Cloning and characterization of the ecdysone receptor and ultraspiracle protein from the water flea Daphnia magna

Abstract

cDNAs encoding the ecdysone receptor (EcR) and ultraspiracle (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.

Roles of ecdysteroids for progression of reproductive cycle in the fresh water crustacean Daphnia magna

“…The final product of the ecdysteroids synthesis pathway, 20E, actively interacts with EcR/USP heterodimeric receptor in D. magna [16]. This indicates that the binding of 20E to EcR in arthropod species can activate the ecdysteroids signalling pathway.…”

“…Sequence of shd of D. magna was available in the NCBI database (AB257771.1). Sequences of EcR and USP were defined in our previous study (AB274819-24) [16].…”

“…The complex of 20E and EcR recruits ultraspiracle (USP) in insects and daphnids or retinoid X receptor (RXR) in decapods, and acts as a heterodimer [15]. EcR and USP have been cloned in D. magna [16]. Three EcR paralogues with five distinct N-terminus sequences and one USP were identified [16].…”

“…EcR and USP have been cloned in D. magna [16]. Three EcR paralogues with five distinct N-terminus sequences and one USP were identified [16]. These genes in the ecdysteroids biosynthesis pathway except for nvd belong to CYP450 encoding hydroxylases.…”

Review of the reproductive biology of amphipods and their endocrine regulation: Identification of mechanistic pathways for reproductive toxicants

“…An increase in 20-hydroxyecdysone concentrations mediates a cascade of gene-regulatory events mediating molting and reproduction. The ecdysone receptor is a ligand-dependent transcription factor, and it activates transcription of target genes by forming a heterodimer with another nuclear receptor, the ultraspiracle protein [118]. Binding to the DNA helix is mediated by the nuclear receptor that encompasses two zinc-finger polypeptides, where Zn complexes four cysteine amino acids, thereby facilitating the folding of a protein domain involved in the DNA-protein interaction [119].…”