Molecular cloning of Clock cDNA from the prawn, Macrobrachium rosenbergii

Abstract

CLOCK, which belongs to the basic helix–loop–helix (bHLH)/PER-ARNT-SIM (PAS) superfamily of transcription factors, is one of the most essential proteins involved in circadian systems of animals. Clock genes have been cloned from several species, including mammals, insects, birds, fish, and amphibians. In the present study, we successfully isolated a Clock homolog (termed Mar-Clock) from the giant prawn, Macrobrachium rosenbergii. The 2949-bp cDNA contained a 2115 bp open reading frame that encoded a putative CLOCK protein of 704 amino acids (termed Mar-CLOCK) exhibiting high identities with CLOCK homologs in other species (30–35%). This is the first report of a circadian clock gene from crustaceans. Mar-CLOCK possessed an exceptionally long glutamine-rich domain (140 amino acids) in its C-terminus, which usually ranges from 14 to 57 amino acids in other known CLOCKs and is supposed to function in transcriptional activation. Using RT-PCR, we observed that Mar- Clock was expressed in all tested tissues. Semiquantitative RT-PCR was performed to investigate the gene expression profile during the light–dark cycle. The results indicated that the expression of the Mar- Clock gene had no significant rhythmicity in central nervous tissues (thoracic ganglia and eyestalk) or peripheral tissues (gill, ovary, hepatopancreas, and muscle). Furthermore, gene expression tended to increase in the central nervous system (brain, thoracic, and abdominal ganglia) of eyestalk-ablated or constant dark (DD) prawns, and in the eyestalk-ablated gill. No expression change was found under constant light (LL) or in heart and muscle.