, 2009, Muskus et al., 2007, Sekine et al., 2008 and Xu et al., 2005), demonstrating that manipulations increasing DBT activity over wild-type levels do not necessarily affect period. Because there are no mutations in the endogenous gene A-1210477 price to allow assessment of loss-of-function phenotypes, we used RNAi knockdown of the endogenous transcript to investigate bdbt function. Wild-type flies maintained in constant darkness exhibit persistent circadian rhythms of activity, with high levels of activity during the daylight hours in a

previous light/dark regimen and low levels of activity during the dark hours in the previous light/dark cycle. However, flies with the genotype timGAL4 > UAS-dcr2; BKM120 ic50 UAS-bdbt RNAi or elavGAL4 > UAS-dcr2; UAS-bdbt RNAi exhibited arrhythmic locomotor activity if allowed to age for 7 days before initiation of the assay ( Table 1; Figure 2A). In a timGAL4 > UAS-bdbt RNAi genotype without a UAS-dcr2 gene, we obtained a weaker but more clearly circadian phenotype exhibiting long periods instead of arrhythmicity ( Figure 2B; Table 1).

Finally, light:dark cycles (LD) drove diurnally cycling behavior that lacked anticipation of the dark-to-light and light-to-dark transitions in timGAL4 > UAS-dcr2; UAS-bdbt RNAi flies ( Figure 2C); cycles of behavior which lack anticipation of the lighting transitions are typical of circadian loss-of-function mutants. Interestingly, the evening peak of activity was largely missing ( Figure 2C), and this also speaks to altered circadian function. The bdbt RNAi behavioral phenotypes, which include long period and arrhythmic activity

in DD and lack of anticipation to lighting transitions in LD, establish bdbt as a Rolziracetam true circadian gene. The parameters for RNAi knockdown were investigated. If assayed immediately after eclosion from pupae, timGAL4 > UAS-dcr2; UAS-bdbt RNAi flies were mostly rhythmic with wild-type periods (data not shown), suggesting that expression of the RNAi from the timGAL4 driver does not reach high enough levels to become effective until the adult stage. The results were obtained with both of the RNAi lines obtained from the VDRC ( Table 1). The efficacy of the knockdown, shown by immunoblot ( Figure 3A, bottom panel), demonstrated partial knockdown of BDBT protein levels; incomplete knockdown with the circadian driver is expected if BDBT is expressed in noncircadian tissues. Consistent with expression in noncircadian neurons, knockdown with the general neuronal driver elav-GAL4 is more complete ( Figure S4C). Consistent with the locomotor activity arrhythmicity, there were effects on PER and DBT expression. High levels of PER with fast mobility on SDS-PAGE, shown in a previous study to arise from hypophosphorylated PER (Edery et al.