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
Vector0.00001M
Wild type5++I
I-A
 +26+S
 +84M
 +1415+S
 +202M
 +261M
 +323++S
 +389+/−M
 +442+/−M
 +50f 7M
 +564M
 +622++I
DAT
 +683++M
 +741+M
 +808+M
 +863++S/M
 +922++M
 +985++M
 +1047++S
 +11011++S
 +1161++S
 +1221++M
 +1286++M
I-B
 +1341+/−M
 +1401+/−M
 +1461+/−M
 +152f 10M
 +1586M
 +1643M
 +1702M
L1
 +1761++I
 +1823++I
 +1880.4++I
 +1941++I
 +2001++I
 +2063++I
 +2120.4++I
 +2180.3++I
 +2241++I
 +2300.4++I
 +2363++I
 +2421++I
 +2481++I
 +2541++I
 +2661++I
 +2722++I
 +2782++I
 +2845++I
 +2903++I
 +2965++I
 +3025++I
 +3084++I
 +3145++I
 +3204++I
 +3266+I
 +3325+I
 +3384++I
 +3445++I
II
 +3508+/−S
 +3563M
 +3628+/−S
 +3683++I
 +3744++I
 +3806+/−M
 +386e 9M
 +3926M
 +39820+S
 +4046M
L2
 +4107++I
 +4164++I
 +4225++I
 +4282++I
 +4346++I
 +4403++I
III
 +4465+/−M
 +4523M
 +4583+/−M
 +4646M
 +4701M
 +4761M
 +4821M
 +4882+/−M
 +4942M
 +5003++I
 +5064+/−M
 +512e 2M
 +5185+/−M
 +5244++I
 +5304M
 +5364M
 +5424M
  • 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.