GAL4-responsive genes in larval salivary glands of late Drosophila prepupae

The yeast transcription factor GAL4 has been reported to cause cell death and to have other biological effects when expressed in Drosophila (Kramer and Staveley, 2003: Genet. Mol. Res. 2, 43; Rezaval et al., 2007: Eur. J. Neurosci. 25, 683). Using heat-shock-induced expression of GAL4 to drive expression of a UAS-senseless responder gene in transcriptional profiling experiments, we found that the underlying cause of these effects might be a genomic response to GAL4. To further characterize this response and to account for GAL4-independent changes caused by the transgene integration, GAL4 was expressed from two copies of the transgene in two independent lines, P{GAL4-Hsp70.PB}89-2-1 (short P{hs-GAL4}89) and P{hs-GAL4}X1. In addition, GAL4 was expressed from only one copy of the transgene in P{hs-GAL4}89 prepupae to account for the dosage dependence of observed effects. Prepupae carrying the hs-GAL4 transgenes were subjected to a 30-min heat shock treatment (37 °C) at 9 hours after puparium formation. RNA was isolated from salivary glands dissected from these and similarly treated w1118 control animals at 14 hours after puparium formation and subjected to microarray analysis with Affymetrix GeneChips. The microarray data identified an overlapping set of 1,009 genes that showed an at least 1.5-fold change in expression in both of the GAL4-expressing lines, defining a core set of GAL4-responsive genes in the salivary glands. This set includes genes involved in the control and execution of programmed cell death and in other important regulatory pathways. Keywords: ectopic expression experiment Experimental RNA samples were obtained from GAL4-expressing salivary glands of the line P{hs-GAL4}X1 in 3 biological replicates (SG_P{hsGAL4}X1_14APF_rep1, SG_P{hsGAL4}X1_14APF_rep2, SG_P{hsGAL4}X1_14APF_rep3); of the line P{hs-GAL4}89 in one replicate (SG_P{hsGAL4}89_14APF_rep1); of animals carrying only one copy of P{hs-GAL4}89 in one replicate (SG_1P{hsGAL4}89_14APF_rep1). The samples were compared to control samples obtained in two biological replicates from the salivary glands of w1118 control animals (SG_w1118(2)_14APF_rep1, SG_w1118(2)_14APF_rep2). All samples were compared using dChip and normalized to the same baseline array (median intensity 145). Statistical analysis of differences between the P{hs-GAL4}X1 and w1118 samples was carried out using GeneSpring (Agilent Technologies) and a false discovery rate (FDR) cutoff of 0.05.