The second ATG of the limitin gene is a major transcriptional initiation codon.

(A) Constructs of the pRL-SV40, 1st-ATG/pRL-SV40, 2nd-ATG/pRL-SV40, mu1st-ATG/pRL-SV40, and mu2nd-ATG/pRL-SV40 plasmids. The relative positions of the open reading frames of the limitin proteins with the translational start codons are shown. The open reading frame of renilla luciferase is in frame when the first ATG of thelimitin gene is used as a translational initiation codon (1st-ATG/pRL-SV40 and mu1st-ATG/pRL-SV40). The open reading frame of renilla luciferase is in-frame when the second ATG of thelimitin gene is used as a translational initiation codon (2nd-ATG/pRL-SV40 and mu2nd-ATG/pRL-SV40). (B,C) BMS2.4 stromal cells (B) and the indicated stromas (C) were transfected with the indicated plasmids, together with pGL-Control vector by lipofectamine transfaction. After 2 days of culture, the transfectants were collected and subjected to luciferase assays. The relative renilla luciferase activities were calculated by normalizing transfection efficiency according to the firefly luciferase activities. Results are shown as means ± SDs of triplicate samples. Similar results were obtained in 3 independent experiments.