Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan.
J Biol Rhythms. 2012 Apr;27(2):126-34. doi: 10.1177/0748730411435997.
Recent studies show that the timeless (tim) gene is not an essential component of the circadian clock in some insects. In the present study, we have investigated whether the tim gene was originally involved in the insect clock or acquired as a clock component later during the course of evolution using an apterygote insect, Thermobia domestica. A cDNA of the clock gene tim (Td'tim) was cloned, and its structural analysis showed that Td'TIM includes 4 defined functional domains, that is, 2 regions for dimerization with PERIOD (PER-1, PER-2), nuclear localization signal (NLS), and cytoplasmic localization domain (CLD), like Drosophila TIM. Td'tim exhibited rhythmic expression in its mRNA levels with a peak during late day to early night in LD, and the rhythm persisted in DD. A single injection of double-stranded RNA (dsRNA) of Td'tim (dstim) into the abdomen of adult firebrats effectively knocked down mRNA levels of Td'tim and abolished its rhythmic expression. Most dsRNA-injected firebrats lost their circadian locomotor rhythm in DD up to 30 days after injection. DsRNA of cycle (cyc) and Clock genes also abolished the rhythmic expression of Td'tim mRNA by knocking down Td'tim mRNA to its basal level of intact firebrats, suggesting that the underlying molecular clock of firebrats resembles that of Drosophila. Interestingly, however, dstim also reduced cyc mRNA to its basal level of intact animals and eliminated its rhythmic expression, suggesting the involvement of Td'tim in the regulation of cyc expression. These results suggest that tim is an essential component of the circadian clock of the primitive insect T. domestica; thus, it might have been involved in the clock machinery from a very early stage of insect evolution, but its role might be different from that in Drosophila.
最近的研究表明，在某些昆虫中， timeless (tim) 基因不是生物钟的必需组成部分。在本研究中，我们使用无翅昆虫 Thermobia domestica 研究了 tim 基因是否最初涉及昆虫生物钟，或者是否在进化过程中作为生物钟的组成部分获得。克隆了时钟基因 tim 的 cDNA，并对其结构分析表明，Td'TIM 包括 4 个定义明确的功能域，即与 PERIOD（PER-1、PER-2）二聚化的 2 个区域、核定位信号（NLS）和细胞质定位域（CLD），与果蝇 TIM 相似。Td'tim 的 mRNA 水平表现出节律性表达，在 LD 中的白天后期到早期夜间达到峰值，并且这种节律在 DD 中持续存在。将 Td'tim 的双链 RNA（dsRNA）（dstim）单次注射到成年火蚁的腹部可有效降低 Td'tim 的 mRNA 水平并消除其节律性表达。大多数注射 dsRNA 的火蚁在注射后 30 天内失去其在 DD 中的昼夜节律运动节律。Cycle (cyc) 和 Clock 基因的 dsRNA 通过将 Td'tim mRNA 敲低至完整火蚁的基础水平，也消除了 Td'tim mRNA 的节律性表达，表明火蚁的分子钟类似于果蝇的分子钟。然而，有趣的是，dstim 还将 cyc mRNA 降低至完整动物的基础水平并消除了其节律性表达，表明 Td'tim 参与了 cyc 表达的调节。这些结果表明，tim 是原始昆虫 T. domestica 生物钟的必需组成部分；因此，它可能在昆虫进化的早期阶段就已经参与了生物钟机制，但它的作用可能与果蝇不同。
