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Fractionation of human H1 subtypes and characterization of a subtype-specific antibody exhibiting non-uniform nuclear staining

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Abstract

Four histone H1 subtypes and H1° were fractionated from human placental nuclei and purified to homogeneity by a combination of Bio-Rex 70 chromatography and reversephase high-performance liquid chromatography (RPHPLC). Polyclonal antibodies were generated in rabbits against one of these subtypes designated H1-3. Antibodies reacted only against this subtype in enzymelinked immunosorbent assays and Western assays; subtype specificity was documented further by Western blotting of cell and nuclear extracts. They crossreacted with monkey H1, but not with H1 from other vertebrates tested. The epitope(s) recognized were mapped by immunoblotting against peptides prepared by cleavage with N-bromosuccinimide (NBS) and α-chymotrypsin; it includes the variant amino-terminal tail of the protein as well as a portion of the globular domain. The antibody stains mitotic chromosomes weakly but uniformly and, unlike antibodies that recognize total H1 which show uniform nuclear staining after indirect immunofluoresence localization, anti-H1-3 exhibits preferential labelling of the nuclear periphery. This non-uniform staining suggests compartmentalization of this subtype which may have functional significance with respect to differential chromatin condensation.

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  1. Robert F. Clark

    Present address: the Department of Biology, Washington University, 63130, St Louis, MO, USA

  2. Loren J. Hauser

    Present address: the Biology Division, Oak Ridge National Laboratory, PO Box Y, 37830, Oak Ridge, TN, USA

  3. Nadja Dvorkin

    Present address: the Department of Biological Chemistry, College of Medicine, University of California, 92717, Irvine, CA, USA

Authors and Affiliations

  1. the Department of Molecular Biology and Biochemistry, University of California, 92717, Irvine, CA, USA

    Missag H. Parseghian, Debra A. Harris & Barbara A. Hamkalo

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Parseghian, M.H., Clark, R.F., Hauser, L.J. et al. Fractionation of human H1 subtypes and characterization of a subtype-specific antibody exhibiting non-uniform nuclear staining. Chromosome Res 1, 127–139 (1993). https://doi.org/10.1007/BF00710036

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