Sedimentation studies of giant DNA molecules present in unsheared whole‐cell lysates of D. melanogaster cells

Abstract

AbstractWe have used band sedimentation in shallow density gradients of CsCl in the preparative centrifuge to analyze the distribution of sedimentation coefficients present in tritium labelled DNA from D. melanogaster cells. The cells were lysed according to the method of Kavenoff and Zimm to preserve very high molecular weight DNA. Sedimentation measurements have been carried out as a function of speed of centrifugation. The resulting distribution functions have been interpreted with the aid of the Zimm‐Schumaker equation for the speed dependence of the sedimentation coefficient of very high molecular weight DNA. Low‐speed centrifugation (3000 rpm) indicates that DNA molecules from the lysate are evenly distributed over values of S20,w from 0 to 514S. This distribution is very sensitive to changes in speed of centrifugation and is transformed into a bimodal distribution at 12,080 rpm. Analysis of this transformation allows us to postulate that perhaps 55% of the DNA in the lysate may have molecular weights in excess of 40 × 109 g/mol. Some of these molecules may also possess a variety of configurations including partially replicated branched structures.