Abstract. The intranuclear position of a set of genes was analyzed with respect to the territories occupied by the whole chromosomes in which these genes are localized. Genes and their respective chromosome territories were simultaneously visualized in three-dimensionally preserved nuclei applying dual color fluorescence in situ hybridization. Three coding (DMD, MYH7, and HBB) and two noncoding sequences (D1Z2 and an anonymous sequence) were analyzed in four different cell types, including cells where DMD and MYH7 are actively transcribed. Spatial analysis by confocal laser scanning microscopy revealed that the genes are preferentially located in the periphery of chromosome territories. This positioning was independent from the activity of the genes. In contrast, the nonexpressed anonymous fragment was found randomly distributed or localized preferentially in the interior of the corresponding chromosome territory. Furthermore, the distribution of the analyzed genes within the territorial peripheries was found to be highly characteristic for each gene, and, again, independent from its expression. The impact of these findings with regard to the three-dimensional arrangement of the linear DNA string within chromosome territories, as well as with respect to a putative nuclear subcompartment confining gene expression, are discussed.'~EtPuIrTeE intensive efforts to decode the linear strucof complex genomes, up to the present day little is known about the three-dimensional genome organization within the cell nuclei of higher organisms. A territorial organization of interphase chromosomes has already been postulated by early cytologists such as Carl Rabl and Theodor Boveri (for review see Cremer, 1985), but this concept was mainly ignored in the 1960's and 70's (see e.g., Comings, 1980). The territorial organization was first shown indirectly by experiments, in which damaged regions of microirradiated cell nuclei, visualized in the subsequently prepared metaphase chromosomes, were found to be locally clustered (Zorn et al., 1979;Cremer et al., 1982). The chromosome territories were later visualized directly by means of in situ hybridization in interspecies somatic hybrid cells (Manuelidis, 1985;Schardin et al., 1985). Application of chromosome painting protocols subsequently proved that territorial organization of chromosomes occurs also in nonhybrid cells of mammalian (Cremer et al., 1988; Lichter et al., 1988a,b; Please address all correspondence to P. Lichter, Abteilung Organisation komplexer Genome, DKFZ, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Pinkel et al., 1988) and plant (Schwarzacher et al., 1989;Leitch et al., 1990) origin. However, the spatial organization of the linear DNA molecule within a chromosome territory remains unclear. Based on the analysis of nuclear distances between DNA segments from the same chromosome, it has been suggested that the spatial position of chromatin segments within a chromosome territory follows a random distribution (van den Engh et al., 1992;Yokota et al., 1995;Sa...
