Figure 4.

Identification of a muscle-specific regulatory region in rm-hsc71m upstream sequences. (A) Examination of the activity of 5′-upstream sequences in muscle based on reporter activity. Schematic drawings of a series of 5′-deletion constructs containing the EGFP reporter gene flanked by variable lengths of rm-hsc71m 5′-upstream sequences, which are shown on the left side. The relative levels of reporter gene expression in liver and muscle tissues are shown on the right side. The constructs were transiently co-transfected into cultured Rivulus muscle or liver tissue, along with a pCMV-lacZ control vector. The level of EGFP expression was monitored 48 h after transfection by RT–PCR. Transfection efficiency was normalized to the level of lacZ expression. In all cases, EGFP/lacZ values obtained from at least two different plasmid preparations and three experiments were normalized to that of the basal construct (pRM79 containing only a TATA box). (B) Position- or orientation-dependency of the rMME. Reporter constructs containing the rMME (1.2 kb PvuII fragment from –2617 to –1418 bp of rm-hsc71m) downstream of the EGFP reporter in a sense or antisense orientation were transiently transfected into cultured muscle or liver tissue. Schematic drawings of each construct and their relative expression levels are shown in the left and right panels, respectively.