Complementary DNA libraries from liver and ovary of an immature female channel catfish were screened with a homologous ER?? cDNA probe. The hepatic library yielded two new channel catfish ER cDNAs that encode N-terminal ER?? variants of different sizes. Relative to the catfish ER?? (medium size; 581 residues) previously reported, these new cDNAs encode Long-ER?? (36 residues longer) and Short-ER?? (389 residues shorter). The 5???-end of Long-ER?? cDNA is identical to that of Medium-ER?? but has an additional 503-bp segment with an upstream, in-frame translation-start codon. Recombinant Long-ER?? binds estrogen with high affinity (Kd = 3.4 nM), similar to that previously reported for Medium-ER?? but lower than reported for catfish ER??. Short-ER?? cDNA encodes a protein that lacks most of the receptor protein and does not bind estrogen. Northern hybridization confirmed the existence of multiple hepatic ER?? RNAs that include the size range of the ER?? cDNAs obtained from the libraries as well as additional sizes. Using primers for RT-PCR that target locations internal to the protein-coding sequence, we also established the presence of several ER?? cDNA variants with in-frame insertions in the ligand-binding and DNA-binding domains and in-frame or out-of-frame deletions in the ligand-binding domain. These internal variants showed patterns of expression that differed between the ovary and liver. Further, the ovarian library yielded a full-length, ER?? antisense cDNA containing a poly(A) signal and tail. A limited survey of histological preparations from juvenile catfish by in situ hybridization using directionally synthesized cRNA probes also suggested the expression of ER?? antisense RNA in a tissue-specific manner. In conclusion, channel catfish seemingly have three broad classes of ER?? 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?? or related proteins that modulate ER?? or ER?? activity. The existence of ER antisense mRNA is reported in this study for the first time. Its role may be to participate in the regulation of ER gene expression. ?? 2000 Academic Press.
Additional Publication Details
Novel transcripts of the estrogen receptor ?? gene in channel catfish