The replacement linker histones H1 0 and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1 0 dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1 0 variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1 0 -2 is the only H1 0 subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.

The binding behavior of the DNA binding dyes 7-aminoactinomycin D (7-AAMD) and 4',6-diamidino-2-phenylindole (DAPI) to human neutrophilic granulocytes and lymphocytes was studied by image cytofluorometry. Peripheral blood leukocytes were prefixed in paraformaldehyde (PFA) and attached to cover glasses. Different fkation, permeabilization, and acid extraction methods were applied before the cells were stained to equilibrium using varying concentrations of 7-AAMD or DAPI. The apparent association constant and number of high afEnity dye binding sites were estimated for the different cell types, dyes, and treatments. Acidextracted cells, supposedly containing nucleosomefree DNA, were chosen to represent maximal dye binding. Only about 10% of the 7-AAMD binding sites remained in the unextracted PFA-fixed cells, and the apparent dye aiEnity was also reduced. We found no major difference in high a f h i t y binding between the cell types, but granulocytes showed more fluorescence from less specitically bound 7-AAMD compared to lymphocytes. DAPI had a much higher alfinity than 7-AAMD, independent of the preparation method. It showed a cooperative binding behavior with an apparent saturation of the high afhity binding sites at a dye concentration of about 50 nM. We conclude that both dyes may be useful as probes for chromatin structure in intact cells and that our new technique may contribute to such studies since it allows determination of dye aEdties and numbers of high aSanity binding sites ill S h . 0 1995 Wiley-Liss, Inc.

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 μM 7‐AAMD or 50 nM DAPI. Lymphocytes stained with 7‐AAMD showed a gradual increase from 11% to 36% of HCl 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. Cytometry 28:212–219, 1997. © 1997 Wiley‐Liss, Inc.

No abstract

Background:It is generally assumed that linker histones contribute to condensation of chromatin and to the repression of genetic activity by binding to chromatin with increased affinity. The aim of this study is to investigate a possible correlation between linker histone binding to chromatin in situ and chromatin condensation using a cytochemical approach that largely preserves nuclear structure. Methods: Ionically bound H1 was extracted with increasing concentrations of sodium chloride. Thereafter, the cells were fixed in formaldehyde and stained with 50 nM 4Ј,6-diamidino-2-phenylindole (DAPI) and fluorescence intensity was measured by image cytofluorometry. Results:The association between linker histones and chromatin was stronger in cultured human fibroblasts and