R-banding produced by DNase I digestion of chromomycin-stained chromosomes.

A distinct reverse (R-) banding pattern was produced on human chromosomes by digesting chromosome spreads with pancreatic deoxyribonuclease I (DNase I) in the presence of an excess of chromomycin A3 (CMA), followed by staining with Giemsa. The banding pattern corresponds with that obtained by chromomycin A3 fluorescence, and bands which fluorescence brightly with chromomycin appear darkly with Giemsa. The same relationship was observed in two plants, Scilla siberica and Ornithogalum caudatum, which have contrasting types of heterochromatin. Chromomycin bright C-bands stained darkly with the CMA/DNase I technique, whereas chromomycin negative C-bands appeared lightly stained. The digestion patterns are thought to reflect the variation in chromomycin binding capacity along the chromosome with R-bands and dark C-bands being sites which preferentially bind the antibiotic.