Molecular cloning and tissue distribution of three estrogen receptors from the cyprinid fish Varicorhinus barbatulus.

We present molecular cloning and tissue expression analysis of three estrogen receptor (ER) subtypes, vbERalpha, vbERbeta1 and vbERbeta2, from liver of the cyprinid fish Varicorhinus barbatulus through reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The sequence alignment and phylogenetic analysis reconfirmed the evolutionary relationship of V. barbatulus within the family Cypriniformes. Directional constraints for subtype-specific substitution of critical amino acids were observed in the E2 binding region. For amino acid substitution, vbERbeta exhibited a M517L change in the ligand-dependent transactivation region. The tissue distributions were investigated using RT-PCR with subtype-distinguishable primers. Both vbERalpha and vbERbeta1 were most highly expressed in liver, while vbERbeta2 was higher in intestine. Here we demonstrate that the identification and cloning of ER subtypes using PCR is feasible in wildlife in that the temporal and spatial observations are consistent with those from phylogeny analysis and crystal structural investigation by others.