Telomerase activity in telomerase reconstitution assays and in primary cells. (A) Wild-type (WT) or the different indicated abnormal TERC sequence variants were expressed either individually (lanes 1-9) or simultaneously in different combinations (lanes 10-12, 15-23) from either the cellular elongation factor 1α (EF1α) or the viral cytomegalovirus (CMV) promoter, which are located within the pBud-CE 4.3 vector (Strategene) in the transfected VA13⁺ TERT cell line. Serial 5-fold dilutions of the transfected cell lysates were used to ensure the linearity of the PCR reactions. (–) is the result of the heat-inactivated sample similar to the one shown in lane 1, whereas (+) shows the PCR products amplified from the positive TSR8 DNA template provided in the TRAPeze kit (Chemicon International). IC shows PCR products amplified from an internal control to ensure equal loading of the samples. (B) TRAP results of the in vitro reconstitutions of the telomerase complexes using the rabbit reticulocyte lysate (RRL; Promega) to translate a wild-type copy of the TERT protein in the presence of the indicated synthetic TERC RNA variants individually or in combinations. Various dilutions of the TERC RNAs (100 ng, 10 ng, and 1 ng) were used for each RNA sample. No TERC indicates similar RRL sample without the TERC RNA; –, heat-inactivated sample containing wild-type TERC; +, PCR products amplified from the positive TSR8 DNA template provided in the TRAPeze kit (Chemicon International). (C) TRAP results of the VA13⁺ TERT cells that were transfected with plasmid DNAs that contain nucleotide substitutions shown in Figure 2F, lines 5 to 9, including the junctional J4/4.1 sequence and sequences predicted to either abolish the formation of the P4.1 helical stem [P4.1(lt) or P4.1(rt)] or restore it [P4.1(ltrt)]. (D) TRAP results of the primary T lymphocytes obtained from either the patient F17 (lanes 1-3) with 2 different TERC abnormalities or an unrelated healthy donor control F19 (lanes 4-6). Lanes 7 to 9 show the PCR products obtained from the positive control K562 cell lysate, indicated as (+). IC shows PCR products amplified from an internal control to ensure equal loading of the samples. (E) Northern blotting analysis of total cellular RNA extracted from VA13⁺ TERT cells that were transfected with a similar set of DNA constructs as shown in panel A. TERC indicates telomerase RNA template transcripts; β-actin, housekeeping β-actin message. The amounts of TERC RNAs and those of the β-actin in all samples were quantified using the ImageQuant analysis software (Molecular Dynamics). The expression levels of the TERC RNA were normalized to those of the housekeeping β-actin gene by dividing the TERC values in each of the lanes with the amounts of the β-actin in the respective lanes. The ratios of TERC gene expression for each of the samples (lanes 1-5) to that of the wild-type TERC sample (lane 6) are shown in the bottom of the gel.