152 ALEXANDER L. DOUNCE 



IV. Addendum 



The major part of the discussion of the chemistry of cell nuclei and nu- 

 cleoli in the preceding review has dealt with resting or interphase nuclei. 

 A treatment of the chemistry of cell nuclei would hardly seem complete, 

 however, without mention of the rather remarkable isolation of the mitotic 

 apparatus of the sea urchin egg by Mazia and Dan,-^^ although this appa- 

 ratus may actually be derived from the cytoplasm rather than from the 

 nucleus. 



The method of isolation is briefly as follows. Before fertilization, the ferti- 

 lization membranes of the eggs were removed by treatment with trypsin 

 and chymotrypsin. The eggs were then fertilized and, when they had de- 

 veloped to the desired stage of the first cell division, they were concentrated 

 by centrifugation and fixed in 30 % ethanol at — 10°. 



A mechanical isolation of the mitotic apparatus was achieved by running 

 the suspension of cells through a No. 25 hypodermic needle, and subse- 

 quently separating the liberated apparatus by differential centrifugation 

 in water. This procedure, however, yielded a product somewhat contami- 

 nated with cytoplasm, and a better method was found to be treatment of 

 the cells with H2O2 followed by an anionic detergent (Duponal D). In this 

 procedure, the liberated mitotic apparatus was centrifuged down and puri- 

 fied by recentrifugation in water. The role of the hydrogen peroxide was to 

 render the protein of the mitotic apparatus resistant to the action of the 

 detergent, possibly through the formation of — ^S — ^S — linkages from — -SH 

 groups. 



The isolated mitotic apparatus consisted of the mitotic spindle, the as- 

 ters, and the centrosomes. In some cases chromosomes were also present, 

 but the absorption spectrum in the ultraviolet region failed to show the 

 presence of any nucleic acid. This is not surprising, however, since the de- 

 tergent very likely rendered the DNA soluble; but it was calculated that 

 even if no DNA had been lost, too little could have been present -relative 

 to the amount of protein to affect the absorption spectrum appreciably. 

 The isolated mitotic apparatus appeared to consist principally of protein, 

 which was found to be quite homogeneous, judging from its behavior in the 

 ultracentrifuge. This protein was soluble in alkali and acid but precipitated 

 near pH 6. 



This very interesting piece of work, which has already demonstrated the 

 chemical nature of the mitotic apparatus, may eventually prove to be of 

 considerable importance in elucidating the mechanism of the mitotic di- 

 vision of cells. 



Further study of nuclear enzymes isolated in sucrose and nonaqueous 



206D. Mazia and K. Dan, Proc. Natl. Acad. Sci. U.S. 38, 826 (1952). 



