118 ALEXANDER L. DOUNCE 



c. Choice of Method for Study of Nucleic Acids 



For work with DNA, it is very satisfactory to isolate nuclei at pH 4.0 

 or lower with dilute citric acid. The DNA can then be isolated by the use of 

 detergent*^ if desired, prior to analysis. However, it should be kept in mind 

 that severe losses of protein may occur if nuclei are isolated in aqueous 

 media, and hence the Behrens-type nuclei would appear to be the best in 

 studies of the percentage of DNA present in the cell nucleus. This statement 

 must be accepted with some caution, however, since the purity of the 

 Behrens-type nuclei may not be as high as that of nuclei isolated in aqueous 

 media. 



For estimating the amount of DNA per nucleus, nuclei isolated at pH 

 4.0* or lower^°-'^-^ can be used, since DNA is not lost from such nuclei. 

 Nuclei isolated at higher pH values also have been used,^ but there prob- 

 ably is no advantage in this procedure. 



It is not yet known what type of nuclei are best for studies of PNA. Work 

 has been done on PNA turnover using nuclei prepared in strong citric acid, 

 but there is a suspicion that some PNA may be lost under these conditions. 

 Such turnover studies therefore might not reflect the turnover of total nu- 

 clear PNA. 



Nuclei prepared according to Hogeboom et air- from rat liver have a 

 relatively high PNA content. Behrens-type nuclei may have a high^* or a 

 low^* PNA content. Thus a low PNA content is not necessarily an indication 

 of nuclear purity, and it is possible that the amount of PNA per nucleus 

 may vary considerably. Tentatively, it is suggested that nuclei isolated near 

 pH 4.0 with dilute citric acid may be reasonably suitable for PNA studies. 

 The calcium chloride-sucrose nuclei isolated according to the reviewer's 

 procedure already described might also be satisfactory if something other 

 than gum arable were used in the final steps of washing, or if the tissue could 

 be perfused to render unnecessary the washing in gum arable. 



d. Choice of Method for Studies of Proteins 



It is probable that no single method for isolating cell nuclei is suitable for 

 studies of all of the protein constituents of the nuclei. If quantitative esti- 

 mations of total nuclear protein are to be made, the Behrens-type of pro- 

 cedure is no doubt the best for isolating the nuclei, provided that some un- 

 certainty as to the purity of the nuclei is taken into account. In the Behrens 

 type of isolation, no protein can be lost or gained by the nuclei except pos- 

 sibly by diffusion during the interval between the death of the animal and 

 the freezing of the livers, but the possibility of an appreciable exchange 

 during this period seems remote. 



Some of the nuclear globulins, at least, can be easily extracted from 



" E. R. M. Kay, N. S. Simmons, and A. L. Dounce, J. Am. Chem. Soc. 74, 1724 (1952). 



