Circadian Rhythm and cDNA Cloning of the Clock Gene period in the Honeybee Apis cerana japonica

ABSTRACT—Isolated individual foragers of Apis cerana japonica could be entrained under a light-dark cycle, and the predominant activity was concentrated to the later part of the photophase. Foragers showed circadian rhythm under conditions of constant light and constant dark with free-running periods of more and less than 24 hr, respectively. These observations indicated that A. cerana possesses a circadian clock con-trolling locomotor activity. To investigate the molecular mechanism underlying the circadian system we cloned cDNA for a homolog of the clock gene period (per) from the honeybee by a PCR-strategy. The cloned per-cDNAs consisted of two types, α and β, encoding a putative protein of 1124 amino acids and 1116 amino acids, respectively. The sequences of types α and β were identical except that the former possessed an additional 24 bp stretch corresponding to 8 amino acids in the conserved C2 block. These two types were assumed to be differentially spliced variants and found also in per cDNA of A. mellifera. In support of this idea, Southern blotting experiments showed that per of A. cerana is a single copy gene. RT-PCR analysis and subcloning of the products revealed that the both types α and β are expressed in the brain of the forager. A quantitative RT-PCR assay by which the level of per mRNA in one single brain can be detected was established. Per mRNA level showed daily oscillation under a light-dark cycle with a change of the ratio of type α to β