The cDNA of the estrogen receptor (ER) has been isolated from red seabream liver and sequenced. The cDNA contains a complete open reading frame encoding 581 amino acid residues. The structural homology indicated that the red seabream ER (rsER) lacks region A in region A/B (trans-activation domain) as well as tilapia, medaka and rainbow trout ERs. In region B, amino acids were conserved also in rsER around a serine residue that is putatively phosphorylated by a mitogen-activated protein kinase. In region C (DNA binding domain), rsER shows a high degree of sequence conservation with other fish ERs, particularly eight cysteins in two zinc-fingers and the P-box were completely conserved. Region E (hormone binding domain) was also highly conserved. On the other hand, in regions D and F, low homology was revealed.
The trans-activation function of this clone was verified by transfection into HeLa cells, which lack endogenous ER. A CMV-based expression vector consisting of rsER cDNA was cotransfected with a chimeric reporter plasmid consisting of an estrogen responsive element fused to the thymidine kinase promoter and the chloramphenicol acetyl transferase (CAT) gene. In the presence of estradiol 17-beta, the CAT activity was stimulated by up to 10-fold. This showed that this clone encoded the functional receptor.