Figure 3.

In vivo expression of iaRNA and in situ visualization of its binding to B52. (A) Transient expression of iaRNA in Drosophila S2 cell cultures. The transcriptional templates BBS (5.2) and BBS (5.12) were driven by a heat shock promoter (Hic) or a metallothionein promoter (Mtn). Steady-state iaRNA level without induction and iaRNA accumulated after induction were compared. Actinomycin D was added to the media after heat treatment, and total RNA was prepared at the times indicated. (B) iaRNA expression in transgenic flies. HicBBS (5.12) is a homozygous strain containing the iaRNA gene described in A. hsGAL4-UASBBSs are heterozygous flies generated by mating different UASBBS transgenic strains with the strain containing the transgene hsGAL4, which is driven by a heat shock promoter. The iaRNA in these samples were measured by RNase protection assay. (C) Subcellular localization of iaRNA. A Texas Red-labeled RNA probe was used to visualize the iaRNA in the nuclei of whole mount salivary glands in late third instar larvae of the HicBBS (5.12) transgenic line with or without heat treatment. DNA was stained with Hoechst 33258. (D) The iaRNA transgene in the HicBBS (5.12) strain was mapped to the locus 12A (Left). After heat induction, the expression of the iaRNA resulted in a medium sized puff (Center). B52 was strongly recruited to this site relative to the heat shock loci (Right), as visualized by immunofluorescence. The images were pseudocolored in red and merged with those of stained chromosomes (cyan) to facilitate the localization. The transgenic insertion site is indicated by a white arrow head in each panel. The major heat shock loci at 87 A and C are indicated by white dots in the right panel.