Structural Analysis and Identification of Novel Isoforms of the Circadian Clock Geneperiodin the Silk Moth Bombyx mori

Abstract

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 alpha and period beta isoforms were then determined by the 3' and 5' RACE methods. Isoform period alpha consists of a 3,324 bp oligonucleotide encoding 1,108 amino acid residues, whereas isoform period beta comprises 3,309 bp corresponding to 1,103 amino acids. Isoforms period alpha and period beta 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 alpha and beta proteins are the isomers that reveal firstly the different PAS domain sequences.

Comparative analysis of circadian clock genes in insects

“…In fact, circadian behavioural rhythmicity in LD as well as in constant conditions has been observed in a number of species (Sauman & Hashimi, 1999; Froy et al ., 2003). Most of the comparative circadian studies conducted on Lepidoptera involve the giant silkmoth, Antheraea pernyi , the domestic silk moth Bombyx mori and the monarch butterfly, Danaus plexippus , (Sauman & Hashimi, 1999; Chang et al ., 2003; Sehadova et al ., 2004; Takeda et al ., 2004; Zhu et al ., 2005, 2008a,b; Iwai et al ., 2006; Reppert, 2006; Trang le et al ., 2006; Iwai et al ., 2008). The latter species is particularly important given the possible link between the circadian clock and annual long distance migrations characteristic of this organism.…”

“…The A. pernyi genome harbors a family of three sex‐linked per genes, of which only one ( ApperZ ) is essential for circadian clock functions (Gotter et al ., 1999). Conversely, B. mori has only a single copy of the per gene in both males and females (Takeda et al ., 2004; Iwai et al ., 2006; Sandrelli et al ., 2007b), that produces two Bm PER isoforms, differing by 5 aa in length (Takeda et al ., 2004) whereas in the monarch butterfly only a single Dpper cDNA has been isolated (Froy et al ., 2003). The PER proteins of these Lepidoptera show the canonical Drosophila PER domains, including the nuclear localization signal (NLS), PAS A and B regions, cytoplasmic localization domain (CLD) and nuclear export signal (NES) (Reppert et al ., 1994; Takeda et al ., 2004).…”

Organization of endogenous clocks in insects

“…So far, PER was isolated from several Dipterean [4,5] and Lepidopterean species [6][7][8][9], two Hymenopterean species [10,11] and two Hemipterean species [7,12] (Figure 1). PER possesses a PAS (PER/ARNT/SIM) domain, split into two structural motifs (PAS-A and PAS-B) which facilitate protein-protein interactions.…”

“…', pp. 911-920 c 1996Elsevier; and[18] c 2003 American Society for Biochemistry and Molecular Biology, with additional data from[9,20].…”

Expression of spliced variants ofperiodmRNA in the Japanese honeybeeApis cerana japonica

“…The silkmoth (Bombyx mori) also possesses two per variants with different PAS domain sequences (Takeda et al 2004). To date, there is no information regarding the functional significance of the plural per variants and no evidence of their presence in other insects.…”