Nuclear organization and gene expression: visualization of transcription and higher order chromatin structure Authors

The way chromatin is organized inside the nucleus of eukaryotic cells is a key factor in gene control. It seems that the cell is able to transcriptionally regulate genomic loci by changing their local environment in the nucleus. 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 fluorescent in situ hybridization (FISH). Fluorescent labelling of specific proteins is achieved by indirect immunolabelling. This contribution 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.