light would explain additional light-responsivity. By contrast, both DmCRY and plant cryptochromes occur in a largely oxidized flavin state in vivo [9,12,56] and require photoreduction by light to produce the radical state proposed for signaling. Interestingly, an apparent Type II cryptochrome from the garden warbler [57], was reportedly isolated in the oxidized form and only transiently accumulated radical in the course of photoreduction, suggesting greater light responsivity than in their mammalian counterparts. In the latter cases, a small proportion of radical that may accumulate intracellularly in the absence of light could explain any residual minor light-independent response characteristics of these and other types of cryptochromes. In summary, we demonstrate that mammalian (Type II) cryptochrome is capable of mediating light-independent pheno- types in transgenic expressing Drosophila related to gene expression and timing of development, some of which can be further stimulated by light. We furthermore provide evidence that DmCRY can mediate novel transcriptional responses in flies in the absence of light, in contrast to the light-dependent mechanism underlying DmCRY clock function. Therefore, the conceptual distinction between light sensing and light-independent crypto- chromes may not imply fundamental differences in mechanism but instead be a reflection of the different equilibrium states reached between activated (anionic radical) and inactive (oxidized) resting states of the receptor that can be further modified by light. In mammals, cryptochromes are expressed in most tissue types and also occur in peripheral locations that are penetrated by light. Therefore, future studies of HsCRY1 signaling pathways may lead to identification of novel light-dependent cryptochrome responses in man that have yet to be discovered. Materials and Methods Constructions and preparation of transgenic flies Full- length Hscry1 cDNA was ampified by PCR and cloned into the pP(UAST) vector [27] downstream of the UAS activator. Primers used for cloning were: N- terminal; ccc ccc gaa tcc ttt ttt atg ggg gtg aac gcc gtg cac and C - terminal; ccc ccc tct aga cta att agt gct ctg tct. For HsCRY1 - Dmcry fusion constructs (Hsdm) the c- terminal domain of Hscry1 was replaced with a short c-terminal fragment of Dmcry. Primers were N-terminal; ccc ccc ctc gag atg ggg gtg aaac gcc gtg cac and C-terminal: ccc ccc tct aga cta cac cac gtc ggc cag cca gaa gaa ctg acc cac ttc ctc ctc gtt tgc cag aag acc ta g tcc. The fusion junction is at A501 of HsCRY1, to the peptide 527NEEEVRQFFWLADVV* of DmCRY. At least two indepen- dent insertion lines for each construct were analyzed for all tests. HsCRY1 antibodies were as described [9]. Circadian actogramm experiments Actogramms of rhythmicity conferred by Hscry transgenic flies in LL and DD. Three day old adult flies expressing Hscry1, Hsdm in both wt and cryb mutant genetic backgrounds were entrained for 72 hours to 12 hr day/night cycles and analyzed for free running period in LL and DD for 10 days. Rhythmicity and period length are shown.The mean values of circadian period (h), associated powers, and activities (number of events per 0.5 h) are given 6 s.e.m. Tests for eclosion 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 mmolm22 sec21) or continuous dark (DD) in a temperature controlled and Human Cryptochrome Light Responsivity humidity controlled plant growth chamber, 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. A. Time to eclosion was plotted for LL (continuous light). Wild type flies were the non-expressing tim-Gal4 parental lines. cryb mutant flies were flies in which the cryb mutation was introduced into the tim-Gal4 parental lines. The genetic back- grounds of wild type and cryb mutant flies are therefore comparable. RNA Isolation and RT-PCR Analysis Total RNA was prepared, using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. RNA (1 mg) was treated with RQ1 RNase-Free DNase (Promega, Madison, WI, USA) and reverse-transcribed using random hexamers and SUPERSCRIPTTM III First-strand kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommenda- tions. The expression levels of Dro3 (CG32283) and WHITE (CG2759), 7906 (CG7906) and End (CG6467), were determined using the corresponding primers: dro3-5: GCACACTGTTTTGG- CACG, dro3-3: GGCGGCACTTTTCTCC; white-5: AGGT GCTGAAGAAGCTGTC, white-3: ACCCACGTAGGAAAA- GAAGTC; 7906-5: TGTGACCAAGGTGATGCAG, 7906-3: GTGTTGTTTCGGATGGTTCAG; end-5: CATCTCCCTGA TCAAGATCCC, end-3: AGATTGGTAGCCACACCAC. Rela- tive mRNA abundance was calculated by using the comparative cycle threshold (DCt) method and normalized to the correspond- ing RP49 (CG7939) gene levels using the following primers: RP49- 5: CGTTTACTGCGGCGAGAT; RP49-3: CCGTTGGGGTT GGTGAG. Equivalent efficiencies between the different probes and our internal standard were observed during the PCRs. Microarray analysis Total RNA was prepared, using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. 500 ng of total RNA from each samples were processed, labeled and hybridized to Affymetrix Genechip Drosophila 2.0 arrays according to the manufacture’s suggestions (39 IVT Express Kit User Manual, Affymetrix, Santa Clara, CA). Three Biological replicates were run for each condition. Gene-level expression values were derived from the CEL file probe-level hybridization intensities using the model-based Robust Multichip Average algorithm (RMA) [58]. RMA performs normalization, background correction and data summarization. An analysis is performed using the LPE test [59]. and a p-value threshold of p,0.05 is used as the criterion for expression. The estimated false discovery rate (FDR) of this analyse was calculated using the Bonferroni approach [60] in order to correct for multiple comparisons. All data is MIAME compliant and the raw data has been deposited in ArrayExpress, UK database (accession number pending, will be received prior to publication). HsCRY1 Protein Purification The gene encoding HsCRY1 was synthesized by GenScript and codon-optimised for expression in Pichia pastoris, hence removal of the EcoR1 in the native HsCRY1 cDNA. It was recloned into the EcoR1 and Xbal sites of the pPICZ B Pichia pastoris expression vector. This was then transformed into the SMD1168H Pichia host strain. Expression trials were analysed by SDS PAGE and immunodetection using the HsCRY1 polyclonal rabbit antibody (Thermo Scientific, Figure 1a). The recombinant protein was expressed by growing 4 L of culture in BMMY media at 30uC and by methanol induction (0.5% v/v) every 24 hours. The cells were PLoS ONE | www.plosone.org 12 March 2012 | Volume 7 | Issue 3 | e31867