title: Heat shock factor binds to heat shock elements upstream of heat shock protein 70a and Samui genes to confer transcriptional activity in Bombyx mori diapause eggs exposed to 5°C.
abstract: To understand the molecular mechanisms of how 5°C-incubation activates mRNA expression of Hsp70a and Samui genes in Bombyx mori diapause eggs, we first searched the 5'-upstream regions of the Hsp70a and Samui genes for heat shock elements (HSEs) and found two regions [Hsp70aHSE-1 (-95 to -58) and -2 (-145 to -121), and SamuiHSE-1 (-84 to -55) and -2 (-304 to -290)] corresponding to HSEs (repeats of nGAAn and/or nTTCn). We cloned four cDNAs encoding heat shock factor (HSF)-a2 (627 amino acids), -b (685 aa), -c (682 aa) and -d (705 aa), which were produced by alternative splicing. When we exposed diapause eggs to 5°C beginning at 2 day post-oviposition to break diapause, HSFd mRNA only increased after chilling for 6-8 days, a pattern very similar to those of Hsp70a and Samui mRNAs. To examine further whether HSFd binds to the respective HSEs, we carried out a gel shift assay using HSFd protein expressed in a cell-free system and the isolated HSEs; migration of the respective digoxigenin(DIG)-labeled HSE-1 and -2 of Hsp70a and Samui was retarded by addition of HSFd; the retarded bands disappeared after addition of the corresponding unlabeled HSE-1 and -2 as competitors, but were not affected by addition of the respective mutated unlabeled HSE-1 and -2. These results indicated that HSFd protein binds to the respective HSEs and may activate mRNA expression of Hsp70a and Samui genes upon exposure of diapause eggs to 5°C.
authors.affiliation: Laboratory of Sericulture & Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
authors.forename: Fukashi
authors.initials: F
authors.lastname: Kihara
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Niimi
authors.forename: Okitsugu
authors.initials: O
authors.lastname: Yamashita
authors.forename: Toshinobu
authors.initials: T
authors.lastname: Yaginuma
mesh_terms: D024506:5' Flanking Region; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System; D003001:Cloning, Molecular; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D010063:Ovum / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D019476:Insect Proteins; D012333:RNA, Messenger; C431690:samui protein, Bombyx mori