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. Author manuscript; available in PMC: 2016 Nov 1.
Published in final edited form as: Clin Genet. 2014 Dec 26;88(5):494–498. doi: 10.1111/cge.12541

Figure 3. Effect of the 5′ UTR GRHPR variants upon in-frame translation of a luciferase reporter gene in transient transfection.

Figure 3

For A, the entire 41-bp GRHPR 5′ UTR (cDNA NM_012203.1) was subcloned immediately upstream of the initiator Met of the luciferase gene in plasmid pGL3-Promoter. This vector has an SV40 promoter upstream of the reporter gene (SV40-P; see diagram). In this assay, only in-frame translation from the canonical luciferase translational start site gives rise to functional luciferase. CHO cells were transfected with empty vector alone (Vector), or with vector containing wild-type 5′ UTR-luciferase reporter gene chimera (-41ATG-WT) or the variant with out-of-frame upstream translational initiation site (-41ATG-Var). P-values: Vector vs. -41ATG-WT, 0.0003; -41ATG-WT vs -41ATGVar, 2 × 10-5. For B, 1.3 kb of the 5′ flanking region/proximal promoter sequence of human GRHPR inclusive of the 41-bp 5′ UTR was subcloned immediately upstream of the luciferase reporter gene in plasmid pGL3-Enhancer. This vector has an SV40 enhancer downstream of the reporter gene (SV40-E; see diagram). CHO cells were transfected with empty vector alone (Vector), or with vector containing wild-type (-1321ATG-WT) or mutant (-1321ATG-Var) GRHPR promoter and 5′ UTR. P-values: Vector vs. -1321ATG-WT, 0.0007; -1321ATG-WT vs -1321ATGVar, 0.0003. Diagrams of constructs are not to scale. Data are mean ± SEM.