Separation of native and denatured fractions from partially denatured pneumococcal DNA

After partial denaturation at temperatures which inactivate only some genetic markers, pneumococcal DNA is shown to consist of a mixture of intact and denatured molecules. Two boundaries are obtained in sedimentation velocity measurements, and two bands are resolved after centrifugation in a cesium chloride density-gradient. These correspond to untreated native DNA and fully denatured DNA. When DNA, partially denatured at 90·0°C, is eluted from a methylated albumin column, with salt solutions of increasing concentration, a native fraction and two denatured fractions are separated. The following properties are observed for the native fraction, as compared with untreated native DNA: the transforming activity of the preserved genetic markers is increased 1·5-fold; the intrinsic viscosity is high and unchanged (85 dl./g); the sedimentation coefficient is slightly lowered ( S 20,w=23s). It is concluded that the resolved native fraction is similar in molecular weight and configuration to intact native DNA, and is enriched in those molecules which are stable at 90·0°C. The two fractions of denatured DNA are similar in physical properties to fully denatured DNA, yet they are shown by rechromatography to be distinctly different fractions. They also differ in capacity to regain transforming activity under optimal conditions for renaturation; the early column fraction shows no increase in activity, and the later fraction can be reactivated as efficiently as the unfractionated denatured DNA. The combined results indicate that “all-or-none” denaturation occurs. There is no evidence for the formation of partially denatured molecules, and all of the molecules which bear a particular genetic marker either remain intact or are fully denatured. On this basis, as well as the molecular homogeneity indicated by the sharp sedimenting boundaries, it is proposed that chromosomal breakage during isolation of pneumococcal DNA occurs at predetermined, regularly spaced intervals.