On the Nature of Recombinants Formed during Transformation in Hemophilus influenzae

Abstract

During the process of transformation in Hemophilus influenzae integration of donor DNA, i.e. the formation of recombinant DNA, involves the incorporation of single-stranded DNA. Evidence was obtained from cesium chloride density gradient centrifugation of DNA from donor-recipient complexes that integration was accompanied by the formation of hybrid DNA with a density intermediate with respect to heavy, 2H, 15N, donor and light, 1H, 4N recipient DNA. On denaturation the position of the heavy donor DNA moved closer to, but not all the way toward, the density position of the original donor DNA. In addition to supporting the idea of single-stranded incorporation, this evidence suggested that the integrated donor DNA was covalently linked to light recipient DNA. The DNA was taken up in the double-stranded form and no detectable amounts of denatured DNA could be found during the transformation process. However, during the process of integration an amount of donor atoms, equivalent to the amount of hybrid DNA formed, appeared in recipient DNA, and indicated that while one strand of DNA was integrated the other was broken down and resynthesized. The density of the hybrid DNA, as well as rebanding of denatured hybrid, indicated that the size of the integrated piece of DNA was large, approximately 6 x 106 daltons.