Nonrandom Segregation of Histones during Chromatin Replication

Abstract

Exponentially growing Ehrlich ascites tumor cells were labelled in vivo during roughly two cell cycles by three different methods: (a) DNA was labelled during the first cell cycle with [14C]thymidine, and during the second cycle with bromodeoxyuridine + [3H]thymidine; (b) proteins were labelled during the first cell cycle with L‐[14C]valine and DNA during the second cycle with bromodeoxyuridine + [3H]thymidine; (c) both proteins and DNA were labelled during the same cell cycle with L‐(14C]valine and bromodeoxyuridine + [3H]thymidine respectively. DNA isolated from cells labelled according to method (a) was also resolved on a CsCl density gradient to show that the two successive labels are incorporated in the same DNA molecules. Chromatin was isolated by salt extraction of nuclei obtained with Nonidet P40 from Ehrlich ascites tumor cells labelled according to methods (b) and (c); 4–8% of the total protein (10–15% of the histones) were chemically bound to DNA by the procedure of Levina and Mirzabekov [Dokl. Akad. Nauk. SSSR, 221, 1222–1225 (1975)]. The unbound proteins were removed by centrifugation in a Cs2SO4/sarcosyl density gradient. The chemically linked nucleohistone was alkali‐denatured and the two DNA strands, together with the bound histones, were separated on a shallow alkaline metrizamide density gradient. The newly synthesized histones were found to be associated with the newly synthesized DNA strand while the old histones remained with the parantal DNA strand. Copyright © 1979, Wiley Blackwell. All rights reserved