Wang et al. 10.1073/pnas.0504635102. |
Supporting Figure 2
Supporting Table 4
Supporting Figure 3
Supporting Figure 4
Supporting Figure 2
Fig. 2. Chromosome orientation FISH analysis of one fourth-generation (G4) mTert-/- splenocyte, showing multiple telomere sister chromatid exchanges (T-SCEs). This was the only cell showing multiple T-SCEs among 100 G4 mTert null cells examined. Some T-SCE-positive chromosome ends had stronger telomeric DNA signals than T-SCE-negative chromosome ends.
Fig. 3.
Sister chromatid exchange (SCE) or chromosome orientation FISH analysis of telomeric DNA recombination or interspersed G+C tracts in murine ES cells after two rounds of BrdUrd/BrdC incorporation. (A) Representative arrows point to the SCE events (color switching between dark or light sister chromatid). (B and C) The pattern of double telomeric signals could distinguish telomeric DNA recombination from nonhomologous end-joining events that ligate extrachromosomal telomeric fragment to chromosome ends (1). The former appears on different sister chromatids (arrow) (B), and the latter remains associated with the same dark sister chromatid (C).1. Londono-Vallejo, J. A., Der-Sarkissian, H., Cazes, L., Bacchetti, S. & Reddel, R. R. (2004) Cancer Res. 64, 2324-2327.
Fig. 4.
Telomeric FISH analysis of a representative metaphase spread of murine splenocytes and ES cells. (A and B) A fourth-generation (G4) mTert-/- splenocyte showed more signal-free ends (SFEs) than a G2 mTert-/- splenocyte. (C-E). Different passages of mTert-/- ES cells, showing an increased in SFEs and chromosome end-to-end fusions during prolonged growth in culture. SFEs were only detected at the site of chromosome end-to-end fusion in an mTert deficient ES cell at passage 55, its enlarged version was shown in F--F". Arrowhead, SFEs; arrow, end-to-end fusion. The telomere images were overexposed to intensify weak telomeric fluorescent signals.Table 4. Frequencies of subpopulations with low or high rate of telomere sister chromatid exchanges (T-SCEs) in wild type, mTert deficient murine splenocytes, and different passages of ES cells
Cell type | No. (%) of cells with low rate of T-SCEs * | No. (%) of cells with high rate of T-SCEs * | Total no. of cells counted |
ES cells | |||
Wild type | |||
p5 | 25 (25.0) | 5 (5.0)† | 100 |
p85 | 19 (19.0) | 2 (2.0) | 100 |
mTert +/- | |||
p5 | 25 (26.9) | 4 (4.3) | 93 |
p85 | 17 (17.0) | 2 (2.0) | 100 |
p150 | 12 (11.7) | 3 (2.9) | 103 |
mTert -/- | |||
p5 | 25 (26.0) | 3 (3.1) | 96 |
p85 | 21 (21.0) | 18 (18.0) | 100 |
Splenocytes | |||
Wild type | 38 (38.0) | 100 | |
G2 mTert -/- | 40 (40.0) | 0 (0) | 100 |
G4 mTert -/- | 54 (54.0) | 1 (1) | 100 |
*Cells were divided into two subpopulations, based on the presence of T-SCE. Low rate, less than two chromosome ends are positive for T-SCE; high rate, multiple chromosome ends are positive for T-SCE.
†
A small population of wild-type murine ES cells showed a high rate of T-SCEs, a phenotype that was not found in wild type murine splenocytes (this table) or wild-type murine embryonic fibroblasts (data not shown).