Fractionation of Pneumococcal Dna following Selective Heat Denaturation: Enrichment of Transforming Activity for Aminopterin Resistance.

The different genetic markers of pneumococcal transforming DNA are inactivated in solution at characteristic temperatures, analogous to melting points. These temperatures are spaced sufficiently to permit inactivation of the lower melting markers while the others are preserved intact.' Such partially inactivated DNA has been considered to be a mixture of native and denatured molecules.1 If this is so, it should be possible to separate the native and denatured fractions because of their different physical properties and thus obtain a native fraction which is enriched for those molecules carrying the surviving markers. The experiments described below show, in fact, that a native DNA fraction is recoverable from selectively heat-inactivated DNA, and that both the specific and plateau transforming activities of the preserved markers are considerably increased. Results and Discussion.-Elution of native and denatured DNA from methylated albumin columns: Heat denaturation brings about a change in a number of the physical properties of native DNA which can be interpreted as arising from the increased flexibility of the molecules due to disruption of the native hydrogenbonded structure. Of interest for this work is the fact that denatured DNA is more tightly bound than native to weakly basic adsorbents, such as methylated albumin-coated kieselguhr (MAK). The stronger binding of denatured DNA to MAK columns was first reported by Mandell and Hershey2 who used continuous salt gradient elution of T2 phage DNA. Sueoka and Cheng3 described stepwise elution of E. coli DNA from MAK columns and recovered native DNA from a prepared mixture of native and denatured. Stepwise salt elution was selected for the fractionation of partially denatured DNA, since it provides several well-defined fractions of moderately high concentration. The greater resolving power of continuous gradient elution is not required when fraction separation is to be based upon native versus denatured DNA. To illustrate this point, a pair of stepwise elution profiles with native and completely heat-denatured pneumococcal DNA are shown in Figure 1. With this particular column material, the major portion of the native DNA was eluted at 0.73 M NaCl, while denatured DNA was not eluted until a salt concentration of 0.9 M was reached. Thus, no denatured DNA is released at a salt concentration just sufficient to elute native DNA. No differences in the ratios of specific transforming activities for any of the genetic markers could be detected in the MAK column fractions obtained from native DNA. Different preparations of 1\IAK varied somewhat with respect both to the absolute molarity of NaCl necessary to elute the DNA fractions and to the total recovery of material. However, the salt concentration required to elute denatured DNA was always about 0.2 molarity units higher than that which eluted native