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4 fractions (6). A modified FISH protocol was developed that minimizes alterations in the normal spatial organization of the nucleus (21) Cells were adhered for 5 min to poly-D-lysine-coated slides, fixed 15 min in 4% paraformaldehyde and 1% methanol in phosphate-buffered saline (PBS), washed twice in 0 3 M glycine and PBS, permeabilized for 10 min in 0 5% Tween and 0.2 N HCl, washed twice in 2x saline sodium citrate (SSC), denatured 3 min in 70% tormamide and 2x SSC, washed twice in 0.5% Tween and PBS and twice in 2x SSC at 4°C, then hybridized and washed as previously described (6) without detection steps D15Z1 (pHSR) and D12Z3 (pA12H8, ATCC) were directly labeled by nick translation with Cy3- and Cy5-labeled deoxycytidine triphosphate (Biological Detection), respectively, and nuclei were counterstained with YOPRO (Molecular Probes). Nuclei were imaged by a Molecular Dynamics CLSM Multiprobe 2001 with an Ar/Kr laser. Imagespace software (Molecular Dynamics) was used for scanning, analysis, and projection images Nuclei were densely distributed (Fig. 1A), and we selected clusters of 6 to 20 nuclei by using only the DNA counterstain filter to avoid bias in selection. Optical sections were scanned with a 60x objective at a 0.21, 0.21, 0.29 voxel resolution and a total image size of 512 by 512 pixels. Hybridization efficiency was 85% (number of nuclei with two red and two green signals out of the total number of nuclei scanned, in an average of four representative experiments). The x, y, and z coordinates were defined at the center of each hybridization signal and used to measure 3D distance by a mouse-driven cursor.
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The distance of 2 0 μm was chosen on the basis of a comparison of the graph of late S phase lymphocytes to that of premeiotic yeast (22). The degree of nonrandomness was similar, but the scale of the graphs was different because the diameter of yeast nuclei was ∼5 μm, whereas lymphocytes are ∼6 to 12 μm (13). Pairing in premeiosis and meiosis may involve a tighter association between homologs, or yeast chromosomes may have fewer spatial constraints than chromosomes in higher eukaryotes, thus explaining the difference in actual distances.
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2), lack of physical spreading of DNA by the altered FISH protocol, and larger probe see (centromeric repeats and 80- to 120-kb P1 clones) In the infrequent cases where replication doublets were observed, the measurement was taken at the point between the two signals Source numbers for P1 clones at Genomesystems are as follows: GABRB3/A5, 2269: D15S113, 6343; D13S104, 6269, D15S128, 7207; and D15S46 (ATCC)
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We thank W. Robinson, K Hirschom, S. Orkin, A. Bottani, B. Horsthemke, R Wharton, J Holden, B White, M. Higgins, A Flint, K. Glatt, O. M Liu, H. Lidov, J Wagstaff, and L M. Kunkel for their help and important contributions to this work Supported by Howard Hughes Medical Institute and NIH grant RO1-NS30628
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