Flow cytometric evaluation of DNA digestion with micrococcal nuclease on isolated HeLa nuclei.
Flow cytometric assessment of DNA digestion with micrococcal nuclease has been performed on isolated HeLa nuclei by determining the relative reduction in stainability with the DNA-specific fluorochrome, propidium iodide. At the nuclease concentrations used, DNA histograms of digested nuclei showed the typical bimodal pattern, when the enzymatic reaction was performed in a medium maintaining chromatin in its native (i.e. condensed) or partially decondensed from. In contrast, when nuclei were digested in a buffer lacking both the mono- and divalent cations K+ and Mg2+, an extensive decrease in fluorescence intensity, with loss of the histogram shape, was observed. In nuclei with native chromatin, DNA stainability decreased as a function of time and enzyme concentration, to reach a lower limit of about 46%, as compared with undigested control samples. Removal of the histone H1 induced a significant increase (approximately by a factor of 2) in the extent of digestion, although only in nuclei with partially decondensed chromatin. These results suggest that the sensitivity of DNA to digestion with micrococcal nuclease can be quantitatively monitored with flow cytometry when appropriate reaction conditions are chosen.
Prosperi
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E
Centro di Studio per l'Istochimica del CNR, Dipartimento di Biologia Animale, Università di Pavia, Italy.
Giangaré
M C
MC
Supino
R
R
Bottiroli
G
G
D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D008836:Micrococcal Nuclease / Q000378:metabolism*
D004247:DNA; D008836:Micrococcal Nuclease