Cloning, in vitro expression, and novel phylogenetic classification of a channel catfish estrogen receptor

We obtained two channel catfish estrogen receptor (ccER) cDNA from liver of female fish using RT-PCR. The two fragments were identical in sequence exc ept that the smaller one had an out-of-frame deletion in the E domain, sugg esting the existence of ccER splice variants. The larger fragment was used to screen a cDNA library from liver of a prepubescent female. A cDNA was ob tained that encoded a 581-amino-acid ER with a deduced molecular weight of 63.8 kDa. Extracts of COS-7 cells transfected with ccER cDNA bound estrogen with high affinity (K-d = 4.7 nM) and specificity. Maximum parsimony and N eighbor Joining analyses were used to generate a phylogenetic classificatio n of ccER on the basis of 18 full-length ER sequences. The tree suggested t he existence of two major ER branches. One branch contained two clearly div ergent clades which included all piscine ER (except Japanese eel ER) and al l tetrapod ER alpha, respectively. The second major branch contained the ee l ER and the mammalian ERP. The high degree of divergence between the eel E R and mammalian ERP suggested that they also represent distinct piscine and tetrapod ER. These data suggest that ERa and ERP are present throughout ve rtebrates and that these two major ER types evolved by duplication of an an cestral ER gene. Sequence alignments with other members of the nuclear horm one receptor superfamily indicated the presence of 8 amino acids in the E d omain that align exclusively among ER. Four of these amino acids have not r eceived prior research attention and their function is unknown. The novel f inding of putative ER splice variants in a nonmammalian vertebrate and the novel phylogenetic classification of ER offer new perspectives in understan ding the diversification and function of ER. (C) 1999 Academic Press.