Structural analysis and identification of novel isoforms of the circadian clock gene period in the silk moth Bombyx mori

The molecular basis of the circadian clock is an autoregulatory feedback loop in which the PAS domain-containing protein PERIOD periodically inhibits its own transcription. In the present study on PERIOD of the silk moth Bombyx mori, we have cloned two distinct period mRNA homologues with different PAS domain sequences either with or without the pentapeptide GTQEK. A period cDNA fragment first amplified by PCR exhibited a 15 bp-deleted nucleotide sequence in the PAS domain, compared with the database sequence. A possible alternative splicing mechanism was examined by PCR analyses, and a 15 bp-inserted clone was also amplified. The entire sequences of these period α and period β isoforms were then determined by the 3′ and 5′ RACE methods. Isoform period α consists of a 3,324 bp oligonucleotide encoding 1,108 amino acid residues, whereas isoform period β comprises 3,309 bp corresponding to 1,103 amino acids. Isoforms period α and period β were found to be exactly identical except for the 15 bp deletion/insertion site. Such a pair of isoforms with a deletion/insertion sequence, namely two splice variants, has previously been reported only for the PERIOD proteins of the two honeybees, Apis mellifera and A. cerana. The occurrence of an alternative splicing mechanism in the B. mori period gene was hypothesized based on the genome structure recently clarified. Bombyx mori PERIOD α and β proteins are the isomers that reveal firstly the different PAS domain sequences.