When normal purified DNA samples from calf thymus, Bacillus subtilis, Escherichia coli or Hemophilus influenzae are denatured, either by heat or by strong alkali, a few per cent of the DNA molecules regain their native structure on return to a non-denaturing environment. In the case of B. subtilis, the native-like character of this “reversibly denaturable” fraction has been demonstrated by measurements of its specific activity in transformation, its buoyant density in a CsCl density gradient, and the sharp hyperchromic change characteristic of its optical melting profile.

A detailed examination shows that this generally occurring, reversibly denaturable DNA fraction behaves identically to nitrous acid-induced cross-linked DNA under a wide variety of test conditions. The only exception is that measurements of sedimentation coefficients for melted cross-linked DNA suggest that the “naturally occurring” cross-link differs in being located at an end of a double-helical DNA molecule. It is concluded that these molecules contain a discrete covalent type of cross-strand linkage, one approximately for every 4.6 × 10⁸ daltons of B. subtilis DNA.

As a byproduct of these studies, the pH of maximum chemical stability of transforming DNA at 100 °C has been determined as approximately pH 11, with pH 13 nearly equivalent to pH 7 as judged by maintenance of the biological activity of cross-linked molecules. In addition, what appears to be a remarkable sensitivity to shear of DNA single strands in alkali is described, and a denaturation method designed to avoid the resulting degradation of cross-linked DNA is presented.