50 S. S. COHEN 



precipitated in isotonic saline to give threads of histone-nucleate. These 

 probably should be regarded as artifacts in which the original spatial 

 relations of histone and DNA no longer exist (Cohen, 1945); in addition, 

 non-histone protein can thus be introduced mto the nucleoprotein when 

 the latter is taken from the dissociated state in M NaCl to 0.11 M 

 NaCl. 



DNA and histone comprise a large proportion of chromosomal substance, 

 e.g., about 80 % of calf thymus chromosomes. After extraction of nucleo- 

 histone with salt, the remainder of the chromosome exists in the form of 

 small coiled threads, which stain red in methyl green-pyronine and consist 

 of tryptophan-containing protein or protems and about 10 % RNA (Allfrey 

 et al., 1955b). The proportion of the tryptophan-containing protein in 

 chromosomes is stated to vary directly as the quantity of cytoplasm 

 in the cell. For instance, placmg DNA content as a constant, Hver 

 chromosomes contain four times as much residual protein as thymus 

 chromosomes. 



The careful analysis of chromosome structure in fixed cells by digestion 

 wdth enzymes of known specificity followed by staining has been described 

 by Kaufmann et al. (1950). Although previous investigators had stressed the 

 solubilization of cliromosomes by trypsin, attributing the integrity of the 

 chromosome to a particular protein continuum, these workers have shown 

 that the subsequent washes, after trypsin action, served to dissociate the 

 degradation products. Although DNAase removed the deeply staining 

 basoj)hihc substance from the bands of certain chromosomes, this did not 

 dissolve the chromosome. However, successive treatments with proteases 

 and nucleases do dismtegrate the chromosomes and it has been concluded 

 that the chromosome represents an integrated fabric in v/hich no smgle 

 protein or nucleic acid may be regarded as the primary structural 

 component. 



However, in studies on isolated liver chromosomes, it has been shown that 

 after extraction of histone at pH 2.8, which leaves the DNA on the stiU 

 intact structure, treatment with DNAase does disrupt the chromosome into 

 minute coiled threads of residual protein (Mirsky and Eis, 1951). It was 

 concluded, contrary to the conclusions of Kaufmann et al. (1950), that the 

 residual protein forms the fmidamental thread of the chromosome, whose 

 gross configuration depends on the association of this protein and DNA. It 

 was proposed that in native chromosomes DNA is bound to both liistone and 

 other protein structures. 



One might expect that the residual protein remaining after removal of 

 nucleic acid and histone consists of many proteins. Unfortunately, this 

 fraction has been difficult to obtain in amounts permitting fractionation and 

 characterization. Nor have immunochemical techniques proven useful since 



