The Nuclear Matrix Prepared by Amine Modification

• Published in: Proceedings of the National Academy of Sciences - PNAS 1999-02, Vol.96 (3), p.933-938
• Main Authors: Wan, Katherine M., Nickerson, Jeffrey A., Krockmalnic, Gabriela, Penman, Sheldon
• Format: Article
• Language: English
• Subjects: Amines; Amino acids; Ammonium Sulfate; Biological Sciences; Cell extracts; Cell Fractionation - methods; Cell Line; Cell nucleus; Cells; Cellular biology; Chromatin; Chromatin - ultrastructure; Deoxyribonucleases, Type II Site-Specific; DNA; DNA - analysis; Elution; Histones - isolation & purification; Humans; Hypertonic Solutions; Microscopy, Electron; Nuclear matrix; Nuclear Matrix - ultrastructure; Nuclear Proteins - isolation & purification; Nuclear structure; Theca cells; Tritons; Tumor Cells, Cultured
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.96.3.933

The nucleus is spatially ordered by attachments to a nonchromatin nuclear structure, the nuclear matrix. The nuclear matrix and chromatin are intimately connected and integrated structures, and so a major technical challenge in nuclear matrix research has been to remove chromatin while retaining a native nuclear matrix. Most methods for removing chromatin require first a nuclease digestion and then a salt extraction to remove cut chromatin. We have hypothesized that cut chromatin is held in place by charge interactions involving nucleosomal amino groups. We have tested this hypothesis by chemically modifying amino groups after nuclease digestion. By using this protocol, chromatin could be effectively removed at physiological ionic strength. We compared the ultrastructure and composition of this nuclear matrix preparation with the traditional high-salt nuclear matrix and with the third nuclear matrix preparation that we have developed from which chromatin is removed after extensive crosslinking. All three matrix preparations reveal internal nuclear matrix structures that are built on a network of branched filaments of about 10 nm diameter. That such different chromatin-removal protocols reveal similar principles of nuclear matrix construction increases our confidence that we are observing important architectural elements of the native structure in the living cell.