Telomere shortening caused by Trt1p mutations. (A) Diagram of hybridization with telomeric probe. In wild-type S. pombe telomeres, EcoRI cuts ≈1 kb from the telomere end. With shortening telomeres, shorter EcoRI-fragments and weaker hybridization signals are expected up to the point at which the whole telomeric repeat sequence is lost. (B) Telomere blot of EcoRI-cut DNA from S. pombe strains CF199 (wild-type), CF797 (starting strain for plasmid shuffle), and frozen cultures of days 1, 3, and 5 from growth curves of strains carrying pKAN1-trt1⁺ (wt) or pKAN1-trt1^mut. To test for equal loading, the blot was also hybridized with a probe to the single-copy gene encoding DNA polymerase α (pol1⁺), which lies on a 5.6-kb EcoRI-fragment.

Trt1p mutants are expressed at wild-type level. Cellular extracts prepared from S. pombe strains expressing untagged Trt1p and wild-type or mutant myc₉Trt1p were separated on an SDS polyacrylamide minigel, were transferred to a nylon membrane, and were probed with polyclonal anti-c-myc antibody. The putative band for the myc₉Trt1p fusion protein is indicated by an arrow. To confirm equal loading, identical samples were run on a second minigel, and proteins were stained with Coomassie blue (data not shown).

In vitro telomerase activity of Trt1p mutants. (A) Extracts from S. pombe cells expressing untagged Trt1p, or wild-type or mutant myc₉Trt1p fusion protein were immunopurified on monoclonal anti-c-myc antibodies conjugated to agarose beads. Extension of primer oligonucleotide PBoli 14 was assayed as in Fig. 5, where the precipitation control and length marker are also described. (B) A darker exposure of a section better illustrates the primer+2 reaction product for mutant R512A. (C) To test whether equal amounts of myc₉Trt1p were bound to the immunoprecipitation beads, the other half of the beads used for the telomerase assay was boiled in Laemmli loading buffer, proteins were resolved on a 6% SDS polyacrylamide minigel, and tagged proteins were revealed by immunoblotting. The arrow and triangle indicate the putative myc-tagged Trt1p species described in the text. (D) Dilution assay to determine the sensitivity of the in vitro telomerase assay. After immunoprecipitation from cells expressing wild-type myc₉Trt1p fusion protein, immunoprecipitation beads suspended in TMG(200) were successively diluted and were used in in vitro activity assays as described in A.

Growth curves of Trt1p mutants. Precultures inoculated with colonies after plasmid shuffle were grown in YES liquid media containing geneticin at 32°C for 24 h. Each day thereafter, liquid cultures were inoculated at a density of 5 × 10⁴ cells/ml, grown at 32°C for 24 h, and the cells were counted. Those cells not used for inoculation were harvested by centrifugation and were stored frozen for DNA preparations. For every mutant, at least two growth curves from independent colonies were recorded. In the case of F444A, the one curve showing a growth defect was subsequently found to be caused by a second-site two-amino acid deletion in Trt1p, whereas those cells carrying only the F444A mutation grew normally. The positions of the mutations in the T-motif and in the RT-domain of Trt1p are indicated in the diagram.

Detection of Cmyc₉Trt1p fusion protein in cell extracts. Immunoblot of extracts prepared from a wild-type S. pombe strain with untagged Trt1p (CF199) and a strain expressing C-terminal myc₉-tagged Trt1p (CF830). Crude extract (50 μg of total protein), immunoprecipitation with monoclonal anti-c-myc-antibody bound to agarose beads, and the supernatant thereof were separated by SDS/PAGE. Tagged proteins were detected by immunoblotting using a rabbit polyclonal anti-c-myc primary antibody (Santa Cruz Biotechnology) and peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Boehringer Mannheim). Putative bands for the myc₉Trt1p fusion protein are indicated by an arrow and open triangle (see text). The band at ≈50 kDa indicated by a dot is unrelated to Trt1p.

Validation of the in vitro telomerase assay. Cell extracts prepared from wild-type cells with untagged Trt1p (CF199) and cells expressing myc₉Trt1p fusion protein (CF830) were subject to immunopurification on monoclonal anti-c-myc antibodies conjugated to agarose beads. After incubation with (+) and without (−) RNase A, telomerase activity was assayed. A ³²P-5′-end labeled 100-mer oligonucleotide (Upper) was added to test that no reaction products were lost during phenol-chloroform extraction or ethanol precipitation. Products were separated by sequencing gel electrophoresis. As length markers, PBoli 14 oligonucleotide was elongated by using [α-³²P]dGTP (M ladder) or [α-³²P]ddGTP (M+1), respectively, by terminal deoxynucleotidyltransferase. Because electrophoretic mobility depends on nucleotide composition, the M ladder does not align with the telomerase extension products.