36 S. S. COHEN 



Several reactions, such, as Millon's reaction for tlie phenolic groups of 

 tjTOsine, or a modified Sakaguchi reaction for argmine, are also useful in 

 detecting protein. A simple staining procedure with the acid dye, fast green, 

 has been described for basic proteins, such as histones and protamines 

 (Alfert and Geschwind, 1953), This reaction is almost specific for these 

 nuclear proteins, since other proteins with high isoelectric points, e.g., 

 cytochrome c, which also stain, do not occur in cells in significant concentra- 

 tion. The procedure requires the prior removal of DNA. Under the conditions 

 described, nucleoli faU to stain. Only the chromosomes take this stain, a 

 result in accord with other results on the distribution of the histones. These 

 histones are most acidophilic in dividing cells and it has been suggested 

 that interphase nuclei increase their content of a nonhistone protein which 

 combines with the chromosomes in such a way as to restrict the combination 

 of histone with fast green (Alfert, 1957). 



The quantitation of these staining reactions for nucleic acids and proteins 

 has been accomphshed through spectrophotometric methods appHed to intact 

 cells, complete nuclei, or extracts. A detailed discussion of these reactions is 

 given by Swift (1955). 



3. The Isolation of Nuclei 



The preparation of nuclei by chemical methods was first accomphshed by 

 Miescher (1897), who isolated the nuclei of pus ceUs after digesting away the 

 cytoplasm with pepsin-HCl. Miescher and Kossel (1928) also isolated the 

 nuclei of fish sperm, and many workers undertook to isolate the nuclei of bird 

 erythrocytes. Nuclei have been liberated from the latter source by such 

 methods as freezing and thawing, or by means of hemolytic agents, such as 

 saponm or lysolecithin. A pecuharly suitable biological source has occasionally 

 been discovered as a source of nuclei; for instance, before cell wall formation 

 the coconut endosperm contains large numbers of syncytial nuclei. These 

 nuclei may be hberated readily and isolated. Cutter and associates (1952) 

 record the isolation of 0.13 gm. of undamaged nuclei from a single coconut of 

 2 kg. and recommend their procedure for certain other plant nuclei. 



Behrens (1932) developed an important method for the separation of 

 nuclei that has been applied to plant and animal tissue. Both this technique 

 and the Feulgen stain were used to demonstrate DNA in plant nuclei, thereby 

 disposing of the old notion that only animal cells contained this nucleic acid 

 (Bounce, 1955). The Behrens procedure consists of drying the frozen tissue, 

 and grinding it to a very fine powder. The anhydrous nuclei are laboriously 

 obtained by repeated centrifugation from mixtures of benzene and carbon 

 tetrachloride of adjusted density. The flotation process therefore separates 

 cytoplasmic constituents as a function of their anhydrous densities. This 

 procedure has been used to avoid the loss of water-soluble autolyzable 



