Molecular cloning and functional analysis of the clock genes, Clock and cycle, in the firebrat Thermobia domestica

Comparative molecular analysis reveals a wide variation of clock mechanisms among insects. In this study, the clock gene homologues of Clock (Td'Clk) and cycle (Td'cyc) were cloned from an apterygote insect, Thermobia domestica. Structural analysis showed that Td'CLK includes bHLH, PAS-A, PAS-B domains but lacks a polyglutamine repeat in the C terminal region that is implicated for transcriptional activity in Drosophila CLK. Td'CYC contains a BCTR domain in its C terminal in addition to the common domains found in Drosophila CYC, i.e. bHLH, PAS-A, PAS-B domains. Unlike in Drosophila, Td'Clk mRNA levels showed no significant daily fluctuation, while Td'cyc exhibited rhythmic expression. A single injection of double-stranded RNA (dsRNA) of Td'Clk or Td'cyc into the abdomen of adult firebrats effectively knocked down respective mRNA levels and abolished the rhythmic expression of Td'cyc. Most Td'Clk or Td'cyc dsRNA-injected firebrats lost their circadian locomotor rhythm in constant darkness up to 30 days after injection, whereas those injected with DsRed2 dsRNA as a negative control clearly maintained it. From these results, it is likely that Td'Clk and Td'cyc are involved in the circadian clock machinery in the firebrat. However, the structure and expression profile of Td'Clk and Td'cyc more closely resembles those of mammals than Drosophila.