ISOLATION AND COMPOSITION OF NUCLEI AND NUCLEOLI 153 



media by Stern and Mirsky^"^ has indicated that most of the protein is 

 retained by thymus nuclei isolated in sucrose solutions, perhaps because of 

 their high DXA content, whereas certain soluble enzymes (and hence pro- 

 tein) were extensively washed out of calf liver nuclei during isolation in 

 aqueous media. The liver cell nuclei have a much lower DXA content than 

 the thymus nuclei. A paper by Alfert and Geschwind^^** describes the use of 

 fast green in a basic medium as a means to measure histone (or other 

 equally basic proteins) in cell nuclei histochemically. 



An inner "chromatic" nuclear membrane has been described by Bren- 

 j^gj. 209 which is said to be composed of segments of chromosomes applied 

 to the inside of the achromatic membrane. The book by A. Hughes-^" con- 

 tains much information concerning cell nuclei. 



In the writer's laboratory it has been found that 15 % sucrose containing 

 0.005 M calcium chloride is an especially favorable medium for isolating 

 nuclei from cells such as certain tumor cells which are difficult to break. 

 The nuclei are isolated by differential centrifugation without overlaying on 

 stronger sucrose solution. Fairly satisfactory preparations of nuclei from 

 Walker tumor 256 have been isolated in this manner, using the new homoge- 

 nizer, and this has previously been impossible without the use of strong 

 citric acid. It has also been found that liver cell nuclei isolated in sucrose- 

 calcium chloride solutions contain a considerably higher DNA content than 

 nuclei isolated at pH 6.0 in sucrose solutions, and hence apparently have 

 lost more protein than the latter, probably because of the higher pH at 

 which the former are isolated. 



In addition, it has been ascertained that the enzyme (or enzymes) re- 

 sponsible for loss of the ability of isolated nuclei to form gels in salt solutions 

 or alkali lies in the mitochondrial fraction, and that this enzyme is trans- 

 ferred to the nuclei if the latter are isolated in such a manner that mito- 

 chondria are ruptured. If mitochondria are discarded while still intact, the 

 enzyme is lost and the nuclei will gel. Thus nuclei isolated at pH 6.0 in 

 sucrose solutions without the addition of calcium chloride will gel if the 

 new homogenizer is used for rupturing the cells, so that the mitochondria 

 are not seriously damaged. Nuclei isolated by the latest procedure of Schnei- 

 der and Hogeboom, which in our experience did not form gels, probably 

 would do so if our new homogenizer were used to break the cells. 



2" H. Stern and A. E. Mirsky, J. Gen. Physiol. 37, 177 (1953). 

 "8 M. Alfert and I. Geschwind, Proc. Natl. Acad. Sci. U.S. 39, 991 (1953). 

 "s S. Brenner, Exptl. Cell Research 5, 257 (1953). 



21" A. Hughes, "The Mitotic Cycle." Academic Press, New York, and Butterworth's 

 Scientific Publications, London (1952). 



