Common fragile sites are chromosomal loci prone to breakage and rearrangement, hypothesized to provide targets for foreign DNA integration. We cloned a simian virus 40 integration site and showed by f luorescent in situ hybridization analysis that the integration event had occurred within a common aphidicolin-induced fragile site on human chromosome 7, FRA7H. A region of 161 kb spanning FRA7H was defined and sequenced. Several regions with a potential unusual DNA structure, including high-f lexibility, lowstability, and non-B-DNA-forming sequences were identified in this region. We performed a similar analysis on the published FRA3B sequence and the putative partial FRA7G, which also revealed an impressive cluster of regions with high f lexibility and low stability. Thus, these unusual DNA characteristics are possibly intrinsic properties of common fragile sites that may affect their replication and condensation as well as organization, and may lead to fragility.

SummaryFluorescence imaging of two independently labelled proteins is commonly used to determine their co-localization in cells. Antibody-mediated crosslinking can mediate the patching of such proteins at the cell surface, and their co-localization can serve to determine complex formation among them. However, manual analysis of such studies is both tedious and subjective. Here we present a digital co-localization analysis that is independent of the fluorescence intensity, is highly consistent and reproducible between observers, and dramatically reduces the analysis time. The approach presented is based on a segmentation procedure that creates binary objects, and then determines whether objects belonging to two different groups (e.g. greenand red-labelled) are co-localized. Two methods are used to determine co-localization. The 'overlap' analysis defines two objects as co-localized if the centre of mass of one falls within the area of the other. The 'nearest-neighbour distance' analysis considers two objects as co-localized if their centres are within a threshold distance determined by the imaging modality. To test the significance of the results, the analysis of the actual images is tested against randomized images generated by a method that creates images with uncorrelated distributions of objects from the two groups. The applicability of the algorithms presented to study protein interactions in live cells is demonstrated by co-patching studies on influenza haemagglutinin mutants that do or do not associate into mutual oligomers at the cell surface via binding to AP-2 adaptor complexes. The approach presented is potentially applicable to studies of co-localization by other methods (e.g. electron microscopy), and the nearest-neighbour distance method can also be adapted to study phenomena of correlated placement.

In multicellular organisms, the higher order organization of chromatin during interphase and the reassembly of the nuclear envelope during mitosis are thought to involve an interaction between the nuclear lamina and chromatin. The nuclear distribution of lamins and of peripheral chromatin is highly correlated in vivo, and lamins bind specifically to chromatin in vitro. Deletion mutants of Drosophila lamin Dm 0 were expressed to map regions of the protein that are required for its binding to chromosomes. Underlying the inner nuclear membrane and abutting the chromatin is the filamentous protein meshwork of the nuclear lamina (reviewed in refs. 1-4). The nuclear lamina is involved in several biological activities, including the regulation of the size, shape, and assembly of the nuclear envelope (5-10); facilitation of higher order chromatin organization (7,8,11); and regulation of DNA replication (12)(13)(14). Changes in the nuclear lamina composition during development point toward a possible role for lamins, which are the major proteins of the nuclear lamina, in cell differentiation (reviewed in ref. 15). The nuclear lamina also is a major substrate for signals that control the cell cycle (16), and lamins are specifically degraded in apoptosis (17).Lamins are classified as type V intermediate filament proteins, and like all intermediate filaments, they contain a helical rod domain flanked by amino (head) and carboxyl (tail) domains (reviewed in refs. 16 and 18). Different eukaryotes possess between one and six lamin genes. Mammalian lamins A and C result from alternative splicing of the same gene product, whereas lamins B1-B3 and C2 are coded for by separate genes (19). The two major lamins in chicken are lamins A and B2 (20). An additional minor species is termed lamin B1. Xenopus laevis has at least five different lamin genes (21,22). Drosophila melanogaster has two lamin genes, termed lamin Dm 0 and lamin C (23, 24). Caenorhabditis elegans probably has only a single lamin gene, termed CeLam-1 (25).Three-dimensional in vivo studies in Drosophila and mammalian cells revealed that lamin fibers are closely associated with chromatin fibers (26). In vitro studies have shown that lamins can specifically bind chromatin fragments and interphase chromatin (27-29), condensed in vitro assembled chromatin (9), or mitotic chromosomes (30,31). Lamins can also bind chromosomal proteins (27-32) and specific DNA sequences, such as M͞SARs (33-36) and telomeric sequences (37). The binding of lamins to chromatin is specific and depends on the integrity of the chromosomes. Human lamin A binds in vitro to polynucleosomes with a dissociation constant of about 1 nM (29). A binding site for mammalian lamins A and B to chromatin was localized at their tail domain (28). In the latter study, the dissociation constant of the tail domain binding to interphase chromatin was estimated to be in the range of 0.12-0.3 M, and the binding was mediated by core histones. The actual association of the lamin filament may be stronger, because ...