Sedimentation equilibrium studies on intact and fragmented bacteriophage DNA

Abstract

The molecular weight calculated from the variance of the gaussian distribution of DNA molecules at equilibrium in a CsCl density gradient appears to be one-half the best estimates of the true molecular weight. This is true for unbroken DNA molecules from T2, T4, T5, P22 and T7 bacteriophages. It follows that, when this factor is taken into account, the calculated molecular weight is a good measure of actual molecular weight for unbroken genetically uniform molecules. This uniform proportionality between calculated molecular weight and the best estimates of the true molecular weight suggests that there is no detectable density heterogeneity in these unbroken molecules, or that there is a contribution by density heterogeneity which increases precisely as 1/ M for these molecules of different origins. This is a rather special requirement which renders this alternative unlikely. Shear-produced fragments of T2 DNA give broader bands than would be expected on the basis of whole molecules of comparable size. The calculated molecular weights of these fragments are approximately one-fourth the best estimates of the true molecular weights. It turns out that the observations are just what one would expect if some of the fragments were slightly richer in GC than others. The magnitude of this compositional heterogeneity, its dependence on fragment size and its possible significance are discussed. The formaldehyde-stabilized polynucleotide chains produced by denaturing the fragments have band-width molecular weights which are two-thirds those of the fragments from which they were derived. This is exactly what would be expected if the two chains of the duplex had the same density and the molecular weight is halved when the duplex fragment is denatured and separated into polynucleotide chains.