Telomerase enzymatic activity in VA13+hTERT cells expressing various hTERC natural variants.
(A) Representative TRAP gels showing the relative telomerase enzymatic activities obtained from the hTERC variants either individually transfected (lanes 1-21) or cotransfected (lanes 22-39). Serial 5-fold dilutions of the transfected cell lysates (indicated by triangles) were assayed for each sample to ensure linearity of the assay. Lanes 40-47 indicate telomerase enzymatic activity reconstituted in vitro using the rabbit reticulocyte lysate as previously described.4 The smallest telomeric band (+4) represents the initial repeat of 4. Repeats shorter than 4 are not observed on the gel due to the length and design of the Cx-ext reverse primer used in the reaction. Primer dimer (PD) PCR products are also observed due to the partial complementarity of the Cx-ext reverse primer and the TS forward primer used in the assay. (B) Northern blot analysis of naturally occurring hTERC sequence variants expressed in transfected VA13+hTERT cells. Lane 6 shows RNA prepared from cells that were transfected with the pcDNA3.1 vector lacking the hTERC coding sequence. Cellular GAPDH mRNA (lower blot) was assayed in parallel using random-primed probe, which is specific to a specific region of the GAPDH gene. (C) Northern blotting analysis of affinity-enriched telomerase complexes assembled in vitro using full-length wild-type hTERC RNA with the selected hTERT sequences (lanes 2-9), or using the full-length wild-type hTERC or its mutant forms with a wild-type copy of the hTERT protein (lanes 10-14). Telomerase RNA-protein complexes were first assembled in the rabbit reticulocyte lysates. The negative control (lane 1) was a lysate that received no hTERT expression vector.
