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Volumn 272, Issue 5262, 1996, Pages 725-728

Homologous association of oppositely imprinted chromosomal domains

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; CELL CYCLE S PHASE; CHROMOSOME 15Q; CHROMOSOME ANALYSIS; CONTROLLED STUDY; FLUORESCENCE IN SITU HYBRIDIZATION; GENE LOCUS; GENOME IMPRINTING; HAPPY PUPPET SYNDROME; HUMAN; HUMAN CELL; PRADER WILLI SYNDROME; PRIORITY JOURNAL; T LYMPHOCYTE;

EID: 0030043993     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5262.725     Document Type: Article
Times cited : (203)

References (37)
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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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    • 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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