Novel transcripts of the estrogen receptor alpha gene in channel catfish

Complementary DNA libraries from liver and ovary of an immature female chan nel catfish were screened with a homologous ER alpha cDNA probe. The hepati c library yielded two new channel catfish ER cDNAs that encode N-terminal E R alpha variants of different sizes. Relative to the catfish ER alpha (medi um size; 581 residues) previously reported, these new cDNAs encode Long-ER alpha (36 residues longer) and Short-ER alpha (389 residues shorter). The 5 '-end of Long-ER alpha cDNA is identical to that of Medium-ER alpha but has an additional 503-bp segment with an upstream, in-frame translation-start codon. Recombinant Long-ER alpha binds estrogen with high affinity (K-d = 3 .4 nM), similar to that previously reported for Medium-ER alpha but lower t han reported for catfish ER beta. Short-ER alpha cDNA encodes a protein tha t lacks most of the receptor protein and does not bind estrogen. Northern h ybridization confirmed the existence of multiple hepatic ER alpha RNAs that include the size range of the ER alpha cDNAs obtained from the libraries a s well as additional sizes. Using primers for RT-PCR that target locations internal to the protein-coding sequence, we also established the presence o f several ER alpha cDNA variants with in-frame insertions in the ligand-bin ding and DNA-binding domains and in-frame or out-of-frame deletions in the ligand-binding domain. These internal variants showed patterns of expressio n that differed between the ovary and liver. Further, the ovarian library y ielded a full-length, ER alpha antisense cDNA containing a poly(A) signal a nd tail. A limited survey of histological preparations from juvenile catfis h by in situ hybridization using directionally synthesized cRNA probes also suggested the expression of ER alpha antisense RNA in a tissue-specific ma nner. In conclusion, channel catfish seemingly have three broad classes of ER alpha mRNA variants: those encoding N-terminal truncated variants, those encoding internal variants (including C-terminal truncated variants), and antisense mRNA. The sense variants may encode functional ER alpha or relate d proteins that modulate ER alpha or ER beta activity. The existence of ER antisense mRNA is reported in this study for the first time. Its role may b e to participate in the regulation of ER gene expression. (C) 2000 Academic Press.