Figures & data
Figure 1 Structure of TYMS 5′-UTR tandem repeats-luciferase reporter constructs having Kozak sequences from firefly luciferase (LK) and human TYMS (HK) are shown. Leucine (CTT) was inserted by site mutagenesis between methionine (ATG) and glutamine (GAA) to generate constructs with native TYMS Kozak sequence GCCATGC in place of the firefly Kozak GCCATGG.
Figure 2 (A) Transient transfection results using reporter constructs having the firefly luciferase Kozak (LK). Reporter activities from seven plasmid DNA preparations were used in transient transfection assays, each constituting two replicates of 2R-LK, 3Rc-LK and 3Rg-LK. In each experiment, the 2R-LK activity was arbitrarily defined as “100”. No meaningful difference was found between reporter activities using the three VNTR structures. The luciferase reporter values were normalized by the renilla readings of the same well. The normalized luciferase value for each plasmid construct is presented as a mean of the seven experiments ± standard error (bars). (B) Transient transfection results using reporter constructs having the human Kozak (HK). Reporter activities from seven plasmid preparations were used in transient transfection assays, each constituting two replicates of 2R-HK, 3Rc-HK and 3Rg-HK. In each experiment, the 2R-HK activity was arbitrarily defined as 100. No meaningful difference was found between the reporter activities using the three VNTR structures. The normalized luciferase value for each plasmid construct is presented as a mean of the seven experiments ± standard error (bars).
Figure 3 Normalized expression of luciferase reporter values in stably transfected, selected pools of cells having firefly Kozak and human TYMS Kozak constructs. For each construct, one transfection was performed to generate a stable clone. Each of the six clones was evaluated in three independent experiments, plotted as three dots, and the mean for each. The normalized luciferase activity was derived from the luciferase enzymatic activity divided by the luciferase RNA values measured in the corresponding cell population by qPCR. The expected systematic difference in the desired direction was not found between the different stable lines having different VNTR structures.
