330 Essays in Biochemistry 



cosity, ultracentrif ligation) revealed that the substance was in highly 

 polymerized form. 



The conclusion that the transforming principle is DNA was criticized 

 mainly on the following grounds: (1) The chemical methods were 

 not sensitive enough to exclude the presence of an impurity (protein?) 

 which might be responsible for the activity. (2) Even accepting the 

 evidence that the destruction of DNA by DNAase destroys the activ- 

 ity, it could still be that DNA is not active alone but merely in 

 combination with such a hypothetical protein so that the destruction 

 of either moiety results in inactivation. (3) The non-destruction of 

 activity by a few proteolytic enzymes, by itself, does not prove the 

 non-protein nature of the transforming principle, since proteins are 

 known which resist many proteolytic enzymes. 



The methods of purification and analysis have undergone consider- 

 able improvement in recent years in partial answer to the first point 

 of criticism. The transforming principle of H. influenzae has been 

 purified to the point where it contains less than 0.4% of protein, 

 immunologically active substance, or ribonucleic acid. 19 * 21 No loss of 

 biological activity occurred during the gradual removal of impurities. 

 The transforming principle of pneumococcus has now been purified to 

 where it contains less than 0.02% of protein. 22 The amount of DNA 

 sufficient to transform one cell of H. influenzae is of the order of 

 10 ~ 8 /xg. according to one estimate. 21 An impurity of 0.01% would 

 correspond to about six molecules of molecular weight 10 5 , or less than 

 one molecule of molecular weight 10 6 . Thus one approaches the situa- 

 tion where the probability of the transforming principle being protein 

 in nature can be excluded on purely analytical grounds. 



Other studies also offer further indications although not absolute 

 proof that the transforming principle is DNA. Crystalline pancreatic 

 DNAase in concentrations lower than 10~ 4 ^g./ml. produces 10-fold 

 decrease of activity within 20 minutes. 21 Upon heating, the tempera- 

 ture at which the transforming principle begins to lose its activity 

 (81°, 1 hour) is the same as the temperature at which the viscosity 

 of the bulk of the preparation begins to decrease. 21 Most known 

 proteins cannot withstand these heating conditions. The pH values 

 (on both acid and alkaline side) at which the activity begins to 

 decrease are again the same as the pH values at which the viscosity 

 begins to decrease. Thus, the active molecule of the transforming 

 principle seems to behave like the average molecule of DNA. 



In summary, the evidence favors the view that the transforming 

 principle is DNA ; no evidence to the contrary has ever been presented. 



