The Role of Nick Formation in Delayed Quinacrine Mustard Fluorescence in the C-heterochromatin of Apodemus argenteus

Chromosomes stained with fluorochromes, including quinacrine mustard (QM), emit the brightest fluorescence immediately after exposure to excitation light, and the fluorescence gradually fades with an increase in exposure time. However, in the QM-stained chromosomes of the small Japanese field mouse Apodemus argenteus, most C-heterochromatic regions emit weak fluorescence immediately after exposure to blue light, and they become brightly fluorescent by prolonged exposure (delayed QM fluorescence). We proposed recently that the delayed QM fluorescence is somehow related to nicks produced in C-heterochromatic DNA by blue light irradiation. To test this possibility, we examined the chromosomal distribution of nicks by in-situ nick translation and changes, if any, in the QM fluorescence pattern after methylene blue (MB) -mediated photooxidation, which is considered to induce nicks in chromosomal DNA. It was found that C-heterochromatic regions fluoresced brightly without any delay after exposure to blue light, and that nicks increased considerably in the same regions after the MB-mediated photooxidation. It seems, therefore, that photooxidation and strand breaks in DNA (including nicks) are responsible for the induction of delayed QM fluorescence. Trypsin digestion, on the other hand, abolished delayed QM fluorescence. Thus, not only DNA but also chromosomal protein(s) are involved in the unusual sequence of QM fluorescence patterns in A. argenteus.