DNA stainability with base-specific fluorochromes: dependence on the DNA topology in situ

Abstract

The influence of DNA topology on stainability with the externally binding fluorochromes Hoechst 33258 (HO) and mithramycin (MI) was investigated in HeLa nuclei in comparison with the intercalating dye propidium iodide (PI). Changes in DNA topology were induced with a mild DNAse I treatment. Stainability properties of untreated and nuclease-treated nuclei were compared with those of the supercoiled-circular and the relaxed-linear forms of the plasmid pBR322. DNAse-treated nuclei stained with HO showed a higher fluorescence intensity than control samples, independently of the dye concentration, in contrast with the findings obtained with PI. Similar behaviour was observed with the relaxed-linear form of pBR322, compared with the supercoiled-circular molecule. With MI, the stainability of HeLa nuclei did not depend on the DNA topology, whereas the stainability of the plasmid was similar to that of HO. In order to assess whether this discrepancy depended on differences in the availability of DNAse-sensitive sites to the fluorochromes, fluorescence resonance energy transfer (FRET) studies were performed in nuclei stained with HO+PI, or with HO+MI dye pairs. After DNAse I digestion, the relative FRET efficiency between donor (HO) and acceptor molecules (PI or MI) was reduced significantly only when MI was the acceptor. This result may be due to greater stainability of DNAse-sensitive sites with HO than with MI. These findings indicate that DNA stainability with base-specific fluorochromes may be affected by the topology of chromatin regions.

Variation of the genome size estimate with environmental conditions in Drosophila melanogaster

“…Fluctuations in genome size estimates of the kind observed in our experiments can thus simply be explained in terms of changes in the topology of chromatin, which modifies the accessibility of DNA to fluorochromes, especially to propidium iodide, which is often used in flow cytometry, as shown in various experiments (23,26,30,31). Changes in DNA accessibility after a temperature treatment have been indeed reported for ethanolfixed cells of rat thymocytes (27).…”

“…A correlation between estimates obtained using different methods is not in itself sufficient, however, to eliminate the possibility of an artifact, because DNA accessibility depends on complex interactions between intercalating or nonintercalating fluorochromes and DNA and various proteins. Variation in DNA accessibility due to the state of condensation of the chromatin has been reported in plants (21,23), during spermatogenesis (24) and in frozen and thawed human spermatozoa (25), in mouse thymocyte nuclei (26), in ethanol-fixed cells (27), in dividing and stationary Euglena cells (28), during differentiation of Friend leukemia cells (29), and in DNAse I treated HeLa nuclei (30,31). The exact impact of external environmental conditions on the genome size estimate therefore needs to be determined before a protocol can be defined that would permit reliable and comparable genome size estimates between individuals and populations within a given experiment and between different experiments.…”

Spatial Distribution Analysis of AT- and GC-rich Regions in Nuclei Using Corrected Fluorescence Resonance Energy Transfer

“…The structural and topological changes of the DNA were interpreted in terms of changes in fluorescence intensity, which is known to be related to local DNA concentration (Bruno et al 1991;Urata et al 1991;Colomb and Martin 1992;Santisteban and Brugal 1995). A few reports have focused on the DNA structure in interphase nuclei, where additional information content of the fluorescence resonance energy transfer (FRET) by steady-state fluorescence measurement was exploited (Bottiroli et al 1989;Prosperi et al 1994;Szollosi et al 2002).FRET is a non-radiative transfer of the excited-state energy from the initially excited donor to an excitable acceptor (Forster 1948;Stryer and Haugland 1967;Stryer 1978;Jovin and Arndt-Jovin 1989;Clegg 1992;Lakowicz 1999;Murata et al 2000b). Because the efficiency of FRET depends on the sixth power of the distance between the donor and the acceptor, the microscopic FRET is an excellent ruler for quantification of distances on the molecular scale in cells (Jovin and Arndt-Jovin 1989).…”

G-banding and chromosome condensation in the ant,Tapinoma nigerrimum

“…Prosperi et al (1994) have observed that the ability to stain D N A with base-specific fluorochromes depends on the D N A topology in situ. In the first hypothesis, we suggest that only one segment of the short arm of chromosome 6 is sufficiently AT rich to produce a differential fluorescence.…”