Review The architecture of interphase chromosomes and nucleolar transcription sites in plants Peter J. Shaw, a, * Rita Abranches, a Ana Paula Santos, a,b Alison F. Beven, a Eva Stoger, a,c Eva Wegel, a and Pablo Gonz alez-Melendi a,d a Department of Cell Biology, The John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK b Instituto de Tecnologia Quimica e Biologica, Apartado 127, 2781-901 Oeiras, Portugal c Institute of Molecular Biotechnology, BiologieVII, RWTH Aachen, Worringerweg 1,52074 Aachen, Germany d Centro de Investigaciones Biologicas, CSIC, Velazquez 144, 28006-Madrid, Spain Received 27 June 2002, and in revised form 20 September 2002 Abstract Fluorescence in situ hybridization (FISH) coupled with confocal microscopy has been used to reveal the interphase chromosome organization in plants. In wheat and several other related species, we have shown that the interphase chromosomes are in a very well-defined organization, with centromeres and telomeres located at opposite sides of the nuclear envelope—a classic Rabl con- figuration. In transgenic wheat lines, FISH analysis of metaphase chromosomes has shown that multiple transgene copies can be integrated along a single chromosome, with large regions of intervening genomic sequence. These multiple copies are often colo- calized in interphase, suggesting either an ectopic association or a highly reproducible interphase chromatin configuration. Bromo- uridine (BrU) incorporation has been used to label transcription sites in the nucleolus. Using pea root tissue, we have combined BrU incorporation with preembedding 1-nm gold detection to image the nucleolar transcription sites by electron microscopy. This has revealed many distinct elongated clusters of silver–gold particles. These clusters are 200–300 nm in length and are thicker at one end than the other. We suggest that each cluster corresponds to a single transcribed gene. Serial sectioning of several entire nucleoli has enabled the reconstruction of all the nucleolar transcription sites, and we have estimated that there are 200–300 transcribed genes per nucleolus. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: 1nm gold; BrU; 3D microscopy; Chromosome territory; Confocal microscopy; Electron microscopy; Fluorescence in situ hybridization; Genomic in situ hybridization; Interphase chromosomes; Nucleolus; Nucleus; Plant; Rabl configuration; rDNA, rRNA; Serial sectioning; Transcription sites; Transgene; Triticum aestivum; Pisum sativum 1. Introduction Much of the fundamental biochemistry of basic nu- clear functions such as transcription, RNA processing, and ribosome biogenesis is common to all known eu- karyotic organisms. On the other hand there is a huge diversity of large-scale structure between and within the different eukaryotic kingdoms—plants are very different organisms from animals. The comparison of subcellular organization between phylogenetically diverse organisms has the potential to provide powerful insights into the relation between biochemical processes and the associ- ated structures and organization. Thus where organiza- tion is similar across phylogeny, this suggests that such organization is necessary for function. Where it differs, this gives clues to the range of viable organization. In this review we shall outline some of our recent results in two areas of subnuclear organization in plants: the structure of interphase chromosomes and the organization of transcription of rRNA genes in the nucleolus. 2. Interphase chromosome organization Bread wheat (Triticumaestivum) has provided a useful plant system for the study of chromosome organization, Journal of Structural Biology 140 (2003) 31–38 www.academicpress.com Journal of Structural Biology * Corresponding author. Fax: +44-1603-450022. E-mail address: peter.shaw@bbsrc.ac.uk (P.J. Shaw). 1047-8477/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S1047-8477(02)00537-3