Full-length sequence and in vitro expression of rainbow trout estrogen receptor cDNA

We previously reported the isolation of a partial cDNA clone encoding the rainbow trout estrogen receptor (rtER). A 0.4 kb 5'-end insert of this cDNA was used to screen the trout liver lambda gt10 cDNA library, and a full-length cDNA was isolated and sequenced. The principal structural characteristics of the complete coding sequence of the rtER are: first a remarkable homology of the DNA binding (C) and hormone binding (E) domains with those of other species, and second the lack of an A region, the function of which is not yet known but which is well conserved in other species. In vitro expression of the full-length rtER cDNA was carried out after transcription by T7 RNA polymerase and translation in rabbit reticulocyte lysate. Translation product analysis shows three major proteins, the largest one of which probably corresponds to the translation of the complete open reading frame of mRNA. The rtER in vitro translation products specifically bind estrogens (estradiol and diethylstilbestrol), without competition from testosterone or cortisol. The equilibrium dissociation constant (Kd), deduced from the Scatchard plot, is in the same order of magnitude as those determined heretofore in salmon livers during classical experiments. The tissue distribution of rtER mRNA shows that the same mRNA size (3.5 kb) is also present in the pituitary and hypothalamus. However, in the pituitary, a smaller sized mRNA (1.4 kb) is also detected.