Drosophila chromatin contains two antigenically distinct H2A histones, H2A.1 and H2A.2. Indirect immunofluorescence analyses revealed that anti-H2A.1 binding was distributed throughout polytene chromosomes, whereas anti-H2A.2 binding was Interband-specific. Thus, H2A.2 probably contributes to the less compacted structure of interbands. Since each band-interband region is thought to contain a single gene, our results suggest that the distribution of H2A.2 echoes the functional organization of the Drosophila genome. Similar H2A histones occur in eukaryotes ranging from protozoa to mammals. Their placement might be an important determinant of chromatin structure.Eukaryotic DNA is compacted into nucleosomes by octamers containing two molecules of each of the four histone types: H2A, H2B, H3, and H4 (reviewed in refs. 1 and 2). Primary sequence histone variants can generate nucleosome diversity. In the sea urchin, sperm-specific histones are replaced by maternal cleavage-stage histones during male pronuclear decondensation (3). In the developing embryos, stage-specific histones (4) affect nucleosomal structure and stability (5). In Tetrahymena, an H2A-like histone, hvl, occurs in the transcriptionally active macronucleus but not in the transcriptionally inactive micronucleus (6). The appearance of hvl in the developing macronucleus coincides with the onset of RNA synthesis (7), suggesting a relationship between gene activity and the distribution of specific histones.In Drosophila melanogaster, only one histone sequence variant has been reported. It was discovered by Alfageme et al. (8) (9), 2 mg of poly(glutamic acid) (Sigma, type III-B) per ml, 10 mM triethanolamine-HCl (pH 8.0), 0.1 M NaCl, and 10 mM 2-mercaptoethanol (17). They were shaken gently (12-14 hr; 20-22°C), diluted 1:10 with 55 mM Na2B407 (pH 9.5), combined with 1/50th vol of dimethyl suberimidate (Sigma; 50 mg/ml in dimethyl sulfoxide) at four 15-min intervals, incubated (15 min), precipitated with 20% trichloroacetic acid (0WC), washed (acetone/0.2% HCl and acetone), and air dried. In some experiments, histone octamers were crosslinked reversibly with dithiobis(succinimidylpropionate) (Lomant's reagent; ref. 18). In these cases, 2-mercaptoethanol was omitted from the reconstitution mixture.Crosslinked histone complexes were separated electrophoretically on 10 x 0.1 cm 5% acrylamide slab gels containing 0.1% NaDodSO4 and 0.1 M sodium phosphate (pH 7.1) (19), stained with Coomassie brilliant blue R, and destained. Bands containing reversibly crosslinked histone octamers, produced with Lomant's reagent, were excised, incubated in 62.5 mM Tris, pH 6.8/5% glycerol/10 mM 2-mercaptoethanol (60 min; 22-23°C), and electrophoresed in 18% acrylamide/NaDodSO4 gels (9).Preparation of Antibodies. H2A.2 was purified from total H2A (9) by electrophoresis in discontinuous gels containing 0.5% Triton X-100, 6% HOAc, and 8 M urea (20), excised from the gels, electrophoresed in 15% acrylamide/NaDod-S04 gels (9), excised, electrophoresed into Tris/glycine tray buff...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.