also, Osawa's data on turnover 

 of nuclei prepared by this method show specific activities in this Nuclear #1 nRNA 

 fraction intermediate between Nuclear #2 nRNA and the cRNA. Also the composition 

 of nRNA #1 is intermediate between #2 and cRNA. 



Dr. Allfrey: The estimates of nuclear contamination are based on DNA and RNA 

 analyses and on tests for cytochrome c oxidase activity (and using thymus nuclei pre- 

 pared in nonaqueous solvents as a standard). These tests led to an upper limit of 10 

 per cent as the possible extent of contamination. It is true that some of the RNA in 

 these nuclear fractions may be cytoplasmic in origin, but there is a simple functional 

 test which, I believe, settles whether a component in this system is nuclear or not. 

 One can test for the effect of DNAase treatment on the labeling or synthesis of the 

 compound. If the uptake of radioactive precursors into the compound is impaired 

 when one removes the DNA from the nucleus, then it can be concluded that in all 

 probability such DNA dependence indicates nuclear localization. This is true for the 

 protein and ribonucleic acid fractions we have considered. Dr. Osawa's experiments on 

 nuclear RNA fractionation follow some of the fractionation procedures we have em- 

 ployed, but he has also substantially modified them, for example, by introducing a strep- 

 tomycin precipitation step. I am not familiar with the relative specific activities of the 

 RNA fractions he has prepared, but I think it can be shown that fractions he has con- 

 sidered to be derived from the nucleus are indeed sensitive to DNAase treatment. 



Dr. Vincent: Will other polyanions than polynucleotides replace in ATP synthesis 

 in the nucleus? 



Dr. Allfrey: Attempts to substitute other polyelectrolytes for DNA have led to 

 some very interesting and surprising results. After finding that polyadenylic acid would 

 restore amino acid uptake to nuclei depleted of their DNA, we tried to extend the 

 observations l(j include polyanions which were not polynucleotides. Among the com- 

 pounds tested were three molecular sizes of polyethylene sulfonate, heparin and chon- 



