Cloning, sequencing and phylogenetic/phenetic classification of an estrogen receptor alpha (alpha) subtype of sheepshead minnow (Cyprinodon variegatus).

The estrogen receptor (ER) is a key component of the reproductive system of both teleosts and tetrapods. In this study, the sequence and evolutionary relationship of sheepshead minnow (Cyprinodon variegatus) ER were examined. Total RNA from livers of adult laboratory-reared gravid female C. variegatus was reverse-transcribed to prepare cDNA. Nested pairs of gene-specific degenerative primers derived from conserved amino acid sequences of the ER DNA binding domain were used to amplify an internal fragment of the ER cDNA of C. variegatus using polymerase chain reaction (PCR) followed by rapid amplification of cDNA ends (RACE). The amplified cDNA products were inserted into pGEM T-Easy Vector for cloning and sequencing. The cloned ER cDNA segments gave a 524-amino-acid ER sequence, which represents approximately 80% of the sequence. The use of PHYLIP for phylogenetic analysis with the maximum parsimony method and for phenetic analysis with the neighbor-joining method, along with bootstrap resampling, using 24 known sequences of alpha and beta ER subtypes (both teleosts and tetrapods) indicated that the ER cDNA sequence of C. variegatus has strong homology to the alpha-subtype (ER alpha) of other teleostean fish, especially the closely related killifish species, Japanese medaka (Oryzias latipes). Because ER alpha was the only subtype found, it appears that the alpha-subtype is predominant in C. variegatus liver.