Nuclease‐induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide

Abstract

A flow cytometric analysis of DNA structural changes induced by cleavage with nucleases was performed on isolated HeLa nuclei by assessing changes in stainability with the DNA-specific fluorochrome propidium iodide (PI). After mild digestion with DNAse I, micrococcal nuclease, or with the single-strandspecific S1 and Neurospora crassa nucleases, fluorescence intensity of nuclei stained with PI increased by about 15-30% above the value of undigested control samples. No significant modifications were observed with the restriction enymzes Eco RI, Alu I, and Not I. The DNAse I-induced increase in fluorescence intensity was also observed with the non-intercalating dye Hoechst 33258, but not with mithramycin. Nucleaseinduced fluorescence intensity changes as determined with PI were found to be dependent on the dye concentration. A constant increase (about 20%) was measured at dye/DNA-P ratios > 0.11. Below this value (2 pg/ml PI), the fluorescence intensity of digested samples was 1530% lower than that of undigested controls. This behaviour towards intercalating dyes is similar to that of the relaxed (nicked) vs. the supercoiled (intact) form of circular DNA. These results suggest that conformation-but not sequence-specific nucleases induce a relaxation of DNA supercoils.

Reference standards for determination of DNA content of plant nuclei

“…However, the two dyes have quite different stain reactions. PI intercalates between base pairs of double-stranded DNA and RNA with little or no base specificity (Properi, Giangare, and Bottiroli, 1991), while DAPI is a nonintercalating stain that binds preferentially and in a complex manner to A-T base regions (Godelle et al, 1993). We show here that PI-based flow cytometry produces results very consistent with those based on Feulgen microspectrophotometry.…”

“…PI intercalates between base pairs of double-stranded DNA and RNA with little or no base specificity (Properi, Giangare, and Bottiroli, 1991), while DAPI is a nonintercalating stain that binds preferentially and in a complex manner to A-T base regions (Godelle et al, 1993). PI is excited by visible light with an absorbancy maximum at 490 nm, while DAPI is excited by UV light at 350 nm.…”

Probing chromatin structure in the early phases of apoptosis

“…Accordingly, more recent reports (Higashikubo et al. 1990al. , Prosperi et al 1991 showed that chromatin alterations, occurring after treatment by DNAse I or micrococcal and S1 nucleases induced an increase in PI fluorescence intensity ranging between 15 and 30%.…”

“…To investigate chromatin modifications without interference by cytoplasmic components (Thornthwaite et al 1980), several nuclear isolation mediums (NIM) were used: 0.5% Nonidet-P40 (NP-40, Sigma) in Ca++-and Mg++-free PBS with or without 1 mM EDTA; 0.5% Triton-X 100 (Sigma) in Caf+-and Mg++-free PBS with or without 1 mM EDTA; 0.1% NP-40 in 0.1% sodium citrate in distilled water with or without 1 mM EDTA. A commercial NIM (Nucleidye, Ylem, Avezzano, Italy) were also used.…”

Flow cytometric DNA analysis of wheat‐rye addition lines

“…PI, being an intercalating dye, does not easily bind to tightly coiled DNA found in heterochromatin (2,13,16). Subtracting the amount of heterochromatin from each chromosome and running the correlation between euchromatin per chromosome and nuclear DNA content, however, does not improve the correlation, r = 0.63.…”