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First published online 13 May 2008
doi: 10.1242/jcs.026989

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Replication-timing-correlated spatial chromatin arrangements in cancer and in primate interphase nuclei

¹ Department of Biology II, Human Genetics, Ludwig-Maximilians University Munich, 82152 Planegg-Martinsreid, Germany
² The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge CB10 1SA, UK
³ Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians University Munich, Munich, Germany

View larger version (79K): [in this window] [in a new window]   Fig. 1. (A-D) Representative FISH experiments with a two-color clone pool for one early-replicating (green) and/or late-replicating (red) locus per chromosome 1-22 and X. (A) Human metaphase chromosomes. (B-G) Dual-color displays of a representative human fibroblast nucleus (maximum intensity projection) after 3D-FISH of the chromosomes 1-22;X clone pools and double-pulse-labeled replication foci. Scale bar: 5 µm. (B) Early-replication foci in green, late-replication foci in red. (C) Early-replicating clone FISH signals in green, late-replicating clone FISH signals in red. (D) Early-replicating clone FISH signals in green, early-replication foci in red. (E) Late-replicating clone FISH signals in green, early-replication foci in red. (F) Early-replicating clone FISH signals in green, late-replication foci in red. (G) Late-replicating clone FISH signals in green, late-replication foci in red. (H) 3D reconstructed human fibroblast nucleus. (I,J) 3D maximum intensity projections of nuclei from hybridized tumor cell lines Mel Juso and SW620. DAPI counterstain in blue. Scale bar: 5 µm. (K-M) Quantitative evaluation of the radial probe distribution in the nucleus (n=number of nuclei) using eADS software for (K) human fibroblasts, together with early-replication and late-replication foci, (L) Mel Juso and (M) SW620.

View larger version (50K): [in this window] [in a new window]   Fig. 2. (A-D) FISH with human chromosome-2-specific pools of large-insert clones representing early-replicating (green) and late-replicating (red) loci to (A) human (schematic of human chromosome 2, together with the mapping position of large-insert clones is shown alongside) and (B) gorilla metaphase chromosomes. (C,D) 3D maximum intensity projections of hybridized human fibroblast and gorilla lymphoblastoid cell nuclei, with early clones labeled green, late clones red, chromosome 2 territories blue and DAPI counterstain gray. Scale bar: 5 µm. (E-J) Quantitative evaluation of radial probe distributions using 3D-RRD or eADS software (n=number of nuclei). The distribution of chromosome 2 clone pools (E) in human fibroblasts, (F) in human lymphoblastoid cell nuclei and (G) in gorilla lymphoblastoid cell nuclei. (H,I) The radial probe distribution with respect to the human chromosome 2 territory and (J) the localization of individual clones 2-3l and 2-6l and the chromosome 2 territory in human fibroblast nuclei.

View larger version (57K): [in this window] [in a new window]   Fig. 3. FISH with pooled large-insert clones representing chromosome 17 early-replicating (green) and chromosome 5 late-replicating (red) loci to (A) human (schematics of human chromosomes 5 and 17, together with the mapping position of large-insert clones, are shown alongside) and (B) Concolor gibbon metaphase chromosomes. 3D maximum intensity projections of nuclei from (C) human, (D) gorilla, (E) Concolor gibbon lymphoblastoids and (F) Mel Juso tumor cells hybridized with these probe pools. In C-F, chromosome 17 early-replicating clones are labeled green, chromosome 5 late-replicating clones red, chromosome 5 and 17 territories blue and DAPI counterstain gray, except for F, in which the wild-type CT17 is shown in blue and the CT der(17) in red. Scale bar: 5 µm. (G-L) Quantitative evaluation of radial probe distributions in the interphase nucleus or the respective chromosome territory, using 3D-RRD or eADS software (n=number of nuclei).

View larger version (66K): [in this window] [in a new window]   Fig. 4. Representative (A) orangutan (schematic of human chromosome 7, together with the mapping position of large-insert clones, is shown alongside) and (B) Karpas 384 metaphases after FISH with human chromosome-7-specific pools of large-insert clones representing early-replicating (green) and late-replicating (red) loci. (C,D,F,G) 3D maximum intensity projections of nuclei from (C) human fibroblasts, (D) Concolor gibbon lymphoblastoids, (F) orangutan lymphoblastoids and (G) Mel Juso cells hybridized with the early-replicating clone pool labeled green, the late-replicating clone pool in red, chromosome 7 territories blue and DAPI counterstain gray. (E) Human and (H) orangutan z-projected fibroblast nuclei after multicolor 3D-FISH with five BAC clones. The clone color code follows panel K. Scale bar: 5 µm. (I-K,O-Q) Quantitative evaluation of radial probe distributions in the interphase nucleus or the respective chromosome territory, using 3D-RRD or eADS software (n=number of nuclei). (L,M) Mean pair-wise clone distance measurements in human and orangutan fibroblast nuclei from the 3D-FISH experiments illustrated in E,H,K. (N) In both species the mean square interphase distance between two clone loci showed a linear correlation with their genomic distance, indicating a `random walk' 3D conformation of these loci.

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