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Spatial relationship between transcription sites and chromosome territories.

Verschure PJ, van Der Kraan I, Manders EM, van Driel R - J. Cell Biol. (1999)

Bottom Line: Transcription sites were found scattered throughout the territory of one of the two X chromosomes, most likely the active X chromosome, and that of both territories of chromosome 19.Strikingly, nascent RNA was almost exclusively found in the interchromatin areas in chromosome territories and in between strongly GFP-labeled chromatin domains.Active loci are located predominantly at or near the surface of compact chromatin domains, depositing newly synthesized RNA directly into the interchromatin space.

View Article: PubMed Central - PubMed

Affiliation: E.C. Slater Instituut, BioCentrum Amsterdam, University of Amsterdam, 1018 TV Amsterdam, The Netherlands. a311pjvx@chem.uva.nl

ABSTRACT
We have investigated the spatial relationship between transcription sites and chromosome territories in the interphase nucleus of human female fibroblasts. Immunolabeling of nascent RNA was combined with visualization of chromosome territories by fluorescent in situ hybridization (FISH). Transcription sites were found scattered throughout the territory of one of the two X chromosomes, most likely the active X chromosome, and that of both territories of chromosome 19. The other X chromosome territory, probably the inactive X chromosome, was devoid of transcription sites. A distinct substructure was observed in interphase chromosome territories. Intensely labeled subchromosomal domains are surrounded by less strongly labeled areas. The intensely labeled domains had a diameter in the range of 300-450 nm and were sometimes interconnected, forming thread-like structures. Similar large scale chromatin structures were observed in HeLa cells expressing green fluorescent protein (GFP)-tagged histone H2B. Strikingly, nascent RNA was almost exclusively found in the interchromatin areas in chromosome territories and in between strongly GFP-labeled chromatin domains. These observations support a model in which transcriptionally active chromatin in chromosome territories is markedly compartmentalized. Active loci are located predominantly at or near the surface of compact chromatin domains, depositing newly synthesized RNA directly into the interchromatin space.

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Preservation of the spatial distribution of centromeres and PML bodies in human primary fibroblasts during the FISH procedure. The spatial distribution of PML bodies and of centromeres were analyzed before and after the FISH procedure in the same nucleus. A, B, and C, Corresponding individual optical sections of the same nucleus labeled with anti-PML antibody are shown in A (before the FISH procedure) and B (after FISH). C shows an overlay of A (red) and B (green). Bar, 0.84 μm. D, E, and F, Corresponding individual optical sections of the same nucleus labeled with anticentromere antibody. D, Before FISH procedure. E, After FISH procedure. F shows an overlay of D (red) and E (green). The FISH procedure results in only small changes in the distribution of PML bodies and centromeres. These changes are in part due to a slight tilting of the nuclei during FISH labeling. Bar, 0.84 μm.
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Figure 2: Preservation of the spatial distribution of centromeres and PML bodies in human primary fibroblasts during the FISH procedure. The spatial distribution of PML bodies and of centromeres were analyzed before and after the FISH procedure in the same nucleus. A, B, and C, Corresponding individual optical sections of the same nucleus labeled with anti-PML antibody are shown in A (before the FISH procedure) and B (after FISH). C shows an overlay of A (red) and B (green). Bar, 0.84 μm. D, E, and F, Corresponding individual optical sections of the same nucleus labeled with anticentromere antibody. D, Before FISH procedure. E, After FISH procedure. F shows an overlay of D (red) and E (green). The FISH procedure results in only small changes in the distribution of PML bodies and centromeres. These changes are in part due to a slight tilting of the nuclei during FISH labeling. Bar, 0.84 μm.

Mentions: Fig. 2 shows the spatial distribution of PML bodies (Fig. 2, A–C) and of centromeres (Fig. 2, D–F) imaged in the same cell before and after FISH. The corresponding optical sections before and after the FISH procedure (PML bodies, Fig. 2A and Fig. B, and centromeres, D and E, respectively) show that nuclear organization is well-preserved during the FISH procedure. Both the integrity and the spatial distribution of the nuclear bodies and of the centromeres are essentially unchanged. The small changes in spatial distribution (see overlay in Fig. 2C and Fig. F) can be attributed largely to a slight tilting of the nucleus relative to the substratum, due to the FISH procedure.


Spatial relationship between transcription sites and chromosome territories.

Verschure PJ, van Der Kraan I, Manders EM, van Driel R - J. Cell Biol. (1999)

Preservation of the spatial distribution of centromeres and PML bodies in human primary fibroblasts during the FISH procedure. The spatial distribution of PML bodies and of centromeres were analyzed before and after the FISH procedure in the same nucleus. A, B, and C, Corresponding individual optical sections of the same nucleus labeled with anti-PML antibody are shown in A (before the FISH procedure) and B (after FISH). C shows an overlay of A (red) and B (green). Bar, 0.84 μm. D, E, and F, Corresponding individual optical sections of the same nucleus labeled with anticentromere antibody. D, Before FISH procedure. E, After FISH procedure. F shows an overlay of D (red) and E (green). The FISH procedure results in only small changes in the distribution of PML bodies and centromeres. These changes are in part due to a slight tilting of the nuclei during FISH labeling. Bar, 0.84 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164981&req=5

Figure 2: Preservation of the spatial distribution of centromeres and PML bodies in human primary fibroblasts during the FISH procedure. The spatial distribution of PML bodies and of centromeres were analyzed before and after the FISH procedure in the same nucleus. A, B, and C, Corresponding individual optical sections of the same nucleus labeled with anti-PML antibody are shown in A (before the FISH procedure) and B (after FISH). C shows an overlay of A (red) and B (green). Bar, 0.84 μm. D, E, and F, Corresponding individual optical sections of the same nucleus labeled with anticentromere antibody. D, Before FISH procedure. E, After FISH procedure. F shows an overlay of D (red) and E (green). The FISH procedure results in only small changes in the distribution of PML bodies and centromeres. These changes are in part due to a slight tilting of the nuclei during FISH labeling. Bar, 0.84 μm.
Mentions: Fig. 2 shows the spatial distribution of PML bodies (Fig. 2, A–C) and of centromeres (Fig. 2, D–F) imaged in the same cell before and after FISH. The corresponding optical sections before and after the FISH procedure (PML bodies, Fig. 2A and Fig. B, and centromeres, D and E, respectively) show that nuclear organization is well-preserved during the FISH procedure. Both the integrity and the spatial distribution of the nuclear bodies and of the centromeres are essentially unchanged. The small changes in spatial distribution (see overlay in Fig. 2C and Fig. F) can be attributed largely to a slight tilting of the nucleus relative to the substratum, due to the FISH procedure.

Bottom Line: Transcription sites were found scattered throughout the territory of one of the two X chromosomes, most likely the active X chromosome, and that of both territories of chromosome 19.Strikingly, nascent RNA was almost exclusively found in the interchromatin areas in chromosome territories and in between strongly GFP-labeled chromatin domains.Active loci are located predominantly at or near the surface of compact chromatin domains, depositing newly synthesized RNA directly into the interchromatin space.

View Article: PubMed Central - PubMed

Affiliation: E.C. Slater Instituut, BioCentrum Amsterdam, University of Amsterdam, 1018 TV Amsterdam, The Netherlands. a311pjvx@chem.uva.nl

ABSTRACT
We have investigated the spatial relationship between transcription sites and chromosome territories in the interphase nucleus of human female fibroblasts. Immunolabeling of nascent RNA was combined with visualization of chromosome territories by fluorescent in situ hybridization (FISH). Transcription sites were found scattered throughout the territory of one of the two X chromosomes, most likely the active X chromosome, and that of both territories of chromosome 19. The other X chromosome territory, probably the inactive X chromosome, was devoid of transcription sites. A distinct substructure was observed in interphase chromosome territories. Intensely labeled subchromosomal domains are surrounded by less strongly labeled areas. The intensely labeled domains had a diameter in the range of 300-450 nm and were sometimes interconnected, forming thread-like structures. Similar large scale chromatin structures were observed in HeLa cells expressing green fluorescent protein (GFP)-tagged histone H2B. Strikingly, nascent RNA was almost exclusively found in the interchromatin areas in chromosome territories and in between strongly GFP-labeled chromatin domains. These observations support a model in which transcriptionally active chromatin in chromosome territories is markedly compartmentalized. Active loci are located predominantly at or near the surface of compact chromatin domains, depositing newly synthesized RNA directly into the interchromatin space.

Show MeSH