SPATIAL DISTRIBUTIONS OF EARLY AND LATE REPLICATING CHROMATIN IN INTERPHASE CHROMOSOME TERRITORIES

The surface area of chromosome territories has been suggested as a pre ferred site for genes, specific RNAs, and accumulations of splicing fa ctors. Here, we investigated the localization of sites of replication within individual chromosome territories, In vivo replication labeling with thymidine analogues IdUrd and CldUrd was combined with chromosom e painting by fluorescent in situ hybridization on three-dimensionally preserved human fibroblast nuclei. Spatial distributions of replicati on labels over the chromosome territory, as well as the territory volu me and shape, were determined by 3D image analysis. During late S-phas e a previously observed shape difference between the active and inacti ve X-chromosome in female cells was maintained, while the volumes of t he two territories did not differ significantly, Domains containing ea rly or mid to late replicating chromatin were distributed throughout t erritories of chromome 8 and the active X In the inactive X-chromosome early replicating chromatin was observed preferentially near the terr itory surface. Most important, we established that the process of repl ication takes place in foci throughout the entire chromosome territory volume, in early as well as in late S-phase. This demonstrates that a ctivity of macromolecular enzyme complexes takes place throughout chro mosome territories and is not confined to the territory surface as sug gested previously. (C) 1998 Academic Press.