Preparation of centromeric heterochromatin by restriction endonuclease digestion of mouse L929 cells

Abstract

When L929 cells in metaphase are digested with either Eco RI or Alu I, chromatin containing about 85% of the DNA is released. DNA from the Alu I- and Eco RI-resistant chromatin is enriched 6.8- and 3.7-fold, respectively, in satellite sequences. Analysis by electron microscopy of these digests reveals the existence of structures containing condensed heterochromatin and kinetochores. When these preparations are incubated with anticentromere serum from a human CREST scleroderma patient and then with rhodamine-conjugated antihuman IgG, fluorescence appears in the form of paired dots, the same pattern found in whole metaphase chromosomes. The fluorescent staining pattern, the electron microscopy, and the enrichment of satellite DNA sequences together support the conclusion that the Eco RI- and Alu I-resistant structures contain centromeres. We anticipate that these preparations will be useful in studies of the interactions between centromeric heterochromatin, kinetochores, and microtubules.