Human Cryptochrome Light Responsivity Figure 5. Purification and characterization of HsCRY1. A. Western blot confirming the expression of HsCRY1 using the Homo sapiens CRY1 polyclonal rabbit antibody. B. Coomassie-stained SDS PAGE of purified HsCRY1 (66 kDa). C. UV-visible spectrum of purified HsCRY1. Inset: Averaged cwEPR spectrum of a ,290 mM sample of HsCRY1. The peak-to-peak linewidth is 15.4 G at a g-value of 2.0042, which indicates an anionic semiquinone. D. UV-visible absorbance spectra representing the photoreduction of HsCRY1 in the presence of 20 mM DTT. The sample was exposed to 100 mmol m22 s21 of 440–460 nm light for a maximum of 85 minutes. Black line - before irradiation; blue line - after 85 min irradiation; red, dashed line – reoxidation in dark (within ,2 min). Inset: the difference spectrum (Light minus dark) showing peak reduction at 450 nm consistent with oxidized flavin. doi:10.1371/journal.pone.0031867.g005 numerous studies. Therefore, if this were indeed the signaling state, there would be no need for light activation to reach a biologically active conformation of Type II cryptochromes and could explain the light-independent role of this receptor. In addition, there is a smaller proportion of receptor with flavin bound in the oxidized form that can undergo light activation to form the radical state. This may explain the additional light resposivity of HsCRY1 in transgenic Drosophila. Finally, there is evidence of an additional bound cofactor, folate, which could contribute to UV sensing. Discussion In the current work, we have taken a transgenic approach to determine the sensitivity to light of a Type II cryptochrome using HsCRY1 expression in Drosophila. A combination of developmen- tal studies and expression profiling analysis, has shown that HsCRY1 has principally light-independent roles in flies, of which a small subset are enhanced in the light. By contrast, Type I (Drosophila) cryptochromes which show numerous light responsive phenotypes as extensively documented for the circadian clock [1] are shown to have significant light-independent signaling roles in addition to those that are responsive to light. These observations suggest an absence of fundamental mechanistic differences between Type I and Type II cryptochromes, and that their different responsivity to light is due to differences in the stability of the radical flavin in the protein which represents the signaling state. It is also intriguing that certain molecular targets of DmCRY may also be recognized by HsCRY1, suggesting that some conservation of cryptochrome signaling pathways may have occurred between such highly disparate organisms as flies and man. Acceleration of Drosophila Development by HsCRY1 We here describe a novel phenotype mediated both by DmCRY and by HsCRY1 in transgenic expressing Drosophila that results in aceleration of larval development. It should be noted that these similar phenotypes are most likely caused by distinct mechanisms (see transcriptome analysis) impacting on different aspects of larval development and should not be construed as direct physiological PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e31867