Fig. 2. The 5.3 kb transvection mediating region (tmr) exhibits orientation-dependent promoter targeting activity. Whole-mount RNA in situ hybridization was performed on 2- to 4-hour-old embryos using anti white or lacZ RNA probes. All embryos are oriented anterior towards the left and dorsal side upwards. Arrows in the posterior regions of the embryos indicate IAB8 activity. (A) IAB8 activates Tp-Z when located 5' proximal to the Tp (Transposase) promoter. (B) IAB8 activates Tp-lacZ from a 3' position, 4.5 kb away from the Tp promoter. (C) The 5.3 kb BamHI-HindIII tmr fragment was inserted just 3' end of the lacZ gene in a C4PLZ vector in the forwards (5'-3') orientation. It consists of 290 bp 5' of the 625 bp PTS, the entire 580 bp Fab-8 insulator, the IAB8 enhancer, a Polycomb response element (PRE) located between Fab-8 and IAB8 (Barges et al., 2000; Zhou et al., 1999), and about 500 bp of additional genomic sequence 3' to the 1.6 kb IAB8. In this orientation, IAB8 is proximally located and activates both the divergently transcribed white (w) and the Tp-lacZ fusion gene. (D) In the reverse (3'5') orientation, IAB8 can direct strong transcriptional activation of either w or lacZ. In two out of 12 lines examined, IAB8 activates only lacZ. Many of these intensely stained embryos also exhibit anterior staining (arrowhead). This is probably caused by the lack of repressor binding sites in the transgenic regulatory regions that normally repress Abd-B enhancer activity in the anterior region of the embryo. (E) In five of these strains, IAB8 activates only w. In the remaining lines, the intervening Fab-8 insulator (data not shown) presumably blocks the IAB8 enhancer.