Comp. Biochem. Physiol. Vol. l13B, No. 2, pp. 341-347, 1996 Copyright © 1996 ElsevierScience Inc. ELSEVIER ISSN 0305-0491/96/$26.00 SSDI 0305-0491(95)02032.G Molecular Cloning of an Ecdysone Receptor (B1 Isoform) Homologue from the Silkworm, Bombyx mori, and its mRNA Expression During Wing Disc Development Manabu Kamimura, Shuichiro Tomita and Haruhiko Fujiwara" NATIONAL INSTITUTE OF SERICULTURAL AND ENTOMOLOGICAL SCIENCE, OHWASHI, TSUKUBA IBARAKI 305, JAPAN ZOOLOGICAL INSTITUTE,GRADUATE SCHOOLOF SCIENCE, UNIVERSITY OF TOKYO, HONGO, BUNKYO-KU, TOKYO l 13, AND JAPAN ABSTRACT. We reported the isolation and sequence of a clone encoding a putative ecdysone receptor B1 isoform of the silkworm, Bombyx mori. The predicted open reading frame encoded 543 amino acids, with 51%, 95% and 71% identities with the Drosophilamelanogaster ecdysone receptor B1 isoform in the N terminal A/ B region, DNA binding domain (C region) and ligand binding domain (E region), respectively. A single 6.2 kb message for the EcR gene was abundant in wing discs and fat bodies at the onset of metamorphosis. At the same stage, however, no or a tiny amount of mRNA was shown in posterior or middle silk glands, respectively. During the final instar, the mRNA expression in wing discs was maximal on the day of wandering. These data suggest the transcription of the Bombyx EcR gene is regulated in tissue-specificand stage-specific manner during metamorphosis. COMP BIOCHEM PHYSIOL 113B, 341-347, 1996. KEY WORDS. Bombyx mori, Silkworm, Ecdysone receptor, EcR, B1 isoform, Ecdysteroid, 5' untranslated region, Wing disc, Silk glands, Metamorphosis INTRODUCTION Steroid hormones coordinate a wide range of developmental and physiological processes in higher organisms. In insects, ecdysteroids, primarily 20-hydroxyecdysone (20E), play a cen- tral role in the orchestration of development during larval molts and metamorphosis (19). It is postulated that ecdyste- roids bind with a receptor protein, namely, ecdysone receptor (EcR), and directly regulate gene expression (1). Recently, the Drosophila melanogaster ecdysone receptor (DmEcR) gene was isolated and shown to be a member of the steroid/thyroid hormone nuclear receptor superfamily (12). There are three isoforms of DmEcR with common DNA and ligand binding domains but different N-terminal regions due to the use of different promoters and alternative splicing (23). These isoforms are expressed in tissue-specific and stage- specific manner and therefore are thought to be important in regulation of these responses (7,23,26). In both Drosophila and the tobacco hornworm, Manduca sexta, the peak stage for the EcR expression in several tissues during the final larval instar, is observed at the onset of meta- morphosis (6,23,26). A large quantitative increase in EcR mRNA at this time suggests that increased levels of receptor Correspondence to: Hamhiko Fujiwara, ZoologicalInstitute, Graduate School of Science, Universityof Tokyo, Hongo, Bunkyo-ku,Tokyo 113, Japan; Fax (03)38161965; e-Mail: haruh@uts.2.s.u-tokyo.ac.jp Received 22 May 1995; revised 12 July 1995; accepted 16 July 1995. are somehow important in programming the metamorphic re- sponses. The small pulses of ecdysteroid during earlier stage in the final instar of Manduca sexta initiate metamorphosis as signalled by the cessation of feeding and the onset of wander- ing behavior. It is suggested that these small rises of ecdyste- roid are correlated with the induction of Manduca EcR mRNA transcription (6). However, in another lepidopteran insect, Bombyx mori, the small peak of ecdysteroid is not detected before the wandering stage, while similar temporal changes are observed during metamorphosis (10,11). In this report, we have characterized a cDNA from the silkworm, Bombyx mori EcR gene that likely encodes a homologue of the Drosophila B1 isoform. Its mRNA expression in wing discs during the final larval instar and in several tissues after the wandering stage was analyzed. MATERIALS AND METHODS Experimental Animals The silkworm larvae of racial F1 hybrid between C145 and J140 were reared on an artificial diet (Yakuruto Co., Tokyo) under 12 hr light: 12 hr dark photoperiod at 25 + 1°C. Since the 4th larval ecdysis occurs during the scotophase, the newly molted last (5th) instar larvae were segregated immediately after the onset of the photophase (21), and this day was desig-