Figure 6.

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.