Figure 5
Expression constructs with various LTSM - TSS distances. (A) Schema of the vector construct pRPL18: the promoter region of RPL18 containing an internal start codon (ATG) was cloned into the gene trap vector pT1β-geo in frame with the β-geo gene lacking its start codon. An Xho I restriction site was added to facilitate the insertion of linker sequences of different lengths. (B, C, D) The cells were transfected with a GFP control construct (pGFP) and the five β-geo constructs, one with the endogenous RPL18 promoter including the Xho I site (pRPL18-Xho) and four with additional linkers of the lengths 4 bp, 29 bp, 53 bp and 117 bp. (B) Northern and Western blotting analyses of β-geo mRNA and protein using a β-geo specific DNA probe and an anti-β-Galactosidase antibody in the respective experiment. (C) One representative X-Gal staining of cells transfected with the different constructs. (D) Overlay of the results of three measures of expression of the construct: mRNA, protein, and enzymatic activity (see B, C). The images were scanned and the signals quantified using ImageQuant. Northern blotting signals were normalized to the rRNA fluorescence intensities of the agarose gels (not shown) and Western blotting signals by reprobing the membranes with an anti-β-actin antibody (not shown). For each experiment the signal of pRPL18-Xho was assigned 100% and the other signals were quantified relative to it.