Figure 6.

Evaluation of boundary activities associated with tyrosinase LCR sequences by transient transfection analysis in mammalian cells. Transient transfection of backbone promoter-only plasmid pTKLuc, and its derivative constructs: pHSTKLuc, pSHTKLuc, pLCRTKLuc, pRCLTKLuc, pLCRΔGTKLuc, pRCLΔGTKLuc, pLCRΔABTKLuc, pRCLΔABTKLuc, pLCRΔABGTKLuc, pRCLΔABGTKLuc in B16 (black bars) and L929 (white bars) cells. Schemes of these plasmids (left) and the result of transfection in B16 and L929 cells (right) are shown. The AB enhancer-core and G-rich sequences are depicted as black boxes. Grey or white boxes correspond to the DNA fragments present or absent, respectively, in each construct. The arrow depicted inside each DNA fragment represent the 5′ to 3′ direction, with respect to the position of these sequences within the endogenous tyrosinase LCR and gene. Crossed and dashed lines indicate the inversion of the same DNA fragment in their corresponding experimental constructs. Results are expressed as relative transactivation, arbitrarily assigning to the backbone plasmid pTKLuc the value of x1 and thereafter referring the activity of each experimental construct to that value. Normalisation of the luciferase reporter values between different transfected constructs is achieved taking into account the activity of a co-transfected lacZ reporter plasmid and the number of pmols of experimental plasmid DNA used in each transfection. See Materials and Methods for plasmid sizes. Relative transactivations are mean values from triplicate experiments (±SD).