Sister chromatid differentiation in 5-bromo-2?-deoxyuridine-substituted chromosomes: A study with DNA-specific ligands and monoclonal antibody to histone H2B

Abstract

Human and Chinese hamster chromosomes were obtained from cells grown in the presence of 5-bromodeoxyuridine (BrdU) for (a) one replicative round, (b) two replicative rounds, (c) one replicative round followed by another round in thymidine and (d) the last period of synthetic phase. Untreated chromosomes and chromosomes treated with UV radiation after previous staining with 33258 Hoechst as photosensitizer were studied in order to investigate the mechanism(s) responsible for BrdU-induced sister chromatid differentiation (SCD). Metaphases were prepared by (1) standard methanol-acetic acid fixative for subsequent investigation with Giemsa or DNA-specific dyes such as ethidium bromide, acridine orange and monoclonal antibodies to double- or single-stranded DNA; (2) the procedure for observation under phase-contrast or electron microscopy; and (3) the cytospin method for subsequent immunoreaction with a monoclonal antibody to histone H2B. Our data exclude the possibility that the presence/absence of BrdU in the template strand might affect chromatin organization and thus resistance, while confirming that UV-induced DNA alteration is not sufficient, by itself, to explain SCD mechanism(s). That molecules other than DNA play a role in explaining SCD production is indicated by the fact that BrdU incorporation induces alterations in DNA-histone H2B interactions which, in turn, seem to produce structural variations in chromatin, possibly at the level of condensation, as monitored by phase-contrast and electron microscopy.