Techniques are reported for the isolation from large quantities of chick intestinal mucosa of 75-80% of the cellular DNA as purified nuclei or nuclear chromatin. The procedure for the isolation of the nuclei involves treatment of the crude nuclear fraction with 0.1% Triton X-100 followed by sedimentation through 1.75 M sucrose at pH 7.5 containing 0.025 M KCl, 0.05 M Tris, and 0.005 M magnesium chloride. Chromatin is prepared from nuclei by gentle breaking of the nuclear membrane with hypotonic EDTA and homogenization. The chromatin is purified by centrifugation in 1.75 M sucrose. Both techniques permit the isolation of other cytoplasmic components such as the mitochondrial and microsomal fractions. Electron micrographic examination of the nuclei and chromatin indicate that they comprise relatively homogeneous and morphologically intact structures. Chemical analysis of crude nuclei, purified nuclei and chromatin revealed an mg ratio of RNA:DNA of 0.80:1.0, 0.30:1.00, and 0.12:1.0 respectively. When assayed for template efficiency in the synthesis of RNA, the chromatin is shown to be biologically active.
ASJC Scopus subject areas
- Cell Biology