Fig. 5. An in vivo transcriptional reporter indicates Da can transactivate the da promoter. Flies were generated that carried a heat shock inducible da+ transgene and a reporter transgene (da.G32), consisting of the da promoter fused to the gal4-coding region. Real-time RT-PCR was used to determine relative levels of gal4 transcript in adult females maintained at 25°C or after heat shock treatment. Both graphs plot the second derivative of SYBR green fluorescence for each replicate (blue, without heat shock; red, with heat shock). The most accurate measure of transcript level is the threshold cycle number (Ct), which is identifiable for each plot as the cycle corresponding to the second derivative peak. The mean threshold cycle numbers are indicated. The gal4 graph shows a significant decrease of 1.48 cycles with heat shock induction of Da protein, indicating at least a two- to threefold increase in RNA. The Mdh1 graph shows no significant change in the threshold cycle number with heat shock: control levels of RNA are unaffected by the treatment.
