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 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.
Takeda
Yukimasa
Y
Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Kyushu University.
Chuman
Yoshiro
Y
Shirasu
Naoto
N
Sato
Seiji
S
Matsushima
Ayami
A
Kaneki
Atsushi
A
Tominaga
Yoshiya
Y
Shimohigashi
Yasuyuki
Y
Shimohigashi
Miki
M
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics*; D002940:Circadian Rhythm / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry* / Q000302:isolation & purification; D005260:Female; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000662:veterinary; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D018076:DNA, Complementary; D019476:Insect Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger