Fluorescence polarization of stretched polytene chromosomes stained with acridine orange.
The molecules of the fluorescent dye acridine orange (AO) bind to DNA in such a way that the absorption and emission dipoles lie on a plane perpendicular to the DNA axis. For this reason, definite fluorescence polarization should correspond to each mode of spatial DNA packing. A chromosome, considered as an axially symmetrical ensemble of DNA, was characterized by two experimental parameters, P parallel and P perpendicular, i.e., by polarizations of fluorescence excited by light polarized parallel and perpendicular to the symmetry axis. In view of the sequential order in the packing levels of DNA fiber in a chromosome, it was suggested that, under mechanical stretching, the highest level is disrupted first, then the others, in the order of their sequence. Isolated chromosomes of Chironomus thummi were stained with AO and stretched with needles of a micromanipulator. From the changes of P parallel and P perpendicular measured during stretching it was concluded the polytene chromosome bands have three, at least, DNA packing levels, tentatively described as 100 A fiber, 250 A coil and chromomere.