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First published online 23 March 2004
doi: 10.1242/jcs.01032

Journal of Cell Science 117, 1961-1970 (2004)
Published by The Company of Biologists 2004
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Sister chromatid separation at human telomeric regions

Michal Yalon, Shoshana Gal, Yardena Segev, Sara Selig* and Karl L. Skorecki

Bruce Rappaport Faculty of Medicine and Research Institute – Technion and Rambam Medical Center, Haifa, Israel 31096



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  		Fig. 1. (A-C) Metaphase singlets in pre-senescent fibroblast cells aged in culture. Fibroblasts from human SR foreskin fibroblast cells at PD 18 (early passage) and PD 65–67 (pre-senescent) were subjected to FISH-IF using probes for the telomeric and control regions listed and an antibody to H3P, which serves as a G2/mitosis marker. The FISH signals are detected with FITC (green) and the anti-H3P antibodies with Cy-3 (red). (A) A pre-senescent cell hybridized to a probe for ß-globin, showing two doublet signals. Bar, 10 µm. (B) A pre-senescent cell hybridized to a 17p telomeric probe showing two singlet signals. (C) The percentage of chromosomes displaying metaphase singlets in pre-senescent cells aged in culture. The mean percentage (±s.d.) of chromosomes displaying a singlet signal is indicated, together with the P value of the binomial distribution probability analysis (*P<0.0001, **P<0.001, ***P<0.01). The number of chromosomes scored for each probe is indicated in the bar.

 


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  		Fig. 2. Metaphase singlets in pre-senescent cells aged in vivo. Fibroblasts derived from three elderly people (y, years) were propagated in culture from cell line establishment until senescence. Pre-senescent cells (PD 15-30) were subjected to combined FISH-IF at 1-2 PDs before senescence, using probes for the telomeric and control regions indicated. The mean percentage (±s.d.) of chromosomes displaying a singlet signal is indicated, together with the P value of the binomial distribution probability analysis (*P<0.0001). The number of chromosomes scored for each probe is indicated in the bar.

 


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  		Fig. 3. Metaphase singlets at telomeres do not occur in cancer cell lines. Metaphase cells from two ovarian cancer cell lines, OVCAR-3 (telomerase positive, short telomeres) and SKOV-3 (telomerase positive, long telomeres), were subjected to combined FISH-IF using probes for the telomeric and control regions listed. The mean percentage (±s.d.) of chromosomes displaying a singlet signal in OVCAR-3 and SKOV-3 in comparison to the corresponding percentage in pre-senescent human fibroblast cells is indicated, together with the P value of the binomial distribution probability analysis (*P<0.0001). The number of chromosomes scored for each probe is indicated in the bar.

 


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  		Fig. 4. (A,B) Expression of hTERT in pre-senescent cells abrogates telomere metaphase singlets. Pre-senescent BJ cells were infected serially with the pBABE-hTERT-eGFP retroviral vector and with a control empty vector. Metaphase cells at pre- and postinfection were subjected to combined FISH-IF using an antibody to GFP and probes for telomeric and control regions. Only GFP-positive cells were scored. (A) Two singlets at telomere 17p are evident in a GFP-positive pre-senescent cell after infection with a control vector. (B) A GFP-positive cell, 4 days after infection with an hTERT-containing vector, displaying two doublet FISH signals at telomere 17. The FISH signal is detected with Cy-3 (red) and the GFP with FITC (green), and the nucleus is stained with DAPI (blue). Bar, 10 µm.

 


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  		Fig. 5. (A-D) Telomeric metaphase singlets hybridize more intensely compared with the single components of a doublet signal. Combined FISH-IF utilizing a 17p telomeric probe and an antibody for H3P was performed on pre-senescent cells aged in culture (A,B) and presenescent fibroblasts from elderly people (C,D). The FISH signal is detected with FITC (green) and the H3P with Cy-3 (red). All four metaphase cells display one singlet signal and one doublet signal. The hybridization signal intensity of the singlet appears stronger than that of each of the two dots composing the doublet. Bar, 10 µm.

 


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  		Fig. 6. (A-G) Nondegradable Scc1 induces the cut phenotype in early passage human fibroblasts with telomeric DNA present on the DNA bridge. Early passage fibroblasts (PD 15-18) were infected serially with ND-SCC1, and PNA-FISH with centromeric and telomeric probes was performed 2-3 days following the last infection. (A-F) Telomere signals are detected with Cy3 (red) and centromeric signals with FITC (green). Nuclei are stained with DAPI (blue). (A) An example of a nucleus with an abnormal morphology induced by the ND-Scc1mutation. In addition to the irregular nuclear shape, additional micronuclei are present in the cell, hybridizing to centromeric and telomeric probes. One of these micronuclei is shown more clearly in the inset. These nuclei are probably the result of lagging chromosomes. (B) A typical cut phenotype. Note that a thin strand of DNA connects the two nuclei most distantly separated in this field. (C) A cut phenotype with a thick DNA bridge containing multiple telomeric signals. The region of the DNA bridge, connecting the two sister nuclei, is enlarged at the bottom left. (D-E) The cut phenotype with a thin DNA bridge. Several telomeric signals are present on the bridge, whereas no hybridization with the centromeric probe is evident. The region of the DNA bridge is enlarged and presented in the bottom frame of each panel. (F) A nucleus containing a torn DNA bridge. An enlargement of the torn DNA strand is present at the bottom frame of the panel. (G) Staining of a GFP-positive cell with the cut configuration with CREST (anti-centromeric) and anti-GFP antibodies. The centromeric regions are detected with Cy3 (red) and the GFP with FITC (green). DNA is stained with DAPI (blue).

 

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© The Company of Biologists Ltd 2004