Scanning electron microscopy of the centromeric region of L-cell chromosomes after treatment with Hoechst 33258 combined with 5-bromodeoxyuridine.

When mouse L-cells were treated with a combination of 5-bromodeoxyuridine (BrdUrd) and Hoechst 33258, the metaphase chromosomes revealed under-condensation of the chromatin fibers in the sister centromeres. The application of the osmium-thiocarbohydrazide technique to the air-dried chromosome preparations made it possible to elucidate the ultrastructure of the under-condensed centromeric region at the level of the 30 nm chromatin fiber. Scanning electron microscopy revealed that the under-condensed region consisted of a coiled fiber with a diameter of about 400 nm, and a gyre diameter of approximately 600 nm. The coiled fiber was composed of the 30 nm chromatin fiber loops. These findings indicate that a continuous coiled structure, which is the final higher order structure of the condensed chromatin fiber, exists throughout the entire length of the mouse L-cell metaphase chromosome.