Human Cryptochrome Light Responsivity Figure 1. HSCRY1 constructs and expression in transgenic Drosophila. Full-length HSCRY1 cDNA was ampified by PCR and cloned into the pP(UAST) vector (Rubin et al. 1985) 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 HSCRY12 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 tag tcc. The fusion junction is at A501 of HsCRY1, to the peptide 527NEEEVRQFFWLADVV* of DmCRY. At least two independent insertion lines for each construct were analyzed for all tests. A. Sequence homology of light-sensing and C-terminal domain segments of HsCRY1 and DmCRY. B. Western blot analysis of expression in flies. Expression was induced from the UAS upstream promoter element under the control of tim-Gal4 and analyzed in F1 heterozygote expressing progeny as described [9]. Anti - HsCRY1 antibody was as described [9]. Approximately 10 flies were used per sample lane and protein concentration was quantitated by Bradford assay before loading - see [9]. Left panels represent Western blots of indicated constructs and right panel indicates quantitation – error bars are SD of three independent measurements. Flies were dark adapted for 48 hours prior to trials. Time course refers to time in white light (150 mmolm22 sec21, fluorescent Philips ‘cool white’ bulbs). Load represents Coomassie stain of total protein from the gels blotted to PVDF transfer membranes for the Western blots. doi:10.1371/journal.pone.0031867.g001 clock. Most likely HsCRY1 does not recognize Drosophila clock proteins. Developmental phenotype of HsCRY1 in expressing transgenic flies As an alternative to clock phenotypes, a developmental phenotype known to be modified by light was investigated for a possible biological effect of HsCRY1 in transgenic Drosophila. It has been previously observed that the time to eclosion in developing flies is significantly reduced when eggs are hatched and maintained in constant light (LL) as compared to darkness (DD) [33]. We determined the role of both insect (DmCRY) and mammalian (HsCRY1) cryptochromes on time to eclosion in expressing flies using wt, Dmcry mutant (cryb), HsCRY1 and HsDM over- expressing transgenic flies (Fig. 2). In continuous light (LL), wt flies developed more rapidly than cryb mutant flies with the peak of eclosion occurring 1 K days earlier, suggesting that cryptochrome promotes this response (Fig. 2a). To test this hypothesis we compared the developmental time of both genotypes in continous darkness (DD). As reported previously, peak eclosion occurred significantly later in wt flies under DD than under LL (after 15.5 days instead of 12 days) (Fig. 2a,b). However, it occurred also later in cryb mutants (after 16 days instead of 13.5 days) (Fig. 2a,b). PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e31867