Annual Report 2003

exogenous genes into a hymenopteraninsect and to analyze these gene functions.Hymenoptera includes many species importantto agriculture as predators and pollinators.It is expected that this system willallow production of valuable hymenopteranstrains that genetically acquired useful features.cDNA cloning and biochemical characterizationof Bombyx mori orphan receptor,BmHR78Takahiro Shiotsuki, Makoto Hirai,Manabu Kamimura, Shuichiro TomitaDevelopmental Biology DepartmnetInsect juvenile hormones (JHs) haveimportant roles in insect physiology, especiallydevelopment and metamorphosis, butstudies of the molecular mechanisms of theJH have not been progressed yet. For theclarification of JH actions, a novel member ofthe nuclear receptor superfamily from thesilkworm Bombyx mori was identified andnamed BmHR78. The DNA binding domainof the BmHR78 shows high similarity tosome other orphan receptors, such asTenebrio molitor hormone receptor 78(TmHR78), Drosophila hormone receptor 78(DHR78) and mammalian testicular receptor2 (TR2), whereas the ligand binding domainis not well conserved. Northern blot analysisshowed that the BmHR78 gene was mostabundantly expressed in the testis in theB. mori. From the 4th to 5th instar, theBmHR78 gene was constantly expressed inthe testis. In the anterior silk gland, thelevel of BmHR78 gene expression was developmentallychanged. From day 10.0 to 11.0in the 5th instar, another BmHR78 transcriptof smaller size appeared. BmHR78forms not only a homodimer but also a heterodimerwith Ultraspiracle (USP), an insecthomologue of RXR, in a yeast two-hybrid invivo assay. USP isoform also appeared atthe same stages in the tesits. The directinteraction between BmHR78 and USP wasalso confirmed by in vitro pull down assay.Deletion mutant analysis showed thatBmHR78 interacts with USP via the 9thheptad repeat in helix 10 of the E region.This repeat is well conserved in RXR and itsheterodimer partners, and was shown to bean interface for their dimerization. Ininsects, only the ecdysone receptor and hormonereceptor 38 (HR38) are known thus farto dimerize with USP. Thus, BmHR78 is athird dimerization partner for USP and maymodulate the molecular action of USP,including the ecdysone signal cascades.Table 1 Quantification of protein-protein interaction by yeast 2-hybrid systemGAL4 DBD GAL4 AD relative activity,% of control USP 0.0 EcR 0.0 BmHR78 0.3BmHR78 BmHR78 2.8EcR USP 155.0BmHR78 USP 128.8p53 SV40 100.0Each combination of baits (pAS2-1, GAL4 DB) and preis (pACT2,GAL4 AD) were transfected into Y187 and GC1945 yeast cells. Controlcombination was performed with p53 and SV40.Annual Report 2003 15