Structure of active chromatin: isolation and characterization of transcriptionally active chromatin from rat liver

Abstract

Rat liver nuclei were isolated in low-ionic-strength buffer in the absence of bi- and multi-valent cations. Digestion of these nuclei by endogenous nuclease, micrococcal nuclease and DNase I revealed that a minor chromatin fraction was preferentially digested into poly- and oligo-nucleosomes. Southern blot hybridization with various active gene probes confirmed that these chromatin fragments represent coding and 5ƀ upstream regions of transcriptionally active chromatin. Active chromatin fragments were released selectively into the medium, with inactive chromatin remaining inside the nuclei, under the above ionic conditions. The inclusion of bivalent cations during the digestion of nuclei reversed the solubility behaviour of active chromatin. Rearrangement and exchange of histone H1 between chromatin fragments was prevented by using low-salt conditions in all steps in the absence of bivalent cations. All histones, including H1, were present in stoichiometric amounts in this active chromatin fraction. Active nucleosomes showed a lower electrophoretic mobility than bulk nucleosomes in an acrylamide/agarose composite gel in the absence of Mg2+, but were selectively bound to the gel in the presence of this ion.

Intermittent fasting prevents the progression of type I diabetic nephropathy in rats and changes the expression of Sir2 and p53

“…Total proteins were isolated from kidney tissue homogenates by sonication and nuclei were isolated according to method described by Tikoo et al [22]. Histones were acid extracted using 0.25 M HCl.…”

Modulation of histone deacetylase attenuates naloxone-precipitated opioid withdrawal syndrome

“…Nuclei, histone isolation and western blotting were performed as described previously (Tikoo et al 1997(Tikoo et al , 2007a(Tikoo et al , b, 2008. Immunoblot analysis was performed using anti-acetylated histone H3 (rabbit, 1:5,000; Millipore, Billerica, MA, USA) and anti-histone H3 (rabbit, 1:5,000, Millipore, Billerica, MA, USA) conjugated secondary antibodies (anti-rabbit) from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA.…”

Differential Analysis of Active and Inactive Genes in Human Neutrophils by Chromosomal In Situ Hybridization

“…Active chromatins have higher sensitivity to DNase I [5,10,15,25,33]. On the Xa-chromatin DNA, the whole CpG island of the DNA is completely unmethylated.…”

“…In the present study, we attempted to explore the protocol of in situ hybridization to discriminate between active and inactive genes by combining use of endogenous Ca/Mgdependent endonuclease and exogenous DNase I, which are known to extract the DNA of inactive and active chromatin parts, respectively [5,10,15,25,31,33]. When the protocol was applied to localize X-chromatin in female neutrophil nuclei, active Xa-chromatin DNA was detected in the inner region and inactive Xi-chromatin DNA was found in the outer region of the nuclei, as was expected.…”