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. 2001 Nov;21(22):7775–7786. doi: 10.1128/MCB.21.22.7775-7786.2001

TABLE 1.

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

Domain and mutanta hTERT expression (105)b Telomerase in vitro activityc Life spand
Vector 0.00001 − M
Wild type 5 ++ I
I-A
 +2 6 + S
 +8 4 − M
 +14 15 + S
 +20 2 − M
 +26 1 − M
 +32 3 ++ S
 +38 9 +/− M
 +44 2 +/− M
 +50f 7 − M
 +56 4 − M
 +62 2 ++ I
DAT
 +68 3 ++ M
 +74 1 + M
 +80 8 + M
 +86 3 ++ S/M
 +92 2 ++ M
 +98 5 ++ M
 +104 7 ++ S
 +110 11 ++ S
 +116 1 ++ S
 +122 1 ++ M
 +128 6 ++ M
I-B
 +134 1 +/− M
 +140 1 +/− M
 +146 1 +/− M
 +152f 10 − M
 +158 6 − M
 +164 3 − M
 +170 2 − M
L1
 +176 1 ++ I
 +182 3 ++ I
 +188 0.4 ++ I
 +194 1 ++ I
 +200 1 ++ I
 +206 3 ++ I
 +212 0.4 ++ I
 +218 0.3 ++ I
 +224 1 ++ I
 +230 0.4 ++ I
 +236 3 ++ I
 +242 1 ++ I
 +248 1 ++ I
 +254 1 ++ I
 +266 1 ++ I
 +272 2 ++ I
 +278 2 ++ I
 +284 5 ++ I
 +290 3 ++ I
 +296 5 ++ I
 +302 5 ++ I
 +308 4 ++ I
 +314 5 ++ I
 +320 4 ++ I
 +326 6 + I
 +332 5 + I
 +338 4 ++ I
 +344 5 ++ I
II
 +350 8 +/− S
 +356 3 − M
 +362 8 +/− S
 +368 3 ++ I
 +374 4 ++ I
 +380 6 +/− M
 +386e 9 − M
 +392 6 − M
 +398 20 + S
 +404 6 − M
L2
 +410 7 ++ I
 +416 4 ++ I
 +422 5 ++ I
 +428 2 ++ I
 +434 6 ++ I
 +440 3 ++ I
III
 +446 5 +/− M
 +452 3 − M
 +458 3 +/− M
 +464 6 − M
 +470 1 − M
 +476 1 − M
 +482 1 − M
 +488 2 +/− M
 +494 2 − M
 +500 3 ++ I
 +506 4 +/− M
 +512e 2 − M
 +518 5 +/− M
 +524 4 ++ I
 +530 4 − M
 +536 4 − M
 +542 4 − M
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.