Isolation and preliminary characterization of histone H1.b allelic variants from quail erythrocytes

Abstract

Our goal was to purify and characterize the allelic variants H1b1 and H1b2 of histone H1.b, one of the seven subtypes of this linker histone extracted from Japanese quail erythrocyte nuclei. These variants are revealed phenotypically as band H1.3 or part of band H1.4 by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). All H1 subtypes together were separated from H5 by gel-permeation chromatography through Bio-Gel P-150. H1 was then fractionated on a column of the cation-exchange resin Amberlite CG-50 by using a shallow guanidine hydrochloride gradient, which enriched subtype H1.b together with H1.z and overlapping with subtypes H1.a and H1.b. Alternatively purification of subtypes was achieved electrophoretically: total H1 fractions from quail with different H1 phenotypes were first resolved into sub-types by PAGE in acetic acid-urea; after staining, the appropriate H1.b bands from several parallel gel pieces were excised and the histone was concentrated by PAGE in SDS. After fragmentation of H1.b in the gel pieces with N-bromosuccinimide (NBS), PAGE in SDS indicated no difference between H1b1 and H1b2 in the C-terminal "half" of the polypeptides. In contrast, limited digestion with endoprotease V8 from Staphylococcus aureus has shown that differences, probably by a few residues in length, reside in the N-terminal part of the molecule, close to the amino-terminus.

Sequence variants of chicken linker histone H1.a

“…For preparative SDS gel electrophoresis, approximately 18 stained gel fragments of the acetic acid–urea gel containing H1.a isoforms and H1.a1‐like protein (H1.y) were separately incubated in adaptation buffer [2% (w/v) SDS, 10% (v/v) 2‐mercaptoethanol (2‐ME), 10% (v/v) glycerol, 0.125 m Tris/HCl, pH 6.8]. The gel pieces were then stacked in a well of one‐dimensional 15% (w/v) polyacrylamide gel containing 0.1% (w/v) SDS [35].…”

“…In our laboratory we have revealed a polymorphism for subtypes H1.a, H1.b, and H1.z in duck [23,31–33] and quail [34,35]. In this work, in addition to well‐known allelic variants of erythrocyte histone H5 [27] distributed differently in distinct chicken breeds [36], we also observed two allelic isoforms of histone H1.a in the chickens with a rare histone H1.a2 in a Rhode Island Red breed only.…”

Linker histone‐like proteins in Muscovy duck (Cairina moschata L) erythrocyte chromatin

“…Further steps were taken to obtain a purified LHL protein to find structural similarities and/or discrepancies between the LHL and selected linker histone subtypes. Enough purified protein was gathered by one dimension SDS polyacrylamide gel electrophoresis of stained pieces cut out of the acetic acid‐urea polyacrylamide gel containing both histone H1.b and H1.c bands (Pałyga and Neelin, 1998) that resulted in the concentrated bands of the individual LHLs. Two slower migrating low abundant LHL protein bands were visible in addition to the fast moving histones H1.b and H1.c (Fig.…”

Modulation of chromatin function through linker histone H1 variants

“…Therefore, a given allelic isoform seems to promote a unique line-specific phenotypic effects by influencing putative allele-related chromatin processes. The allelic isoforms of histone H1 subtypes (Pałyga and Neelin, 1998;Kowalski et al, 1998Kowalski et al, , 2011aKowalski et al, , 2011bKowalski et al, , 2014Górnicka-Michalska et al, 2006) may individually, or together, contribute to the interactions with DNA and chromatin proteins to modulate the physiological processes and phenotypic traits.…”