Kinetics of DNase I digestion of interphase chromatin in differentiated cell nuclei of the mouse: a flow cytometric study.
The process of DNA digestion with DNase I was monitored in interphase chromatin of differentiated cells by flow cytometry after DNA staining with either the intercalating dye propidium iodide (PI) or the AT specific dye Hoechst 33258 (HO). Nuclei from the liver, kidney and spleen of the mouse were studied after different digestion times (0 to 120 min). During the first 30 min of treatment, a tissue specific digestion pattern was found after PI staining; from 60 min onward, the digestion curves ran parallel, with minor quantitative differences among the cell types. After HO staining, the digestion kinetics appeared to be similar for all the cell types; this is likely due to the peculiar base composition of the mouse genome, where inactive c-heterochromatin is exceptionally AT-rich. No quantitative correlation was found between interphase "heterochromatin" and chromatin DNA which is resistant to DNase I cleavage, while the amount of DNase-I-sensitive DNA does not correspond to the interphase "euchromatic" component. It was confirmed that the flow cytometric approach is a tool for quantifying relative changes in the functional state of chromatin in differentiated cell systems.
Pellicciari
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C
Dipartimento di Biologia Animale CNR, Pavia, Italy.
Marchese
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Bottone
M G
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Manfredi Romanini
M G
MG
D000818:Animals; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000032:analysis* / Q000187:drug effects; D002469:Cell Separation; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000494:pharmacology*; D005434:Flow Cytometry; D007399:Interphase; D007668:Kidney / Q000032:analysis / Q000166:cytology; D007700:Kinetics; D008099:Liver / Q000032:analysis / Q000166:cytology; D051379:Mice; D009691:Nucleic Acid Denaturation / Q000187:drug effects*; D013154:Spleen / Q000032:analysis / Q000166:cytology
D002843:Chromatin; D003850:Deoxyribonuclease I