Sedimentation Equilibrium in a Density Gradient

Sedimentation equilibrium in a density gradient has recently been developed into a reliable method for the determination of molecular weights of homogeneous DNA samples. The procedure and calibrations required for this method are outlined in this paper. In 1957, Meselson, Stahl and Vinograd' introduced a remarkable analytical technique for studying the properties of DNA and viruses. Sedimentation equilibrium in a density gradient has since that time been a most important tool of the molecular biologist, playing a role in many of the definitive experiments of the past decade. The resolution and the experimental accuracy which it provides in the determination of buoyant density and amount of DNA in a band have placed this method in a singularly important role for molecular biology. Before discussing some of the detailed features of this method, I would like to list just a small number of the important experiments which have used this technique. Meselson and Stahl2 proved in 1957 that the replication of DNA in E. coli was semi-conservative by following the history of density labelled ('5N) DNA in cells which had been transferred to light medium. This classical experiment was apparently the major motivational factor in the discovery of the density gradient method. The method provided something unique in labelling techniques in that it made possible the physical separation of isotopically labelled material from unlabelled material. This feature of density gradient sedimentation has made numerous similar transfer experiments on other cell components possible as well. Weigle, Meselson and Paigen3 in 1959 showed that a whole class of 2transducing phages have different densities indicating their different DNA content. The controversy between the copy choice and the breakage and rejoin mechanisms of crossing over was resolved in 1961 when it was demonstrated by Meselson and Weigle4 and by Kellenberger, Zichichi and Weigle5 that some phage which arose from a genetic cross contained primarily parental DNA. This showed that DNA replication was not required in large amounts for a cross and that mechanism was likely to be breakage and rejoin. The first demonstration of DNA renaturation and hybridization was made using density gradient sedimentation equilibrium by Schildkraut, Marmur 513 JOHN E. HEARST and CARL W. SCHMID and Doty6 in 1961. This phenomenon is of crucial importance today for those of us who are studying the DNA of the higher organism. Brenner, Jacob and Meselson7 in 1961 presented definitive evidence for rapidly turning over messenger RNA in the T2 phage infection process and for the absence of the production of new ribosomes in this process. Sinsheimer, Starmen, Nagler and Guthrie8 a year later proved the existence of the replicative form of 4iX 174 DNA during the infection process, again using sedimentation equilibrium in a density gradient. More recently, Birnsteil, Speirs, Purdom and Jones9 in 1968 isolated ribosomal DNA from X. laevis and showed it to have G—C rich spacers. This DNA was found in a heavy satellite in a CsCl gradient. Finally, the pure lac Operon DNA was isolated by Shapiro, Machattie, Eron, Ihler, Ippen and Beckwith1° in 1969. Most of these experiments would not have been possible without the technique of density gradient sedimentation equilibrium. The more formal aspects of the development of the density gradient technique as a reliable analytical tool from the point of view of the physical chemist are the topic of this paper. This development is largely the result of the work of Hearst and Vinograd11 and of Hearst, lift and Vinograd'2 in 1961. The use of the bandwidth as a measure of molecular weight has been fraught with problems and therefore subject to much commentary. These problems have been finally resolved, the two major sources of difficulty in the past being: (1) the inadequacies of the photographic record, which has been cured by the double beam optics and the photoelectric scanner now available for the ultracentrifuge, and (2) the failure to extrapolate to zero polymer concentrations, correcting for virial effects. This last factor was demonstrated by Schmid and Hearst13' 14 and with its inclusion and a recalibration of the necessary density gradients, some very good numbers for the molecular weights of homogeneous DNAs have been obtained. The description of the equilibrium distribution of DNA in a density gradient is readily calculated from thermodynamics. Since the choice of neutral components in the three-component electrolyte solution is arbitrary we arbitrarily choose Cs DNA as our neutral macromolecular component. This choice in no way influences the conclusions regarding molecular weight or distribution. Although thermodynamics is more general than the following descriptive approach, it is useful to visualize a neutral Cs DNA molecule with v 'DNA + gv2 v EDNA + 9'H2o 1+g 1+g'