Figure 2

Micrococcal nuclease (MNase) digestion of chromatin within fission yeast nuclei and recovery of protected DNA. (a) DAPI fluorescence/phase contrast micrograph of log-phase S. pombe cells flash-frozen to -196°C then thawed in INCA buffer (see Materials and Methods). (b) Cryo-ground log-phase cells suspended and thawed in INCA buffer. The arrow marks an intact cell adjacent to a group of discrete liberated nuclei. (c) Oil-immersion micrograph of similarly-prepared stationary-phase nuclei. Arrows denote in-focus, liberated nuclei displaying the typical S. pombe tri-lobed DAPI fluorescence pattern. The ratio of liberated nuclei to cell debris seems lower here than in (B) because the microscope is focused on a thin (due to high magnification) plane containing settled cell walls, but many of the released nuclei are floating above that plane. (d) Gel-electrophoretic analysis of DNA recovered from MNase-digested nuclei of native ("Unfixed") and formaldehyde-treated ("HCHO-Fixed") stationary phase cells. "EF" indicates exogenous-Enzyme-Free control incubations. "+MNase" denotes incubation at 25°C with 150 units of MNase per ml for the indicated times. "Mkr bp" is a commercial 50-bp DNA-size-marker ladder. "Mkr Kbp" is a commercial 0.2- to 10-kbp DNA-size-marker ladder. (e) Gel-electrophoretic analysis of "mononucleosomal" DNA recovered from a band excised from a preparative-scale gel ("explant"). Twice as much DNA was loaded into the third and fourth lanes as into the second lane. "Dgst" indicates total DNA recovered from the parent MNase digest (12 min) of unfixed, stationary-phase nuclei; "t", "d" and "m" respectively mark the tri-, di-, and mononucleosomal DNA bands. "+Δ" signifies heat-denaturation of sample immediately prior to loading as an assay for internal nicking. (f) Gel-electrophoretic analysis of DNA recovered from a broad explant centered on the "mononucleosomal" band of a preparative-scale gel ("LFB": Log-phase, Fixed cells, Broad explant), then run on a second preparative gel from which the "mononucleosomal" band was re-sampled as a narrow explant ("LFN": Log, Fixed, Narrow).