Human Cryptochrome Light Responsivity Figure 2. Analysis of developmental time (until eclosion) in HsCRY1 expressing transgenic Drosophila. For all tests, parent flies were maintained for 24 hours in a 50 ml conical falcon tube containing 10 ml of growth medium in order to lay eggs. Adult flies were then removed and tubes transferred to constant white fluorescent light (200 mmol m22 sec21) or continuous dark (DD) in a temperature controlled and humidity controlled plant growth chamber at 22uC. Time to eclosion was measured every 12 hours and eclosed flies counted and plotted for each time point as a percentage of total eclosed flies. Typically between 100 and 500 flies were counted per Drosophila line. Experiments were performed in triplicate showing qualitatively similar results (peak of eclosion); the data from a representative experiment is shown. A. Time to eclosion was plotted for LL (continuous light). Wild type controls were the non-expressing tim-Gal4 parental lines. cryb mutant flies carried the cryb mutation in the genetic background of the tim-Gal4 parental lines. B. Same as for ‘A’, in constant darkness (DD). C. Relative time to eclosion (x-axis) under differing wavelengths of light at 3 mmol m22 sec21 blue light (B), red light (R) and UV/A light (peak 380 nm) (UV). Y-axis is number of flies pooled from three independent experiments. Hscry1(A) flies expressed HsCRY1 under control of the tim-Gal4 promoter, Hscry1 (B) flies expressed HsCRY1 under the da- Gal4 promoter; cry02 is the mutant allele (Dolozelova et. al. 2007). doi:10.1371/journal.pone.0031867.g002 therefore likely to also bind to promoter elements and provide transcriptional phenotypes in heterologous systems such as Drosophila. Therefore, expression profiling was performed in Drosophila using pupa no older than 24 hours post - 3d instar larva; this developmental stage was chosen since HsCRY1 has a profound effect on the time to eclosion (see above). Microarray PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e31867