Eur. J. Biochem. 84, 189-195 (1978) Restriction Enzyme Analysis of Satellite DNA Components from the Bovine Genome Helena KOPECKA, Gabriel MACAYA, Jordi CORTADAS, Jean-Paul THI ERY, and Giorgio BERNARD1 Laboratoire de Gtnetique Moleculaire, Institut de Recherche en Biologie MolCculaire, Paris (Received July 2X/November 21, 1977) A restriction enzyme analysis was performed on satellite DNA components, isolated, as described in the preceding paper, from the bovine genome by a combination of Cs2S04/BAMD and CszS04/Ag' density gradient centrifugation. Such an analysis has led to the unambiguous identifica- tion of eight satellite DNA components and to new information on their repeat units; this indicates that identical repeat lengths are shared by them, a fact strongly suggesting a common origin. Investigations from our laboratory [l - 61, as well as from a number of other laboratories, have shown that buoyant density gradient centrifugation techni- ques are an extremely powerful tool for the analysis of mammalian genomes. In particular, the combina- tion of Cs2S04/BAMD and Cs2S04/Agf density gradients has recently allowed us to prepare eight satellite and 11 minor DNA components from the bovine genome [5,6]. Several of these dG + dC-rich DNA components exhibit the same buoyant density in CsCl and the same behavior in Cs2S04/BAMD or CszSO4/Ag+ density gradients. This encouraged us to use another method, namely the electrophoretic analysis of the fragments produced by restriction enzymes, to assess the identity of the DNA compo- nents. We report here the results obtained so far on the satellite components. Although our initial aim in this investigation was simply to identify in an un- ambiguous way different satellite components, our data have provided interesting new information on the repeat units of some satellites and show that identical repeat lengths are found in several of them. This finding supports the idea of a common origin for the satellite DNA components present in a given species. Ahhreviutions. BAMD, 3,6-bis(acetato-mercurimethyl)dioxane. Satellite DNA components are indicated by their buoyant density in CsCl density gradient, as determined in the preceding paper [6]. Restriction enzymes are indicated according to Smith and Nathans ~71. Enzymes. Restriction endonucleases (EC 3.1.4. -). MATERIALS AND METHODS DNA Samples The DNA samples investigated here were fractions obtained from the combined Cs2S04/BAMD, CszS04/ Ag + density gradient centrifugations previously de- scribed [5,6]. The fractions are identified by their buoyant density in CsCl and their code number [6]. Restrict ion Enzyme Digestions Restriction endonucleases EcoRI, HueIII, Hhal were used; Hue111 and HhaI were prepared according to a method developed in our laboratory [8]; EcoRI was prepared following the method of Yoshimori [9]. DNA samples (5- 15 pg) were digested to complete- ness using conditions already described [lo]. Gel electrophoresis, ethidium bromide staining, photog- raphy of the gel, and scanning of the negative pictures were done as in previous work [lo]. Band stoichio- metry was estimated by integration of the areas under the peaks of the scans. The molecular weight of the fragments obtained by electrophoresis of digested DNA samples was deter- mined using as a secondary standard the fragments of total calf thymus DNA digested with the enzyme used; this standard was calibrated against primary standards formed either by EcoRI fragments of 1"- phage DNA or by Hind11 + 111 or HaeIII fragments of SV40 DNA [lo]. RF values were normalized ac- cording to Prune11 et al. [lo].