Eur. J. Biochem. 76, 13-19 (1977) An Analysis of the Bovine Genome by Density Gradient Centrifugation : Fractionation in Cs2 S0~/3,6-Bis(acetatomercurimethyl)dioxane Density Gradient Jordi CORTADAS, Gabriel MACAYA, and Giorgio BERNARD1 Laboratoire de Gtnetique Moltculaire, Institut de Recherche en Biologie Moleculaire, Paris (Received January 6, 1977) The fractionation of calf thymus DNA by centrifugation in density gradients of CszS04/BAMD, where BAMD = 3,6-bis(acetatomercurimethyl)dioxane, is described. A large-scale separation of (dG + dC)-rich DNA fractions has been obtained, allowing the relative amounts of minor and satellite components in the bovine genome to be precisely assessed. Density gradient centrifugation is one among several possible experimental approaches to the study of sequence heterogeneity in DNA. As such it is potentially very useful for analyzing and fractionating eukaryotic DNA, particularly when applied to DNA complexes with sequence-specific ligands. Centrifuga- tion in CszS04/Ag+ density gradients, for instance, has proved to be a very powerful tool not only to separate simple-sequence ('satellite') DNA from main- band DNA [I], but also to fractionate the latter [2-41. In the present series of investigations we have pushed much further our previous analysis [2,3] of the bovine genome, by using two density gradient centrifugation steps. In a first step, described in this paper, we fractionated calf DNA in Cs2S04/BAMD density gradients. BAMD, or 3,6-bis(acetato-mer- curimethyl) dioxane, preferentially interacts with (dA+dT)-rich DNA [5] and increases its density (H. Biinemann, personal communication) ; this new fractionation technique is particularly suited for the large-scale purification of (dG +dC)-rich DNA com- ponents, which are present in small amounts in eukaryotic genomes. The fractions obtained in the first step were analyzed by analytical centrifugation in CsCl in order to assess the buoyant densities and the relative amounts of the DNA components they contained. The second step, to be presented in a future paper, consisted of applying the Cs2S04/Ag+ density gradient centrifugation technique to the frac- tions obtained from the CszS04/BAMD step, in order to prepare the DNA components recognized here. Abbreviations. BAMD, 3,6-bis(acetato-mercurimethyl)dioxane; rf, molar ratio of BAMD to DNA phosphate. MATERIALS AND METHODS DNA Preparation Calf (Bos taurus) thymus DNA was preparation CTR2, already described elsewhere [6], and charac- terized by Filipski et al. [2]. The sedimentation co- efficient of this preparation was re-determined at different concentrations by band centrifugation using split-beam optics and found to be equal to 26 & 2 S; this value is higher than that (22.6 S) previously reported [2] and corresponds to a molecular weight of 13.106, if the s vs M, relationship of Eigner and Doty [7] is used. DNA ' BAMD Complex Formation The complex between DNA and 3,6-bis(acetato- mercurimethy1)dioxane (BAMD) was formed by mix- ing at room temperature, under vigorous mechanical stirring, equal volumes of DNA and BAMD solutions in 0.1 M Na2S04, 0.005 M Na2B407, pH 9.2 [5], of such concentrations as to obtain the desired final DNA concentration and BAMDIDNA-P molar ratio (o). The DNA-P concentration was calculated from the absorbance at 260 nm (Axo), using a mean molar absorption coefficient of 6600 M-' cm-'. When the calf thymus DNA preparation was used at high con- centrations, precipitation due to local concentration effects occurred upon complex formation ; to avoid this, the complex was formed in dilute solution and then concentrated in a rotary evaporator, under re- duced pressure. The synthesis of BAMD was de- scribed by Edsall et al. [8].