Hoechst 33258 fluorescent staining of Drosophila chromosomes

Abstract

Metaphase chromosomes of D. melanogaster, D. virilis and D. eohydei were sequentially stained with quinacrine, 33258 Hoechst and Giemsa and photographed after each step. Hoechst stained chromosomes fluoresced much brighter and with different banding patterns than quinacrine stained ones. In contrast to mammalian chromosomes, Drosophila's quinacrine and Hoechst bright bands are all in centric heterochromatin and the banding patterns seem more taxonomically divergent than external morphological characteristics. Hoechst stained D. melanogaster chromosomes show unprecedented longitudinal differentiation in the heterochromatic regions; each arm of each autosome can be unambiguously identified and the Y shows eleven bright bands. The Hoechst stained Y can also be identified in polytene chromocenters. Centric alpha heterochromatin of each D. virilis autosome is composed of two blocks which can be differentiated by a combination of quinacrine and Hoechst staining. The distal block is always Q⁻H⁻ while the proximal block is, for the various autosomes, either Q⁻H⁻, Q⁺H⁻ or Q⁺H⁺. With these permutations of Hoechst and quinacrine staining, D. virilis autosomes can be unambiguously distinguished. The X and two autosomes have H⁺ heterochromatin which can easily be seen in polytene and interphase nuclei where it seems to aggregate and exclude H⁻ heterochromatin. This affinity of fluorochrome similar heterochromatin was best seen in colcemid induced multiple somatic non-disjunctions where H⁺ chromosomes were distributed to one rosette and H⁻ chromosomes were distributed to another. Knowing the base composition and base sequences of Drosophila satellites, we conclude that AT richness may be a necessary but is certainly an insufficient requirement for quinacrine bright chromatin while GC richness may be a sufficient requirement for the absence of quinacrine or Hoechst brightness. Condensed euchromatin is almost as bright as Q⁺ heterochromatin. While chromatin condensation has little effect on Hoechst staining, it appears to be “the most important factor responsible for quinacrine brightness”. All existing data from D. virilis indicate that each fluorochrome distinct block of alpha heterochromatin may contain a single DNA molecule which is one heptanucleotide repeated two million times.