Table 1.

hTERT expression and catalytic and biologic telomerase activity for the panel of N-terminal hTERT mutants

Domain and mutantahTERT expression (105)bTelomerase in vitro activitycLife spand
Wild type5++I
 +50f 7M
 +152f 10M
 +386e 9M
 +512e 2M
  • a Each mutant is named after the starting position of the substitution in the native hTERT peptide sequence (i.e., +2 is 2PRAPRC to 2NAAIRS). Essential domains (I-A, I-B, II, and III), biologically essential domain (DAT), and nonessential linker regions (L1 and L2) are shown left of columns for mutant positions.

  • b Fold overexpression of hTERT transcripts compared to vector control after samples were normalized for RNA content with the transcript level of the housekeeping gene PBGD.

  • c In vitro telomerase activity for each mutant was determined by normalizing the activity to the internal standard and then expressed as a percentage of wild-type FLAG-hTERT activity as follows: ++ (>60%), + (60 to 15%), +/− (<15%), and − (extremely low or no detectable activity). At least two separate lysates were tested for each mutant.

  • d Polyclonal HA5 cells overexpressing FLAG-hTERT mutants were serially passaged to determine the life span. Lifespan was defined by similarity to growth of vector (negative control) and wild-type FLAG-hTERT (positive control) cell lines. Mutants that grew like vector eventually underwent crisis and are termed M (mortal), mutants that continually divide at a rate similar to that of the wild-type FLAG-hTERT are termed I (immortal), and mutants that continually divide at a slower rate compared to that of the wild type are termed S (slow growth).

  • e Mutants shown to have reduced hTR binding.

  • f Mutants shown to have wild-type hTR binding.