Fluorescence characterization of the structural heterogeneity of polytene chromosomes.

Studies on the physical nature of the structural heterogeneity of chromatin in their native states are few. The eukaryotic chromatin as observed by dye staining studies is of heterogeneous intensity when observed by fluorescent stains, where less and more bright regions apparently correspond to euchromatin and heterochromatin respectively. These are also associated with differential gene expression where it is believed that euchromatin is transcriptionally more active due to increased flexibility. Unfixed squashed preparations of polytene chromosomes of Drosophila were stained with a dsDNA specific dye PicoGreen and fluorescence lifetimes as well as fluorescence anisotropy decay kinetics were measured. Here we report a positive correlation between fluorescence lifetimes and fluorescence intensities, and show that less bright regions corresponding to euchromatin have shorter lifetimes, whereas more bright regions corresponding to heterochromatin have longer lifetimes. We interpret this as less bright regions being more dynamic, a conclusion also supported by fluorescence anisotropy decay kinetics. We infer that the comparatively higher flexibility associated with euchromatin can be directly measured by fluorescence lifetimes and fluorescence anisotropy decay kinetics.