Fig. 2. Residues 965-981 of the TERT polypeptide are an active NTS sequence. (A) Alignment analysis shows that the sequence aa965-981 of TERT is highly conserved in its counterparts from mouse and Xenopus, and that it is enriched with positively charged residues. (B) Substitution of the three conserved positively charged residues indicated in A with alanines abolished nucleolar localization of the fragment GFP-TERT_aa965-1132 in both human (HeLa) and mouse (NIH3T3) cells. Arrows indicate nucleoli. (C) Fusion of the aa965-981 sequence of TERT to GFP leads to nucleolar accumulation of the expressed fusion protein in both HeLa and NIH3T3 cells. The fluorescence signal shows the localization patterns of the GFP fusion proteins in their corresponding transfected cells. Arrows indicate nucleoli that were visualized by using an optic microscope. 400x magnification. The percentage of cells with the represented GFP staining patterns in the corresponding transfected cell populations are shown to the right (>400 expressing cells counted for each group).