18 Essays in Biochemistry 



tion of the gel with NaCl solutions of increasing concentration. 13 

 When freed of protein, corresponding DNA fractions are obtained 

 which show decreasing proportions of guanine and cytosine and in- 

 creasing amounts of adenine and thymine. (Similar fractions may 

 be obtained " by successive extractions of the gels with salt solutions 

 of constant concentration.) An analysis of the 5-methylcytosine con- 

 tents reveals a disproportionate distribution of this pyrimidine among 

 the fractions, and this 13 constitutes newer, but striking, evidence of 

 the heterogeneity of DNA. 



Despite the fact that twenty-one preparations of calf-thymus DNA 

 afforded by several procedures showed 13 very similar compositions, 

 this similarity obscured the large differences in the constitution of the 

 components of the mixture which is known as calf-thymus DNA. 



Almost simultaneously, work was described 15 in which histone (from 

 calf nucleohistone) was immobilized on columns of kieselghur. Solu- 

 tions of DNA from either calf thymus, Escherichia coli, or human white 

 blood cells were placed upon such columns and then eluted with NaCl 

 solutions of increasing concentration. Convincing evidence of the 

 heterogeneous character of these DNA preparations was obtained from 

 an examination of the fractions in the eluates. A gradation in the 

 base composition was also observed in several fractions from calf- 

 thymus DNA. 



Although it furnished a clue to the heterogeneous nature of DNA, 

 the fractionation procedure which yielded the soluble and insoluble 

 fractions DNAi and DNA 2 could, at best, be described as crude. Better 

 methods were therefore sought. If DNA were indeed composed of 

 different individuals, a DNA preparation should consist, in neutral 

 solution, of a number of polyelectrolytes anionic in character due to 

 phosphate dissociation. Accordingly, columns of anion exchangers 

 were employed in attempts * to effect a separation of individual poly- 

 anions, or groups thereof. Initial attempts with commercially avail- 

 able strong and weak base anion exchanger resins furnished a few 

 encouraging results. In one experiment with the chloride form of the 

 Amberlite IR-4B, over one hundred chromatographic fractions were 

 obtained with calf-thymus DNA. But the experiments were difficult 

 to repeat, and the various resins showed many undesirable properties. 



The success in the fractionation of proteins 16 by means of anion 

 (and cation) exchangers prepared from cellulose prompted an investi- 

 gation of their suitability for the fractionation of DNA. A cation 



* Unpublished experiments, with J. R. Fresco and H. R. Rosenkranz. 



