Fig. 1. A telomere consisting solely of vertebrate telomeric DNA can be stably maintained in yeast. (A) To generate a completely vertebrate-sequence telomere at chromosome VII-L, EcoRI–SalI-digested pUT-H was integrated at the ADH4 locus on chromosome VII-L in a tlc1h strain, in a manner that deletes the terminal ∼20 kb from the chromosome (Gottschling et al., 1990). The 60 bp C₃TA₂ sequence (black box) acts as a seed for telomere formation. The tlc1h telomerase can add only vertebrate repeats to this end, resulting in a VII-L telomere that contains no yeast telomeric repeats. A unique sequence (striped block) between the telomere seed and the URA3 gene was used as a PCR primer site for telomere sequencing and chromatin immunoprecipitations. The arrow above the spotted box indicates the URA3 promoter. The indicated PstI site is located upstream of the URA3 start codon. (B) Sequencing results for modified VII-L telomere. VII-L telomeres from the 499UT-H strain were cloned and sequenced. Clones were made ∼125 cell divisions after transformation in three independent integrants of the UT-H construct. Representative sequences for three clones, one from each integrant, are shown; the centromere-proximal end of the sequence is to the right. Seven modified telomeres were sequenced; none contained yeast telomeric DNA. While none of the sequenced telomeres contained the heterogeneous C_1–3A repeats characteristic of yeast telomeres, the VII-L telomere in the 499UT-H2 transformant contained one copy each of two variants of the vertebrate repeat: C₄TA₂ and C₂TA₂. The 499UT-H1 strain, with 245 bp of pure vertebrate repeats, was used for all subsequent experiments. (C) Telomere length is stable in the 499UT-H strain over many generations. Telomere length in the 499UT-Y and 499UT-H strains was assayed after one and ten serial restreaks on YC plates. Genomic DNA was digested with PstI and XhoI; the Southern blot was probed sequentially with URA3, C_1–3A and C₃TA₂. The upper band in the URA3-probed blot (*) is the endogenous ura3-52 allele. Two to three independent colonies from each strain are shown. Black arrowheads indicate the 499UT-Y VII-L telomere; white arrowheads indicate the 499UT-H C₃TA₂ VII-L telomere. (D) Effect of rad52Δ on telomere length. Southern blots of PstI-digested genomic DNA from four serial restreaks of rad52 and RAD52 versions of the 499UT-Y and 499UT-H strains were probed with URA3 which detects the VII-L telomere fragment. The upper band in the URA3-probed blot (*) is the endogenous ura3-52 allele. Arrows indicate the VII-L (URA3 probe) telomere band.