Meeting Abstract

P2.32  Monday, Jan. 5  Structure and Expression of the Uca pugilator E75 Nuclear Receptor: a Primary Ecdysteroid Response Gene DURICA, DS*; JOHNSON, JB; DAS, S; LEE, K; HOPKINS, PM; Univ. of Oklahoma; Univ. of Oklahoma; Univ. of Oklahoma; Univ. of Oklahoma; Univ. of Oklahoma ddurica@ou.edu

The E75 nuclear receptor gene was one of the first gene loci shown to be transcriptionally up-regulated in response to ecdysteroid exposure. It has been cloned in a number of insect species, where several isoforms, arising through differential splicing and alternative promoter usage, have been characterized. Among crustaceans, E75 homologs have been identified in the shrimp Metapenaeus ensus, where multiple isoforms were identified, and in the brachyuran crab, Gecarcinus lateralis, where a single isoform was discovered. Using specific primers, we first isolated a 431 nucleotide segment of the Uca pugilator E75 homolog using RT-PCR, encompassing the conserved ligand binding domain and a portion of the hinge region. This subclone was then used to screen a late proecdysial oligo-dT primed cDNA library derived from limb bud mRNA. Four independent isolates were recovered from library screenings; overlaps within these clones revealed a single open reading frame of 797 amino acids. The Uca E75 homolog shows 94% nucleotide identity and a probable 99% amino acid sequence identity to the Gecarcinus sequence, using Clustal alignments to other E75 genes and EST sequences from insects and other crustaceans. Nucleotide sequence conservation (>75% identity) also extends into the non-coding regions. The E75 gene is expressed during embryogenesis and early larval development, and during blastemal organization and hypertrophic growth of the regenerating limb bud. Hormonal induction experiments of newly hatched zoal larvae, using three endogenous crustacean ecdysteroids (ponasterone A, ecdysone and 20-hydroxyecdysone) show a dose-dependent increase in transcript accumulation following a 3 hour exposure to ponasterone A, indicating that the E75 transcription unit is an ecdysteroid primary response gene in crustaceans.