Abstract
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.
Highlights
Eukaryotic chromatin is a dynamic entity exhibiting different levels of compaction, and its intrinsic flexibility entails reversible switches between decondensed and condensed states for proper transcription and replication
Cytofluorometry—Fluorescence was confined to nuclear chromatin, and background fluorescence was low in relation to nuclear fluorescence intensity
The fluorescence measurements were performed on whole nuclei, and the results obtained represent an average affinity of linker histones for chromatin within individual nuclei for a small population of cells
Summary
DAPI, digitonin, 3-aminobenzoic acid ethyl ester (MS-222), protamine sulfate, sucrose, N␣-p-tosyl-L-lysine chloromethyl ketone (TLCK), and Trizma (Tris base) were purchased from Sigma. Acetonitrile, aprotinin, E-64, hydroxypropylmethyl cellulose (HPMC; 4000 centipoise) and trifluoroacetic acid were purchased from Fluka (Buchs, Switzerland). Leupeptin, and pepstatin were purchased from Becham (Switzerland), ethylene glycolmonomethylether (EGME) was from Aldrich (Steinheim, Germany), and sequencing grade chymotrypsin was from Roche Molecular Biochemicals. All other chemicals were purchased from Merck (Darmstadt, Germany)
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