Segregation of transcription and replication sites into higher order domains

Science

Volumn 281, Issue 5382, 1998, Pages 1502-1505

  Wei, Xiangyun ^a   Samarabandu, Jagath ^a,b   Devdhar, Rakendu S ^a   Siegel, Alan J ^a   Acharya, Raj ^a   Berezney, Ronald ^a  

^a UNIVERSITY AT BUFFALO   (United States)

^b Life Imaging Systems Inc   (Canada)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; CELLULAR DISTRIBUTION; DNA REPLICATION; DNA TRANSCRIPTION; GENOME; HUMAN; MOLECULAR DYNAMICS; NONHUMAN; PRIORITY JOURNAL;

3T3 CELLS; ANIMALS; CELL LINE; CELL NUCLEOLUS; CELL NUCLEUS; DNA REPLICATION; GENOME; GENOME, HUMAN; HUMANS; IMAGE PROCESSING, COMPUTER-ASSISTED; MICE; MICROSCOPY, CONFOCAL; MODELS, BIOLOGICAL; S PHASE; TRANSCRIPTION, GENETIC;

MAMMALIA;

EID: 0032483372     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.281.5382.1502     Document Type: Article

References (28)

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13. 2, 150 mM NaCl, 25% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, RNasin (25 U/ml), 1.8 mM ATP, 0.5 mM CTP, GTP, BrUTP, 0.1 mM dATP, 0.1 mM dCTP, 0.1 mM dGTP, and 25 μM digoxigenin-11-dUTP] at room temperature for 30 min to label transcription sites and replication sites simultaneously, then were fixed in 100% methanol at -20°C for 20 min followed by 3% paraformaldehyde in phosphate-buffered saline on ice for 5 min.
14. For labeling RNA sites, rat monoclonal antibody to BrdU (Sera-Lab) was used followed by biotin-conjugated goat anti-rat immunoglobulin C and Texas red-conjugated stepavidin incubation. Fluorescein isothiocyanate (FITC)-conjugated sheep anti-digoxigenin Fab · fragments (Boehringer Mannheim) were used for labeling DNA sites. Images from 0.5-μm optical sections were collected with a Bio-Rad MRC-1024 confocal microscope equipped with a krypton argon laser to excite FITC and Texas red simultaneously at 488-nm and 568-nm wavelength, respectively.
15. DNA replication is temporally controlled in the S phase, with transcriptionally active genes usually replicated in early S phase and nonactive genes in late S phase [ M. A. Goldman, G. P. Holmquist, M. C. Gray, L. A. Caston, A. Nag, Science 224, 686 (1984); K. A. Hatton et al., Mol. Cell. Biol. 8, 2149 (1988); S. Selig, K. Okumura, D. C. Ward, H. Cedar, EMBO J. 11, 1217 (1992)]. Also, DNA replication sites show different patterns in different periods of S phase (5).
16. DNA replication is temporally controlled in the S phase, with transcriptionally active genes usually replicated in early S phase and nonactive genes in late S phase [ M. A. Goldman, G. P. Holmquist, M. C. Gray, L. A. Caston, A. Nag, Science 224, 686 (1984); K. A. Hatton et al., Mol. Cell. Biol. 8, 2149 (1988); S. Selig, K. Okumura, D. C. Ward, H. Cedar, EMBO J. 11, 1217 (1992)]. Also, DNA replication sites show different patterns in different periods of S phase (5).
17. DNA replication is temporally controlled in the S phase, with transcriptionally active genes usually replicated in early S phase and nonactive genes in late S phase [ M. A. Goldman, G. P. Holmquist, M. C. Gray, L. A. Caston, A. Nag, Science 224, 686 (1984); K. A. Hatton et al., Mol. Cell. Biol. 8, 2149 (1988); S. Selig, K. Okumura, D. C. Ward, H. Cedar, EMBO J. 11, 1217 (1992)]. Also, DNA replication sites show different patterns in different periods of S phase (5).
18. Visual comparison of replication sites and transcription sites was used to determine the number of replication sites that are colocalized with transcription sites. The total numbers of replication and transcription sites were calculated, using a segmentation algorithm specifically developed in our laboratory for three-dimensional confocal images [J. Samarabandu, H. Ma, R. Acharya, P. C. Cheng, R. Berezney, Proc. SPIE 2434, 370 (1995)].
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20. A. B. Hassan, R. Errington, N. White, D. Jackson, P. Cook, J. Cell Sci. 107, 425 (1994).
21. Using Microsoft Power Point, nuclear regions with three or more sites of one and only one activity were contoured as preferential cluster regions. Regions with mixed distribution patterns of replication and transcription sites were displayed as yellow. The relative sizes of the corresponding areas were directly measured.
22. If we include nucleolar regions in the transcription cluster category, the area occupied by clustered replication and transcription sites will increase to about 80% of the total contoured areas.
23. 1/S border showed that 64% of the contoured area is occupied by replication and transcription site clusters.
24. Cluster analysis was performed on "Sn" number of clusters that were generated using the "random ( )" library function. Of the total number of sites, some were randomly chosen to be transcription sites (red) and the remaining were marked as replication sites (green). Computation of radii of clusters is based on a Gaussian distribution function with a standard deviation of 1.7.
25. Three-dimensional reconstruction of the contours was performed from the confocal optical sections using the drawing tool of IPlab (Signal Analytics).
26. C. Zeng et al., Proc. Natl. Acad. Sci. U.S.A. 94, 6746 (1997); C. Zeng et al., ibid. 95, 1585 (1998).
27. C. Zeng et al., Proc. Natl. Acad. Sci. U.S.A. 94, 6746 (1997); C. Zeng et al., ibid. 95, 1585 (1998).
28. R. Summers and A. Stonebraker (Confocal Microscopy and 3D Imaging Facility of the School of Medicine and Biomedical Sciences, SUNY at Buffalo) provided valuable assistance with confocal microscopy. Computer image analysis was performed at the Microscopic Imaging Facility (Department of Biological Sciences, SUNY at Buffalo). NHF-1 normal human fibroblast cells were kindly provided by D. G. Kaufman, University of North Carolina, School of Medicine. Supported by NIH grant GM 23922 (R.B.) and a Mark Diamond Graduate Student Research Grant from SUNY at Buffalo (X.W.) (45F97).