B'tochemical Properties of the Isolated Nucleus 



V. ALLFREY and A. E. MIRSKY 



The Rockefeller Institute 

 New York, N. Y. 



THE EXPERIMENTS ABOUT TO BE DESCRIBED are Concerned with synthetic reac- 

 tions carried out by isolated cell nuclei — with particular emphasis on amino 

 acid uptake into nuclear protein, the incorporation of purine and pyrimidine 

 precursors in nuclear RNA, and nuclear ATP synthesis. Some of the experiments 

 will emphasize the role of deoxyribonucleic acid in these nuclear activities, others 

 will relate the state of the nucleus to methods of its isolation and its chemical 

 environment.^ 



In earlier work in this laboratory we had been concerned with the chemical 

 composition of cell nuclei isolated in different ways. Nuclei prepared in citric acid, 

 sucrose or saline solutions were compared with those isolated in nonaqueous media 

 by a modified Behrens' procedure (7, 8). The latter type of isolation was selected 

 as a 'standard' because it prevents water-soluble materials from moving between 

 nucleus and cytoplasm during the course of the isolation. Its success depends on 

 rapid freezing followed by lyophilization to remove water from the tissue, which 

 is then ground and fractionated in nonaqueous solvents such as cyclohexane and 

 carbon tetrachloride. The nucleus has a characteristic density range which often 

 permits its separation from other elements of the ground cell suspension by 

 differential centrifugation. The separation of the nuclei is facilitated by selecting 

 a medium of density lower than the nuclear specific gravity but higher than that 

 of possible cytoplasmic contaminants. Centrifugation then yields a nuclear sedi- 

 ment which can often be shown by chemical, immunological and enzymatic tests 

 to be essentially free of cytoplasmic contamination. 



Nuclei isolated in nonaqueous media constitute a 'standard' against which 

 nuclei prepared in aqueous media can be compared. For example, liver or kidney 

 nuclei prepared in sucrose or citric acid solutions were shown to have lost much 

 of their protein during isolation simply by comparing them with the correspond- 

 ing Behrens type nuclei (8, 10). 



However, when such comparisons were carried out in the case of calf thymus 

 nuclei, it was found that thymus nuclei isolated in isotonic sucrose solutions were 



1 The following abbreviations are used throughout: RNA, ribonucleic acid; ATP, adenosine- 

 triphosphate; DNA, deoxyribonucleic aciil; AMP, adenosinemonophosphate; ADP, adenosine- 

 diphosphate; TCA, trichloroacetic acid. 



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