FIG. 4.

Mapping the 3′ boundary of hTR required to produce active telomerase with hTERT that was synthesized in intact human cells. Truncation mutants of hTR were analyzed in assembly reactions in which hTERT was synthesized in intact VA13 cells that lack endogenous hTR and hTERT. (A) RT-PCR was performed to demonstrate that the VA13 human fibroblast cell line immortalized with simian virus 40 large T antigen lacks both hTR and mRNA for hTERT. The telomerase-positive strain HL-60 served as a positive control for amplification. (B) Truncation mutants of hTR were incubated with S100 extracts of VA13 cells that synthesize exogenous hTERT. A fraction of the assembly reaction was analyzed by the TRAP assay. Relative quantitation is shown for this representative TRAP assay and reflects the ratio between the abundance of extended products versus the 36-bp internal standard.