Chromatin replication in a higher plant, Vicia faba

Abstract

Embryo axes of Vicia faba seeds were collected and pulse-labeled with [ 3H]thymidine for 5–6 min at the first DNA synthesis period (34 h stage of germination) and chromatin was isolated. About 67% of the pulse-labeled DNA in the chromatin was converted to acid-soluble material after extensive digestion with micrococcal nuclease. On the other hand, bulk chromatin uniformly labeled with [ 14C]thymidine for a 12-h period showed acid-solubility of about 56% under identical conditions of digestion. Sedimentation analyses of the nuclease digests of chromatin and extracted DNA samples showed that the nucleosome structure was already organized in the chromatin containing 10–11-S single-strand DNA fragments of an intermediate molecule during DNA replication. These fragments were detected as a major component after labeling for 10 to 15 min with [ 3H]thymidine. Further-more, the result of the pulse-labeling for 5–6 min indicated the presence of the nucleosome structure in the chromatin containing 5–6-S and 8–9-S nascent DNA fragments as the main labeled components, thus suggesting the involvement of at least the 8–9-S intermediate fragment in the reorganization of the nucleosome structure. Electrophoresis of DNA fragments obtained after micrococcal nuclease digestion of chromatin showed that after a pulse of 5–6 min with [ 3H]thymidine, labeled dimer and trimer DNA fragments were significantly shorter than corresponding [ 14C]thymidine-labeled bulk dimer and trimer DNA fragments, though the peaks of pulse-labeled and uniformly labeled monomer DNA fragments were coincident with each other in 3.5% gels. The difference in size of dimer and trimer DNA fragments between the newly formed and non-replicating bulk chromatin disappeared after a prolonged labeling for 15 min, irrespective of the fact that the major part of labeled DNA was still the 10–11-S intermediate fragment. The mode of chromatin assembly in the replicating chromatin in higher plant cells is discussed.