second CA/AA time course of y w. Drosophila melanogaster

Circadian clocks are temporally aligned to the environment via signals, or Zeitgebers, such as daily light and temperature cycles, food availability, and social behavior. In this study, we show that genome-wide expression profiles from temperature-entrained flies show a dramatic difference in the presence or absence of a thermocycle. Whereas transcription appears to be modified globally by changes in temperature, there is a specific set of transcripts that continue to oscillate in constant conditions following temperature entrainment. These transcripts show a significant overlap with a previously defined set of transcripts oscillating in response to a photocycle. Further, these overlapping transcripts maintain the same mutual phase relationships after entrainment by temperature or light. Comparison of the collective temperature- and light-entrained circadian phases indicates that natural environmental light and temperature cycles cooperatively entrain the circadian clock. These findings suggest that a single transcriptional clock in the adult fly head is able to integrate information from both light and temperature. Keywords: circadian time course Overall design: y w flies that had been reared in constant darkness initially at 25 C and later in a 12-hr 18 C/ 12-hr 25 C thermocycle for more than 4 days were harvested every four hours during an additional 18 C/25 C thermo cycle (also indicated as a cold/ambient or CA cycle) or an additional day at constant 25 C (also indicated as ambient/ambient or AA) . Relative to time CA0 as the time of the onset of the 18 C cryophase or AA0 as the time of the onset of the subjective cryophase during constant 25 C, samples were collected in a CA2- CA6-CA10-CA14-CA18-CA22 and AA2-AA6-AA10-AA14-AA18-AA22 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

生物钟（circadian clock）通过各类信号（或称授时因子，Zeitgeber）实现与环境的时间同步，这类信号包括每日光照与温度周期、食物可获得性以及社会行为等。
本研究显示，经温度授时的果蝇的全基因组表达谱，在有温度周期与无温度周期的条件下存在显著差异。尽管温度变化可在全局范围内调控转录过程，但经温度授时后，仍有一类特定的转录本在恒定条件下持续呈现节律性振荡。这类转录本与此前报道的受光照周期调控而呈节律振荡的转录本集合存在显著的重叠。此外，这些重叠的转录本在经温度或光照授时后，仍保持一致的相互相位关系。
对温度授时与光照授时的集体昼夜节律相位进行比较后可知，自然环境中的光照与温度周期可协同授时生物钟。上述研究结果表明，成年果蝇头部内存在的单一转录生物钟，能够整合光照与温度两类信号输入。
关键词：昼夜节律时间序列
整体实验设计：本研究使用的y w（黄身白眼）果蝇，初始在25℃恒暗条件下饲养，随后置于12小时18℃/12小时25℃的温度周期中饲养超过4天。之后将其分别置于额外的18℃/25℃温度周期（又称低温/常温循环，即CA循环）或恒定25℃条件（又称常温/常温循环，即AA循环）中，每4小时收集一次样本。以CA0作为CA循环中18℃低温相的起始时间，AA0作为恒定25℃条件下主观低温相的起始时间，样本收集时序为CA2、CA6、CA10、CA14、CA18、CA22以及AA2、AA6、AA10、AA14、AA18、AA22。
通过破碎冰冻果蝇并经一系列筛网分选，分离得到果蝇头部。总RNA通过异硫氰酸胍法提取，并经氯化铯密度梯度离心纯化。后续通过RNeasy纯化柱（Qiagen公司）对RNA样本进行进一步纯化。取25μg纯化后的RNA合成生物素标记的cRNA探针，杂交方法参照此前文献（Wijnen H、Naef F与Young MW, 《Methods in Enzymology》, 2005; 393: 341-365）。更多信息可参阅：http://biorhythm.rockefeller.edu