Fig. 2
Comparison of sense and antisense promoter activities for two-monomer mouse L1 5’UTR consensus sequences in NIH/3T3 cells. A Schematic of the dual-luciferase L1 promoter reporter assay vectors. An L1 promoter, cloned in via flanking SfiI sites, drives the firefly luciferase (Fluc) expression. A built-in Renilla luciferase (Rluc) expression cassette is used to normalize transfection efficiency. Each reporter cassette ends in a polyadenylation signal (illustrated as letter A in a hexagon). Amp, ampicillin resistance gene; HSV-TK, herpes simplex virus thymidine kinase promoter; Puro, puromycin resistance gene. Not drawn to scale. B Titration of plasmid DNA for the cell-based reporter assay. Amount of plasmid DNA is titrated in NIH/3T3 cells in quadruplicate using the control vector pCH117 in which the promoter of human L1RP drives Fluc expression. The mean and standard error are shown for both Fluc and Rluc signals in raw relative luminescence units (RLU). For example, at 5 ng, the mean Fluc and Rluc signals from pCH117 are 124,238 and 25,159 RLUs, respectively. As a reference, the raw background Fluc and Rluc signals are ~ 100 RLUs in the dual-luciferase assay. C The calculated ratio of Fluc/Rluc from above titration experiment. Mean and standard error are shown. D Normalized activity of two-monomer consensus promoter sequences from six mouse L1 subfamilies. Sequence organization of the promoters is illustrated on the left side. The length of M2, M1, and tether (T) for each promoter is annotated (in base pairs). For each subfamily, the promoter activity was tested in both sense (S) and antisense (AS) orientation. The x-axis indicates the normalized promoter activity (i.e., the Fluc/Rluc ratio of a control no-promoter vector, pLK037, was set to 1). Note a broken x-axis is used to contrast sense and antisense promoter activities. E Inverse relationship between the sense promoter activity and subfamily age. A simple linear regression line was shown along with individual data points