1. Short Telomeres in Yeast Are Highly Recombinogenic
Michael J McEachern, Shilpa Iyer 
Molecular Cell 
Volume 7, Issue 4, Pages (April 2001)
DOI: /S (01)

2. Figure 1
Loss of Telomeric Polymorphisms in ter1 Mutants with Short Telomeres
(A) The 30 nucleotide sequence of the wild-type K. lactis template region of the ter1 gene is shown along with the positions of mutations used in this study. The strand shown matches the 25-nt telomeric repeat copied and is complementary to the strand present in the ter1 RNA molecule. The lower sequence shows the template of ter1-Dup21–25, which is wild-type except for a 5-nt shift in its permutation. Arrows indicate the positions of the five nucleotide repeats that border the template. The solid line indicates the position of the Rap1 protein-binding site within a unit telomeric repeat.
(B) Left, shows wild-type K. lactis hybridized with a telomeric (T) or subtelomeric (ST) probe. The number of telomeres normally present in each band and the designations of the telomeric EcoRI bands are shown.
(C) Clones of ter1 template mutants five streaks (∼100–125 cell divisions) after their construction: lanes 1 and 2; phenotypically wild-type TER1-Bcl clones (shown less exposed than neighboring lanes); lanes 3–6; ter1-Taq clones; lanes 7–9; ter1-Bgl(fill) clones; lanes 10–12; ter1-Dup21–25 clones; and lanes 13–16; ter1-Sna clones.
(D) Some of the same mutant clones after 20 streaks (400–500 cell divisions). Clones of ter1-Sna were not grown beyond five streaks. “WT” indicates clones with wild-type TER1. Panels C and D are Southern blots of EcoRI-digested genomic DNA after hybridization to a K. lactis telomeric probe. Molecular weight markers (kb) are indicated for panels C and D. Faint band at ∼3 kb in panel D is a non-telomeric cross-hybridizing fragment
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2
Telomeric Fragment Changes in Two ter1-Taq Clones over Long-Term Growth
(A) Shown is a Southern blot of EcoRI-digested DNA hybridized to a K. lactis telomeric probe. Individual clones of ter1-Taq cells were passaged for 60 streaks (1200–1500 cell divisions) and periodically sampled for preparing genomic DNA. The time course for clone 1 is shown for just 30 streaks as no further changes occurred between streak 30 and 60. Note that at the first streak, telomeres in the mutant had not fully shortened down to the short stable size that characterizes ter1-Taq. A wild-type sample (WT) is also shown. The position of the internal (non-telomeric) fragment carrying the ter1 gene is indicated near the top. It provides an internal control for a single copy telomeric repeat sequence. Molecular weight markers are indicated at the left and the designation of telomeric bands is provided at the right.
(B) Schematic representation of how gene conversions between homologous subtelomeric regions might result in sequential loss and reappearance of telomeric fragments. The top panel depicts four homologous telomeric fragments that share subtelomeric homology but have restriction-site polymorphisms (EcoRI restriction sites indicated by vertical lines) that result in four bands in EcoRI-digests (“Gel pattern”). For clarity, not all K. lactis telomeric fragments are shown. The middle panel depicts the four telomeres after two (R8–9 and R10, marked with arrows) have undergone gene conversions by copying a region (shown in bold) of the telomere present in the smallest band (R1–6). Only two bands would now be visible in a Southern gel pattern. The bottom panel shows the consequences of a third gene conversion, where one telomere (marked with arrow) copies a region (shown in bold) from the uppermost telomere (R11). Such an event could regenerate a band (R8–9) that had previously disappeared, as indicated by the new Southern gel pattern. Gray boxes in diagram indicate telomeric repeat tracts. The positions of EcoRI sites other than those nearest the telomeric repeats are hypothetical
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3
Use of a Subtelomeric URA3 Insert for the Detection of Telomeric Gene Conversions
A DNA restriction fragment containing a K. lactis telomere and a URA3 gene integrated into native subtelomeric sequence is first transformed into K. lactis cells. Resulting transformants (STU clones) contain one telomere replaced by the URA3-marked telomere. Gene conversions between the URA3-marked telomere and another telomere sharing subtelomeric homology (at least 11 of the 12 telomeres in the 7B520 strain of K. lactis) can result in the URA3-marked telomere either being replaced (lower left) or duplicated (lower right). Loss of URA3 can be selected for, and therefore quantified, by plating on medium containing 5-FOA. White boxes indicate telomeric repeats and gray rectangle represents URA3
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4
Southern Blot of ter1-Δ and ter1-Taq STU Clones Immediately after Their Construction
Genomic DNA of STU transformants that has been digested with PvuII are shown hybridized to a telomeric probe (left panel) and to a URA3 probe (right panel). Telomeric fragments that have acquired a URA3 insert are marked by arrowheads. Most transformants appear to contain a single telomere with the URA3 gene. Presence of URA3 leads to telomeric fragments increasing in size by ∼1.2 kb. Some differences in the pattern of telomeric bands had occurred between the ter1-Δ and ter1-Taq strains prior to them being transformed with the URA3-telomere fragment. Size markers are shown to the left. Some traces of residual signal from the prior telomeric probe are visible with the URA3 probe
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5 Spread of URA3 to Multiple Telomeres in ter1-Δ Mutants
Southern blotting and URA3 quantitation data from six independent lineages of ter1-Δ STU cells (5–7, 3–6, 2–3, 5–8, 6–12, and 6–10) are shown. Cells were passaged for eight streaks alternating between SC plates lacking uracil and YPD plates. Numbers above lanes indicate the number of streaks. Left and central sections of each panel show EcoRI-digested DNA hybridized with telomeric and URA3 probes, respectively. Signal near 1 kb represents the major group of EcoRI telomeric fragments (R1–R7 in wild-type cells, see Figure 2) and do not contain URA3. Telomeric fragments with URA3 are typically shifted to ∼2 kb in size. A STU transformant of wild-type cells is shown in the panel that includes ter1-Δ STU 5–8. Complete or near complete loss of the ∼1 kb telomeric bands is apparent in five of the six clones shown by the eighth streak. Loss of the ∼1 kb group of telomeres is accompanied by increase in the number of telomeres carrying URA3. Lower right sections of each panel show Southern blots of XhoI + PstI digested DNA hybridized to URA3 (U) and RAD52 (R). This digest separated URA3 from telomeric repeats and led all copies of URA3 to run identically in gels, as two bands of ∼1 kb and ∼0.2 kb (only the larger is shown). By using the signal level from RAD52 as an internal control, URA3 copy number was estimated by PhosphorImager analysis, results of which are shown in graphs in upper right segments of each panel. URA3 copy number of the ter1-Δ STU clones was not necessarily one at the start of the growth course shown, as a few streaks of the cells had already occurred between the isolation of the clones and the start of the experiment
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6 Loss of the URA3 Gene from K. lactis STU Clones
Lineages of ter1-Δ STU clones were passaged by alteration between being streaked onto plates lacking uracil and plates of rich medium. Cells from rich medium streaks were assayed for URA3 loss from by plating serial dilutions on 5-FOA plates. Data derived from ten lineages of ter1-Δ STU cells are shown pooled together where the percent of colonies found to be Ura3− is plotted against the measured number of telomeric copies of URA3 present at that particular point in the lineage's growth course. Considerable scatter results from the variability inherent in any measurement of a “mutation” rate. Further scatter is likely due to the fact that the populations of cells used for the 5-FOA assays and those used to prepare DNA were not identical, each having been scooped from separate parts of the rich medium plate
Molecular Cell 2001 7, DOI: ( /S (01) )