Structural Organisation of the Interphase Genome
Investigations of the spatial and functional organisation of the genome in vivo must consider the particular characteristics of the intranuclear milieu: macromolecules are unusually concentrated (400 mg/ml in dense chromatin: 1), designed to bind to others by interactions exquisitely sensitive to environmental conditions, and constrained within the meshwork formed by the nuclear lamina. Current models of genome organisation are influenced by these factors; for example they cause nuclear-targeted reporter proteins (GFP, lacZ), enzymes extractable from nuclei in low ionic strength buffers (topoisomerase 2, RNA polymerase 2), and putative attachment regions in DNA (MAR/SARs) to become insolubilised during preparation of the nuclear "matrix" (2,3, reviewed in 4). Further, the concept that genomic DNA forms a series of loops may need reevaluation because the DNA loops seen in microscopical and biochemical studies to radiate from matrix or halo preparations could be entrapped by extrusion through the lamina meshwork, rather than attached. In alternative approaches, we are using nuclei prepared and fractionated in physiological conditions (5) to examine if macromolecular crowding and phase partitioning effects can provide new models for the compartmentalisation of proteins and functions into different intranuclear domains and of chromosomes into territories, and characterising periodically-spaced regions in genomic DNA which frame genes and are specifically accessible in vivo to single-strand nucleases and to topoisomerase 2, which could represent regions of folding of chromatin. (Supported by the Medical Research Council of Canada).
1. B. Bohrmann, B, Haider, and E. Kellenberger, Ultramicroscopy 49, 235 (1993)
Laval University Cancer Research Centre, Quebec, Canada