DNA binding fluorochromes as probes for histone H1-chromatin interactions in situ.
We have investigated using the DNA binding fluorochromes 7-aminoactinomycin (7-AAMD) and 4',6-diamidino-2-phenylindole (DAPI) as cytochemical probes for linker histone (H1)-chromatin interactions in situ. Human lymphocytes, permeabilized with digitonin, were exposed to increasing concentrations of sodium chloride to remove ionically bound H1 from the nuclei. The cells were stained to equilibrium with 1 microM 7-AAMD or 50 nM DAPI. Lymphocytes stained with 7-AAMD showed a gradual increase from 11% to 36% of HC1 treated cell fluorescence intensity when the salt concentration was increased from 0.15 to 0.7 M. The corresponding increase for DAPI was 53-68%. The 7-AAMD obviously showed higher sensitivity for H1-chromatin interactions that DAPI but had disadvantages such as high background fluorescence and an affinity that was dependent on the preparation procedure. DAPI had negligible background fluorescence, and its fluorescence intensity resembles the number of available high-affinity dye-binding sites when used at 50 nM. We conclude that both fluorochromes can be used as probes for H1-chromatin interactions in situ and that our method has a potential to provide new information on such interactions.
Loborg
H
H
Department of Cell Biology, Faculty of Health Sciences, University of Linköping, Sweden.
Rundquist
I
I
D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000378:metabolism; D008856:Microscopy, Fluorescence
D002843:Chromatin; D005456:Fluorescent Dyes; D006657:Histones; D007211:Indoles; D003609:Dactinomycin; C007293:DAPI; C025942:7-aminoactinomycin D; D004247:DNA