The Structure of Chromatin Replicated in vitro

Abstract

Nuclei from concanavalin-A-activated [bovine retropharyngeal lymph node] lymphocytes were used to study the replication of chromatin in vitro. Micrococcal nuclease was employed to obtain information about the structure of the replicated chromatin. The nuclease digestion products were examined by sucrose gradient sedimentation and by gel electrophoresis. Experiments indicate that DNA replicated in vitro is organized into chromatin whose structure is similar to that of bulk chromatin. This conclusion is based on the following observations: DNA replicated in vitro is associated with typical chromatin subunits (nucleosomes) even after short replication times, when the newly replicated DNA consists almost entirely of Okazaki fragments; and the length of internucleosomal spacer DNA in part of the replicated chromatin corresponds to that in bulk chromatin. Evidence which suggests that the structure of nucleosomes is transiently altered in the vicinity of the replication fork is presented.