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. 2009 Apr;15(4):546–559. doi: 10.1261/rna.1194009

FIGURE 6.

FIGURE 6.

CS2a is essential for budding yeast telomerase function in vivo. (A) Deletions of the bulge (B-del) or the apical stem–loop (SL-del) or transition substitutions of the bulge (B-sub) or CS2a in the internal loop (IL-sub) were introduced into the Est1 binding arm of K. lactis TER1. Indicated in dark gray are residues conserved in at least 18 of 20 yeast TERs (>90%). Inserted nucleotides are in black and deletions are boxed. The rest of the sequence is in light gray. The wild-type and mutant TER1 genes, both containing the BclI template mutation (see main text), were introduced into K. lactis cells on CEN-ARS plasmids, replacing the WT TER1 gene, as described previously (Roy et al. 1998). gDNA was prepared from the sixth passage (∼90–120 generations). Two micrograms of gDNA samples were digested with EcoRI or EcoRI+BclI, electrophoresed on a 1% agarose gel, vacuum blotted onto a membrane, and hybridized first with a BclI-specific telomere probe (B) and then with a WT telomeric probe (C), as described previously (Shefer et al. 2007). (D) Deletions of the apical stem–loop and the internal loop (SL&IL-del) or only the apical stem–loop (SL-del) or transition substitution of 7 nt in CS2a (CS2a-sub) were introduced into an otherwise WT S. cerevisiae TLC1 gene on a plasmid (pTLC1TRP; Bah et al. 2004). The resulting plasmids were used to replace the WT TLC1 gene by plasmid shuffling. (E) Southern blot of gDNA derived from the resulting S. cerevisiae strains. DNA was isolated after the indicated numbers of generations of outgrowth, digested with XhoI, subjected to Southern blotting, and probed with an S. cerevisiae specific telomeric repeat DNA probe. DNAs used were as follows: M: end-labeled MW marker DNA; WT: strain RWY10 harboring pAZ1 (wtTLC1) before the plasmid shuffle; Ku-del: a S. cerevisiae strain harboring a deletion of the YKU70 gene as a control for short terminal restriction fragments; pTLC1: two independent clones of RWY10 harboring pTLC1 (WT) after the plasmid shuffle and grown for 100 generations; CS2a-sub: RWY10 harboring pTLC1-CS2a-sub and grown for 25, 45, 85, and 105 generations; SL&IL-del: RWY10 harboring pTLC1-SL&IL-del and grown for 100 generations; and SL-del: RWY10 harboring pTLC1-SL-del and grown for 105 generations. Where two independent clones are shown, clones 1 and 2 are on the left and right lanes, respectively.