Flow cytometric correlation between BrdU/Hoechst quench effect and base pair composition in mammalian cell nuclei

Abstract

72 h after growth stimulation in BrdU supplemented media Hoechst fluorescence histograms of mammalian euploid cell cultures display up to five distinct peaks which correspond to a consecutive series of cell maturity classes. This pattern is due to differential quenching of the Hoechst fluorescence by BrdU-substituted chromatin. There are constitutive differences in the efficacy of fluorescence quenching among species: Human and bovine cells display more quenching at unifilary as apposed to bifilary substitution. The reverse holds for the AT-rich genome of the murine species micromys minutus. The theoretical residual fluorescence computed for an infinite number of replications in the presence of BrdU amounts to 14% in case of micromys cells, but to 19% for human and bovine cells. Flow cytometric estimates of the AT-content, employing base pair specific dyes, yielded 62.0% for micromys, 60.6% for human and 53.2% for bovine cells. While these figures are in accordance with published data, they do not sufficiently explain the concordant behavior of human and bovine cells, and the discordant behavior of micromys cells, as regards the quenching of Hoechst fluorescence. Additional factors such as differences in DNA-sequence organisation (eg. base pair specific interspersion pattern, lateral asymmetry) must be invoked.