Related Experiment Video
Updated: Aug 16, 2026
A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice
Published on: April 19, 2020
Nucleosomes from normal and regenerating rat liver
Abstract:
Micrococcal-nuclease digestion of rat liver nuclei selectively released mononucleosomes associated with ADP-ribosylated [Caplan, Ord & Stocken (1978) Biochem. J.174, 475-483] histone H1. Two classes of mononucleosome were detected, those that leaked out during digestion and those that were subsequently released by 5mm-sodium phosphate buffer (pH6.8)/0.2mm-NaEDTA. The former, from which histone H1 had been dissociated, contained 140-base-pair-length DNA and core histones;the latter contained core particles and mononucleosomes with histone H1 and 200-base-pair-length DNA. When normal liver nuclei were phosphorylated with [gamma-(32)P]ATP, dissociated histone H1, which could be separated from core particles with Sephadex G-200, showed (32)P uptake. (32)P uptake into histones H2A and poly(ADP-ribosyl)ated H3 was appreciable in core particles, but was less evident in nucleosomes still containing histone H1. When [(3)H]-thymidine was given to partially hepatectomized rats in S-phase, 5-10min pulses in animals of over 300g body wt. showed the presence of high-specific-radioactivity DNA in released core particles and mononucleosomes compared with DNA retained in the nuclear pellets. Mononucleosomes from rat livers in S-phase with new, [(3)H]lysine-containing histones, had higher (32)P incorporation in histones H1 and their core histones, than for di- or tri-nucleosomes. Thermal-denaturation properties of control and phosphorylated mononucleosomes and core particles were very similar; removal of histone H1 and non-histone chromosomal proteins in 0.5m-NaCl markedly increased the proportion of DNA ;melting' below 70 degrees C.
Insights
Micrococcal nuclease digestion of rat liver nuclei releases mononucleosomes. Histone H1, ADP-ribosylated and phosphorylated, is associated with specific nucleosome fractions, indicating its role in chromatin structure and DNA accessibility.
Area of Science:
- Biochemistry
- Molecular Biology
- Chromatin Structure
Background:
- Histone H1 plays a crucial role in higher-order chromatin structure.
- Post-translational modifications of histones, such as ADP-ribosylation and phosphorylation, can influence chromatin organization and function.
- Nucleosome structure and DNA accessibility are key determinants of gene regulation.
Purpose of the Study:
- To investigate the association of modified histone H1 with specific nucleosome fractions released by micrococcal nuclease digestion.
- To examine the phosphorylation and ADP-ribosylation status of histones within different nucleosome particles.
- To assess the impact of histone H1 on DNA accessibility and thermal stability of chromatin.
Main Methods:
- Micrococcal nuclease digestion of isolated rat liver nuclei.
- Separation of nucleosome fractions using gel filtration (Sephadex G-200) and buffer extraction.
- Analysis of histone modifications (ADP-ribosylation, phosphorylation) using radiolabeling ([gamma-(32)P]ATP, [(3)H]-thymidine, [(3)H]lysine).
- DNA length determination and thermal denaturation studies.
Main Results:
- Micrococcal nuclease selectively released mononucleosomes associated with ADP-ribosylated histone H1.
- Two distinct mononucleosome populations were identified: those with dissociated H1 (140 bp DNA) and those with H1 (200 bp DNA).
- Phosphorylated histone H1 was observed, and phosphorylation of core histones (H2A, H3) was less evident in H1-containing nucleosomes.
- Newly synthesized histones and DNA were rapidly incorporated into released nucleosomes during S-phase.
- Removal of histone H1 and non-histone proteins increased DNA's susceptibility to thermal denaturation.
Conclusions:
- ADP-ribosylated and phosphorylated histone H1 is selectively associated with specific nucleosome fractions.
- Histone H1 content influences DNA accessibility and chromatin stability.
- These findings highlight the dynamic nature of histone modifications and their role in regulating chromatin structure during DNA replication.
Related Concept Videos
02:55The Nucleolus
01:24Liver Regeneration
Cells of Liver
The liver comprises four major types of cells— hepatocytes, stellate, Kupffer, and sinusoidal endothelial cells. The hepatocytes are large...