timeless Is an Essential Component of the Circadian Clock in a Primitive Insect, the Firebrat Thermobia domestica

• Published in: Journal of biological rhythms 2012-04, Vol.27 (2), p.126-134
• Main Authors: Kamae, Yuichi, Tomioka, Kenji
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites - genetics; Circadian Clocks - genetics; Circadian Clocks - physiology; Circadian rhythm; Circadian Rhythm - genetics; Circadian Rhythm - physiology; Cloning, Molecular; DNA, Complementary - chemistry; DNA, Complementary - genetics; Drosophila; Entomology; Evolution & development; Gene Expression Profiling; Gene Expression Regulation - radiation effects; Genes; Insect Proteins - classification; Insect Proteins - genetics; Insect Proteins - physiology; Insecta - genetics; Insecta - physiology; Insects; Light; Molecular Sequence Data; Motor Activity - physiology; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Thermobia domestica; Time Factors
• ISSN: 0748-7304; 1552-4531
• 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.