Little is known about the dynamics of chromosomes in interphase nuclei. By tagging four chromosomal regions with a green fluorescent protein fusion to lac repressor, we monitored the movement and subnuclear position of specific sites in the yeast genome, sampling at short time intervals. We found that early and late origins of replication are highly mobile in G1 phase, frequently moving at or faster than 0.5 micrometers/10 seconds, in an energy-dependent fashion. The rapid diffusive movement of chromatin detected in G1 becomes constrained in S phase through a mechanism dependent on active DNA replication. In contrast, telomeres and centromeres provide replication-independent constraint on chromatin movement in both G1 and S phases.

Little is known about how chromatin folds in its native state. Using optimized in situ hybridization and live imaging techniques have determined compaction ratios and fiber flexibility for interphase chromatin in budding yeast. Unlike previous studies, ours examines nonrepetitive chromatin at intervals short enough to be meaningful for yeast chromosomes and functional domains in higher eukaryotes. We reconcile high-resolution fluorescence in situ hybridization data from intervals of 14 -100 kb along single chromatids with measurements of whole chromosome arms (122-623 kb in length), monitored in intact cells through the targeted binding of bacterial repressors fused to GFP derivatives. The results are interpreted with a flexible polymer model and suggest that interphase chromatin exists in a compact higher-order conformation with a persistence length of 170 -220 nm and a mass density of Ϸ110 -150 bp͞nm. These values are equivalent to 7-10 nucleosomes per 11-nm turn within a 30-nm-like fiber structure. Comparison of long and short chromatid arm measurements demonstrates that chromatin fiber extension is also influenced by nuclear geometry. The observation of this surprisingly compact chromatin structure for transcriptionally competent chromatin in living yeast cells suggests that the passage of RNA polymerase II requires a very transient unfolding of higher-order chromatin structure.higher-order structure ͉ 30-nm fiber ͉ nucleosomes G enetic studies indicate that the spatial positioning of the genome in interphase contributes to the regulation of nuclear functions, yet the principles that govern the organization of interphase chromosomes (Chrs) are largely unknown. At the simplest level, DNA is folded through interaction with histones forming the nucleosome core particle (NCP), which yields a 6:1 or 7:1 compaction ratio depending on linker length. Arrays of nucleosomes are further condensed by Ϸ6-fold into a higherorder structure, the so-called 30-nm fiber, whose in vivo architecture is unresolved. Several models have been proposed for this structure (ref. 1 and reviewed in ref. 2), yet species' specific variation in linker histones and nucleosome repeat lengths may lead to variation in fiber characteristics. How interphase Chrs fold beyond this level of organization is even less well understood, although in addition to fiber compaction, local looping and͞or anchoring to subnuclear elements may influence chromatin conformation (3, 4).To analyze chromatin compaction ratios in interphase nuclei, laboratories have generally applied fluorescence in situ hybridization (FISH), using differentially derivatized probes. These data were interpreted as identifying mega bp-sized loops (averaging 3,000 kb) with the bases of the loops being distributed in a random walk throughout the nucleoplasm (5-7) or as a chain of chromosomal subcompartments each comprising Ϸ10-20 loops of Ϸ120 kb (8). The random distribution of the chain may reflect local chromatin dynamics, which have been recently well documented in living cells by rapi...

The centromere-specific histone variant CENP-A (CID in Drosophila) is a structural and functional foundation for kinetochore formation and chromosome segregation. Here, we show that overexpressed CID is mislocalized into normally noncentromeric regions in Drosophila tissue culture cells and animals. Analysis of mitoses in living and fixed cells reveals that mitotic delays, anaphase bridges, chromosome fragmentation, and cell and organismal lethality are all direct consequences of CID mislocalization. In addition, proteins that are normally restricted to endogenous kinetochores assemble at a subset of ectopic CID incorporation regions. The presence of microtubule motors and binding proteins, spindle attachments, and aberrant chromosome morphologies demonstrate that these ectopic kinetochores are functional. We conclude that CID mislocalization promotes formation of ectopic centromeres and multicentric chromosomes, which causes chromosome missegregation, aneuploidy, and growth defects. Thus, CENP-A mislocalization is one possible mechanism for genome instability during cancer progression, as well as centromere plasticity during evolution.