Cytological studies have suggested that chromosomes may be nonrandomly arranged in the nucleus, which raises two questions. First, to what extent is the position of a given chromosomal locus determined within the nucleus, and second, to what extent can a given chromosomal locus move around within the nucleus. This work addresses these two questions. Fluorescence in situ hybridization (FISH) and three-dimensional microscopy were used to determine the position of 41 different DNA probes within the nucleus in Drosophila melanogaster embryos. Every locus was found to reproducibly occupy a distinct subregion of the nucleus. In particular, by using a Monte Carlo statistical test, a set of loci was identified that associate reproducibly with the nuclear envelope (NE). These NE association sites are distributed throughout the genome, spaced at intervals of roughly 1 Mb. NE association sites do not correspond to binding sites of known Drosophila chromatin proteins, to known boundary elements, or to scaffold attachment regions (SARs). NE association sites include both euchromatic and heterochromatic regions, and not all heterochromatin is NE associated. The NE associations defined by this work are not seen in telophase and are established later in interphase, demonstrating that this interaction does not play a role in post-mitotic NE reassembly.; The highly specific positioning observed by FISH raises the second question, is chromatin mobile within the interphase nucleus. To answer this, strategies were developed for measuring the diffusion of interphase chromatin in Drosophila and Saccharomyces cerevisiae. In both cases it was found that chromatin can indeed diffuse within the nucleus, with a diffusion constant of approximately 10{dollar}sp{lcub}-12{rcub}{dollar} cm{dollar}sp2{dollar}/s. In yeast, but not Drosophila, the diffusion was constrained, implying that the chromatin is tethered to an internal nuclear structure. This diffusive motion is likely to be Brownian as it is unaffected by treatment of cells with azide, a metabolic inhibitor. The diffusion constant was found to be surprisingly size independent, such that a small plasmid diffused slower, rather than faster, than an entire yeast chromosome. This behavior is also consistent with a tethering model. This work together with the FISH experiments in Drosophila embryos, suggest a picture of nuclear architecture in which chromosomes are held in precisely defined positions by being tethered to an immobile internal structure. These results have important functional consequences for processes such as meiosis that involve large-scale chromosome motion.
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