Differences in telomere length between homologous chromosomes in humans - PubMed (original) (raw)

Comparative Study

Differences in telomere length between homologous chromosomes in humans

J A Londoño-Vallejo et al. Nucleic Acids Res. 2001.

Abstract

Telomeres are important structures for DNA replication and chromosome stability during cell growth. Telomere length has been correlated with the division potential of human cells and has been found to decrease with age in healthy individuals. Nevertheless, telomere lengths within the same cell are heterogeneous and certain chromosome arms typically have either short or long telomeres. Both the origin and the physiological consequences of this heterogeneity in telomere length remain unknown. In this study we used quantitative telomeric FISH combined with a method to identify the parental origin of chromosomes to show that significant differences in relative telomere intensities are frequently observed between chromosomal homologs in short-term stimulated cultures of peripheral blood lymphocytes. These differences appear to be stable for at least 4 months in vivo, but disappear after prolonged proliferation in vitro. The telomere length differences are also stable during in vitro growth of telomerase-negative fibroblast cells but can be abolished by exogenous telomerase expression in these cells. These findings suggest the existence of a mechanism maintaining differences in telomere length between chromosome homologs that is independent of telomere length itself.

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Figures

Figure 1

Figure 1

Telomere fluorescence intensities obtained through PNA-FISH reflect telomere length. (A) Regression of mean fluorescence intensities, measured by PNA-FISH on PBL metaphase spreads, on age (in years) (n = 25). Vertical bars represent standard deviations from the mean metaphase intensity (n = 15–25, exposure time 2 s). (B) Regression of mean fluorescence intensities, measured on metaphase spreads from 10 lymphoblastoid cell lines, on the sizes (in kb) of telomere restriction fragments as detected by Southern blot. Vertical bars represent standard deviations from the mean (n = 15–25, exposure time 2 s). Fluorescence intensity is expressed as mean pixel values.

Figure 2

Figure 2

Identification of homolog-specific telomere signals. Metaphase spread after hybridization with telomeric PNA (top) followed by a second hybridization step with polymorphic subtelomeric probes, f7501 and ICRF10 (bottom). Chromosomes are counterstained with DAPI. In this particular donor, the second step allows distinction between homologs for chromosome pairs 7, 8, 9 and 11. Homologs for chromosome pair 1 can be distinguished by the size of their heterochromatin.

Figure 3

Figure 3

Relative fluorescence intensity differences between homologous telomeres detected in short-term stimulated human PBLs are consistent. The circles represent mean relative fluorescence intensities measured on telomeres for both homologs of chromosomes 7p and 11p from the same donor. PBL samples were obtained over a period of 4 months. P values from paired _t_-tests between homologous positions are also shown. The number of metaphases analyzed in each sample varied from 25 to 40. Vertical bars indicate the 95% confidence intervals.

Figure 4

Figure 4

Extended lymphocyte proliferation following in vitro stimulation leads to homogenization of telomere relative intensities. (A) The box plots represent the distribution of mean fluorescence intensities of metaphase spreads (n = 25–30) from two different donors (top and bottom), 48 h and 11 days after stimulation of PBLs in vitro. The five horizontal lines in each box indicate the 10th, 25th, 50th, 75th and 90th percentiles. Fluorescence intensity is expressed as mean pixel values. Exposure time 1 s. (B) The circles represent the mean relative intensities obtained for the telomeres on chromosomes 9q and 11q in the same metaphases measured in (A). The results of paired _t_-tests carried out between homologous positions are also shown. n. s., not statistically significant. (C) Box plot representing the distribution of relative intensity measurements obtained for all telomeres in the same metaphases. Small circles represent the observations below the 10th and above the 90th percentiles. In each case, the distributions are significantly different as revealed by the Kolmogorov–Smirnov two-sample test.

Figure 5

Figure 5

Overall and individual telomere fluorescence intensities in WI-38 cells during growth. (A) The box plot represents the distribution of mean fluorescence intensities measured on metaphase spreads of WI-38 cells at three different points (population doublings) during in vitro growth. (B) Mean fluorescence intensities obtained for individual telomeres on chromosomes 8 and 9 in the same metaphases. Vertical bars represent standard errors for the data. Fluorescence intensities are expressed as mean pixel values. The number of measured metaphases analyzed varied from 40 (for PD40/42) to 100 (for PD34). Exposure time 2 s.

Figure 6

Figure 6

Differences between the relative fluorescence intensities of homologous telomeres are stable in WI-38 fibroblasts during in vitro growth. The circles indicate the mean relative intensities obtained for each telomere on both homologs of chromosomes 8 and 9, at different population doublings. The bars represent the 95% confidence limits. The results of paired _t_-tests carried out between homologous positions are also shown. n. s., not statistically significant.

Figure 7

Figure 7

Expression of telomerase in WI-38 cells leads to homogenization of relative telomere intensities. (A) Detection of telomerase activity by a TRAP assay in WI-38+hTERT cells (lane 4) but not in wild-type WI-38 cells (lane 2). In lanes 3 and 1 samples were heat inactivated (negative control). (B) Circles indicate the means of relative fluorescence intensities obtained for each telomere on both homologs of chromosomes 8 and 9, in wild-type WI-38 (PD38) and WI-38+hTERT (PD60) cells. The differences in relative telomere length between all identified telomeres are highly significant in the wild-type cells (analysis of variance, P < 10–15) whereas they are not significant (n. s.) in cells expressing telomerase. The same result was observed with WI-38+hTERT cells at PD50 and 70. (C) Box plot representing the distribution of relative intensity measurements obtained for all telomeres in ∼25 metaphases. Circles represent observations below the 10th and above the 90th percentiles. Both distributions differ significantly, as revealed by the Kolmogorov–Smirnov two-sample test.

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