Mutations of the hypervariable region or the CR4-CR5 or box H/ACA domains show variable degrees of telomerase activity. (A) Telomerase enzymatic activity in VA13+hTERT cells expressing various hTERC variants involving the hypervariable paired region or the CR4-CR5 or box H/ACA domains. Natural sequence variants and engineered mutations are indicated in bold. The telomerase activity for each variant is expressed in comparison to that of the wild-type (+++, 20%-100%; ++, 2%-20%; +, 1%-2%; −, undetectable) based on 2 or 3 independent determinations. (B, C) Representative gels showing the relative telomerase enzymatic activities obtained from informative variants. Serial fivefold dilutions of the transfected cell lysates (indicated by triangles) were assayed for each sample to ensure linearity of the assay. Lanes 16 and 27 of panel B and lane 13 of panel C indicate negative controls in which the wild-type cell lysates were denatured at 95°C for 5 minutes before analysis. Lanes 17 of panel B and 14 of panel C show PCR products amplified from the control TSR8 DNA template supplied in the kit. Lanes 18 of panel B and 15 of panel C show cells transfected with the pcDNA3.1 vector lacking the hTERC coding sequence. (D) Northern blotting analysis of affinity-enriched telomerase complexes assembled in vitro using wild-type P6.1 stem (spanning TERC nucleotides 239 to 332) or its mutants. Telomerase RNA-protein complexes were first assembled in the rabbit reticulocyte lysates. The negative control (lane 4) was a lysate that received no hTERT expression vector.