BIOCHEMICAL PROPERTIES OF THE ISOLATED NUCLEUS 187 



the equivalent of the nonaqueous nuclei in many respects. The DNA analyses, 

 protein composition and enzymatic constitution ot the two preparations were 

 strikingly in agreement (24). Once it had been established that thymus nuclei 

 could be isolated in sucrose solutions without leaching out their water-soluble 

 components, it was decided to test whether such nuclei had maintained their 

 capacity for complex synthetic reactions. 



The isolation procedure employed (6) is a modification of that introduced by 

 R. M. Schneider and M. L. Petermann (22). The fresh tissue is minced with 

 scissors and then homogenized in a low-speed blendor in 0.25 m sucrose-0.003 m 

 CaClo solution. The homogenate is filtered through gauze antl through double- 

 napped flannelette to remove clumps of tissue and fiber. The nuclei are separated 

 by differential centrifugation and washed with sucrose-calcium chloride solution. 



Under the light microscope, stained or unstained, such thymus nuclei seem 

 remarkably clean. Contamination consists of a few whole cells, occasional red 

 cells and some cytoplasm which adheres to some of the nuclei. The extent of 

 cytoplasmic contamination can be estimated chemically (e.g. by nucleic acid 

 analyses or tests for cytochrome c oxidase activity), and is found to be below 

 10 per cent. The number of whole cells cannot be estimated with accuracy in the 

 light microscope because of the difficulty in distinguishing between free nuclei 

 and very small thymocytes, but electron microscopy made it evident that whole cell 

 contamination is also very small. For example, in two preparations examined we 

 observed less than 50 cells per thousand nuclei. (We are indebted to Drs. M. 

 Watson and G. Palade of the Rockefeller Institute for their cooperation in these 

 tests.) In a recent paper, Ficq and Errera(ii) report that nuclear fractions 

 prepared by our procedure contain 3 per cent intact cells and that 8 per cent of the 

 nuclei contain a small amount of adhering cytoplasm. In addition to these micro- 

 scopic tests for purity of the nuclei there is other convincing evidence for the 

 absence of appreciable whole cell contamination. This will be given below in 

 connection with studies of deoxyribonuclease action on free nuclei. 



AMINO ACID INCOPORATION INTO PROTEINS OF ISOLATED NUCLEI 



When thymus nuclei are suspended in a buffered sucrose medium and in- 

 cubated at 37°C in the presence of isotopically labeled amino acids, there is a 

 rapid and considerable incorporation of the isotope into the proteins of the nu- 

 cleus. Figure I shows the time course of incorporation of lysine, alanine antl 

 glycine. Curves similar to that shown for alanine or lysine are obtained with 

 C"-leucine, phenylalanine, tryptophan and \aline. In these curves, and in most 

 of the experiments to be described below, the uptake was followed by measuring 

 the radioactivity of the total mixed proteins ot the nucleus. The protein was pre- 

 pared by first extensively washing the nuclei with trichloroacetic acid (TCA), re- 

 moving the nucleic acids with hot T("A and finally removing the lipids with warm 



