TG1-3 Repeat Length I-

Figure 17.3. Monitoring telomere length in budding yeast. A. The telomeres in S. cerevisiae end with two types of middle repetitive elements, Y' and X. The conserved Y' repeat has an Xho I site a defined distance from the TG]_3 repeat tract, while the less conserved X element contains upstream Xho I sites whose distance from the TG]_3 repeat tract is more variable. Thus, a Southern blot of Xho I digested DNA probed with TGi-3 or TG probe shows several telomeres at once in the Y' 1.1-1.3 kb band and individual X telomeres in the 2-4 kb size range (reviewed by Louis, 1995). B. Telomere PCR method to measure TG]-3 length of a unique telomere. Genomic DNA is tailed with terminal transferase, which tails telomeres and fragmented DNA. A specific primer is then used with an oligo dG primer to amplify the TG]-3 repeats of an individual telomere. Because the sequence of the DNA flanking the TGi-3 repeats is known, the size of the TGi-3 repeats can be easily determined by agarose gel electrophoresis.

et al., 2000) (Figure 17.3). Briefly, genomic DNA is treated with terminal transferase and dCTP to add an oligo-dC tail to all free DNA ends. This mix is then PCR amplified using an oligo-dG primer and a unique primer internal to the TG1-3 repeats. The resulting product has TG1-3 repeats flanked by DNA of known sequence, so the range of TG1-3 lengths can be easily determined on agarose gels. In the first demonstration of this technique, a synthetic telomere constructed in vitro was used to replace a chromosomal telomere and thereby place unique sequences adjacent to the TG1-3 repeats (Forstemann et al., 2000). In subsequent uses, primers that hybridize to X and Y' elements have been used to monitor telomere-telomere fusions (Mieczkowski et al., 2003), and a primer that hybridizes to the unique sequences on the right telomere of chromosome VI has been used to monitor the length of that telomere (Hector et al., submitted).

The use of Chromatin Immunoprecipitation (ChIP) and the serial dilution method for observing cell senescence allows one to monitor the components of telomeric chromatin as the telomere shortens. If the original cell lacking a gene for a telomerase component is grown for 31 divisions, one can obtain —2 x 109 cells, or one liter of cells at 2 x 106 cells/ml. This number of cells is sufficient to provide DNA for multiple Southern blots and cells for a standard ChIP experiment and to seed a second one-liter culture for 5 generations of growth. In most ChIP protocols, cells are cross-linked with formaldehyde, the chromatin is isolated and fragmented, chromatin fragments are immunoprecipitated with Abs to the protein of interest, purified, de-crosslinked and amplified by PCR using primers to the region of interest (Strahl-Bolsinger et al., 1997). Because of its unique sequence, the right telomere of chromosome VI has been studied by ChIP to monitor the assembly of silenced chromatin (Luo et al., 2002) and the association of TEL1 and MEC1 proteins with shortening telomeres (Hector et al., submitted).

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