Volume 167, number 1 FEBS 1222 February 1984 Probing with DNase I of nucleosomal core particles assembled in vitro in the presence of polyglutamic acid Jacques D. Retief, B. Trevor Sewell, H. Johann Greyling, Sylva Schwager and Claus von Holt* Chromatin Research Unit, Council for Scientzfic and Industrial Research, Department of Biochemistry, University of Cape Town, Private Bag Rondebosch 7700, Cape Town, Republic of South Africa Received 7 November 1983 DNase I Polyglutamic acid Core particle assembly 1. INTRODUCTION Reconstitution of chromatin fibers in vitro from histones and random or unique DNA will become of increasing importance for the execution of structure-function studies on the eukaryotic genome. The reconstitution of nucleosome core particles at low ionic strength in the presence of polyanions introduced in [l] offers potentially great advantages over other methods proposed (review [2]) because in the former method ill- defined tissue extracts or high concentrations of urea and salts are not required to achieve assembly. The properties of the 145 base pair (bp) DNA- histone octamer particle, assembled in [I], such as the sedimentation coefficient of 11, the ability to introduce supercoils in SV40 DNA and the electron microscopic appearance resemble very closely the natural core particle. We also regard it as necessary to establish the DNase I susceptibility of a reconstituted particle. Not only would a 10 bp ladder indicate that one face of the DNA is protected by the underlying histone-octamer but an investigation of the kinetic parameters at each of the susceptible sites would be a sensitive probe of the identity or otherwise of the reassembled and natural particles. Polyanion assisted assembly may also lead to the formation of a particle resembling very closely the natural * To whom correspondence should be addressed 170 Published by Elsevier Science Publishers B. V. 00145793/84/$3.00 0 1984 Federation of European Biochemical Societies core but consisting only of the histones H3 and H4 as protein component [3]. It is therefore essential to establish whether during polyglutamic acid- assisted assembly such an alternative particle is formed. Should it be octameric with a molecular mass virtually identical to that of the natural oc- tamer it would be indistinguishable from the natural one on cross-linking and SDS gel elec- trophoresis. Furthermore, the interpretation of structural and functional properties of artificial chromatin fibers depends on whether the polyglutamic acid remains associated with the final product. It would be desirable to assemble cores over shorter periods and not over 16 h under con- ditions allowing enzymatic degradation of the component DNA and histones by fortuitously con- taminating enzymes. This report deals with these aspects. 2. MATERIALS AND METHODS Chicken erythrocyte nuclei were isolated [4] us- ing the buffer in [5] and briefly digested with micrococcal nuclease (100 units/mg DNA). Solu- ble chromatin was taken up in 0.25 mM EDTA-Tris at pH 8.0. Long chromatin was isolated from this on S-20070 (w/v) sucrose gra- dients in 550 mM NaCl, 10 mM TrisLHCl (pH 7.6) and 0.1 mM PMSF as a fraction larger than tetrasomes. The former was collected and concen- trated on an Amicon PM 30 membrane. To pro- duce cores this material was redigested in 20 mM