Telomerase enzymatic activities of additional naturally occurring hTERT variants as determined in VA13+hTERC cells or in primary cells.
(A) Representative TRAP gels showing the relative telomerase enzymatic activities obtained from the substitution mutations and the wild-type hTERT constructs either individually transfected (lanes 1-9) or cotransfected (lanes 10-15). Serial 5-fold dilutions of the transfected cell lysates (indicated by triangles) were assayed for each sample to ensure linearity of the assay. Lanes 16 and 18 show negative controls composed of cell lysate denatured at 95°C for 5 minutes prior to assaying. Lanes 17 and 19 show PCR products amplified from the non-hTERC control TSR8 DNA template supplied in the TRAP kit. IC indicates PCR products amplified from an unrelated DNA template, which is included as an internal control for PCR amplification efficiency in each reaction. (B) Western blot of wild-type and various natural hTERT variants isolated from transfected cell lysates and probed with an anti-HA probe against the N-terminal HA tag at each of the hTERT constructs. (C) Telomerase enzymatic assay of cell lysates prepared from about 106 primary lymphocytes collected from a patient with the hTERT K570N variant (lanes 23-25) or a healthy age-matched individual (lanes 20-22). (D) Northern blot analysis of hTERT gene expression in lymphocytes of a patient with the hTERT K570N variant (lane 2), a healthy age-matched individual (lane 1), or in the VA13+hTERC cells (lane 3) that is known to lack hTERT gene expression.
