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Generation and characterization of novel antibodies highly selective for phosphorylated linked histone H1 in Tetrahymena and HeLa cells

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Abstract

Phosphorylated forms of Tetrahymena macronuclear histone H1 were separated from each other and from dephosphorylated H1 by cation-exchange HPLC. A homogeneous fraction of hyperphosphorylated macronuclear H1 was then used to generate novel polyclonal antibodies highly selective for phosphorylated H1 in Tetrahymena and in human cells. These antibodies fail to recognize dephosphorylated forms of H1 in both organisms and are not reactive with most other nuclear or cytoplasmic phosphoproteins including those induced during mitosis. The selectivity of these antibodies for phosphorylated forms of H1 in Tetrahymena and in HeLa argues strongly that these antibodies recognize highly conserved phosphorylated epitopes found in most H1s and from this standpoint Tetrahymena H1 is not atypical. Using these antibodies in indirect immunofluorescence analyses, we find that a significant fraction of interphase mammalian cells display a strikingly punctate pattern of nuclear fluorescence. As cells enter S-phase, nuclear staining becomes more diffuse, increases significantly, and continues to increase as cells enter mitosis. As cells exit from mitosis, staining with the anti-phosphorylated H1 antibodies is rapidly lost presumably owing to the dephosphorylation of H1. These immunofluorescent data document precisely the cell cycle changes in the level of H1 phosphorylation determined by earlier biochemical studies and suggest that these antibodies represent a powerful new tool to probe the functions(s) of H1 phosphorylation in a wide variety of eukaryotic systems.

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Authors and Affiliations

  1. Department of Biology, Syracuse University, 13244, Syracuse, NY, USA

    M. Janice Lu, Christopher A. Dadd & C. David Allis

  2. Department of Biology, Boston College, 02167, Chestnut Hill, MA, USA

    Carolyn A. Perry & Anthony T. Annunziato

  3. Center for Research in Neurodegenerative Diseases, University of Toronto, M5S 1A8, Toronto, Canada

    Craig A. Mizzen & Donald R. McLachlan

Authors
  1. M. Janice Lu
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  2. Christopher A. Dadd
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  3. Craig A. Mizzen
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  4. Carolyn A. Perry
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  5. Donald R. McLachlan
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  6. Anthony T. Annunziato
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  7. C. David Allis
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Lu, M.J., Dadd, C.A., Mizzen, C.A. et al. Generation and characterization of novel antibodies highly selective for phosphorylated linked histone H1 in Tetrahymena and HeLa cells. Chromosoma 103, 111–121 (1994). https://doi.org/10.1007/BF00352320

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  • DOI: https://doi.org/10.1007/BF00352320

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