Nuclear Organization and Gene Expression: Visualization of Transcription and Higher Order Chromatin Structure

Excerpt

The way chromatin is organized inside the nucleus of eukaryotic cells is a key factor in gene control. It seems that the cells are able to transcriptionally regulate genomic loci by changing their local environments in the nuclei. Such environment may inhibit, permit, or boost gene activity. To unravel underlying molecular mechanisms, it is essential to develop techniques that allow the simultaneous microscopic visualization of specific DNA sequences and of components of the protein machineries in the nucleus that are involved in the synthesis, processing, and transport of RNA and in the replication and repair of DNA. Visualization of DNA sequences can be achieved by fluorescence in situ hybridization (FISH). Fluorescent labelling of specific proteins is achieved by indirect immunolabelling.

This report will concentrate on the combination of both techniques under conditions that the structure of the cell nucleus remains largely intact. Applications will be presented in the context of investigation of the functional organization of the cell nucleus. The approach that is taken and the protocol that is presented can be generalized for labelling of essentially any DNA sequence and protein antigen.