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Related Experiment Videos

Temperature-induced chromatin changes in ethanol-fixed cells.

P B Jacobsen1, T Stokke, O Solesvik

  • 1Department of Biophysics, Norwegian Radium Hospital, Oslo.

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
|December 1, 1988
PubMed
Summary
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Elevated temperatures during ethanol fixation release core histones from rat thymocyte chromatin. This temperature-induced histone loss alters chromatin structure, increasing DNA accessibility and dye binding.

Area of Science:

  • Cell Biology
  • Biochemistry
  • Molecular Biology

Background:

  • Chromatin structure is crucial for DNA regulation.
  • Histones play a key role in organizing DNA within chromatin.
  • Ethanol fixation is a common method for preserving cellular structures.

Purpose of the Study:

  • To investigate the impact of temperature during ethanol fixation on chromatin structure.
  • To determine how temperature affects histone association with DNA.
  • To elucidate the relationship between chromatin conformation and DNA accessibility.

Main Methods:

  • Flow cytometry using DNA-specific dyes (mithramycin, Hoechst 33258).
  • Antibody labeling (X-ANA) for core histones.
  • Gel electrophoresis (PAGE) for histone extraction and quantification.

Related Experiment Videos

  • Emission anisotropy measurements.

    • Main Results:

    • Increased temperature (44°C vs 0°C) during ethanol fixation significantly altered chromatin structure.
    • Mithramycin binding increased by 93%, indicating greater DNA accessibility.
    • Core histone labeling decreased by 65%, with unequal loss of histone pairs (H3/H4, H2A/H2B) and H1.
    • Emission anisotropy indicated increased DNA mobility, not denaturation, suggesting removal of short-range constraints.

    Conclusions:

    • Temperature elevation during ethanol fixation causes biphasic changes in chromatin structure.
    • Core histone release is the primary mechanism for increased DNA accessibility at higher temperatures.
    • These findings highlight the sensitivity of chromatin structure to fixation conditions.