LD/DD time course of y w Drosophila #3

Background. The transcriptional circuits of circadian clocks control physiological and behavioral rhythms. Light may affect such overt rhythms in two ways: (1) by entraining the clock circuits and (2) via clock-independent molecular pathways. In this study we examine the relationship between autonomous transcript oscillations and light-driven transcript responses Methodology/Principal Findings. Transcript profiles of wild-type and arrhythmic mutant Drosophila were recorded both in the presence of an environmental photocycle and in constant darkness. Systematic autonomous oscillations in the 12-48 hr period range were detectable only in wild-type flies and occurred preferentially at the circadian period length. However, an extensive program of light-driven expression was confirmed in arrhythmic mutant flies. Many lightresponsive transcripts are preferentially expressed in the adult compound eye and the phospholipase C component of phototransduction, NO RECEPTOR POTENTIAL (NORPA), is required for their light-dependent regulation. Conclusions/Significance. Although there is growing evidence for the existence of multiple molecular clock circuits in plants and fungi, Drosophila appears to possess only one such system. The sustained photic expression responses identified here are partially coupled to the circadian clock and may reflect a mechanism for flies to modulate functions such as visual sensitivity and synaptic transmission in response to seasonal changes in photoperiod. For more information see also http://biorhythm.rockefeller.edu. Keywords: Time course y w flies that had been kept in a 12-hr light/ 12-hr dark cycle for three days were harvested every four hours during an additional light/dark day (ZT) and a subsequent day in constant darkness (CT). Relative to Zeitgeber time 0 (ZT0) as the time of lights on during the LD cycle and Circadian time 0 (CT0) as the time corresponding to subjective lights-on during freerun in DD, time courses were collected in a ZT2- ZT6-ZT10-ZT14-ZT18-ZT22-CT2-CT6-CT10-CT14-CT18-CT22 schedule. Heads were isolated by breaking up frozen flies and passing them through a set of sieves. RNA was prepared using guanidine-thiocyanate extraction followed by purification over a CsCl gradient. Additional purification of the RNA samples was achieved by applying them to Rneasy columns (Qiagen). Biotin-labeled cRNA probe was generated from 25 μg of purified RNA and hybridized as described previously (Wijnen H, Naef F, and Young MW, Methods Enzymol. 2005; 393: 341-365). For more information see also http://biorhythm.rockefeller.edu