Cloning and characterization of the ecdysone receptor and ultraspiracle protein from the water flea Daphnia magna.
cDNAs encoding the ecdysone receptor (EcR) and ultra spiracle (USP) protein were cloned from the water flea Daphnia magna (Crustacea: Cladocera). The deduced EcR and USP amino acid sequences showed a high degree of homology to those of other crustaceans as well as insects. We isolated three isoforms of EcR that differ in the A/B domain. Quantitative PCR analysis indicated differing temporal expression patterns of the EcR isoforms during the molting period and demonstrated that the expression of one subtype correlated well with the timing of molt. Using cDNAs encoding EcR and USP, we constructed a Daphnia EcR/USP reporter based on a two-hybrid system. The gene fusions encoded the EcR ligand-binding domain (LBD) fused to the Gal4 DNA-binding domain, and the USP-LBD fused to the Vp16 activation domain. These chimeric genes were transfected with a luciferase reporter gene. Dose-dependent activation of the reporter gene could be observed when transfectants were exposed to Ec and other chemicals known to have Ec-like activities. This two-hybrid system may represent a useful reporter system for further examination of hormonal and chemical effects on Daphnia at the molecular level.
Kato
Yasuhiko
Y
Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Okazaki, Aichi 444-8787, Japan.
Kobayashi
Kaoru
K
Oda
Shigeto
S
Tatarazako
Norihisa
N
Watanabe
Hajime
H
Iguchi
Taisen
T
D000595:Amino Acid Sequence; D000818:Animals; D033361:Cladocera / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003445:Crustacea / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis*; D004440:Ecdysone / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D007313:Insecta / Q000235:genetics; D008018:Life Cycle Stages; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020033:Protein Isoforms / Q000235:genetics* / Q000378:metabolism; D011987:Receptors, Steroid / Q000235:genetics* / Q000378:metabolism; D017385:Sequence Homology; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D019476:Insect Proteins; D020033:Protein Isoforms; D011987:Receptors, Steroid; C055469:ecdysone receptor; D004440:Ecdysone