Novel transcripts of the estrogen receptor alpha gene in channel catfish.
Complementary DNA libraries from liver and ovary of an immature female channel catfish were screened with a homologous ERalpha cDNA probe. The hepatic library yielded two new channel catfish ER cDNAs that encode N-terminal ERalpha variants of different sizes. Relative to the catfish ERalpha (medium size; 581 residues) previously reported, these new cDNAs encode Long-ERalpha (36 residues longer) and Short-ERalpha (389 residues shorter). The 5'-end of Long-ERalpha cDNA is identical to that of Medium-ERalpha but has an additional 503-bp segment with an upstream, in-frame translation-start codon. Recombinant Long-ERalpha binds estrogen with high affinity (K(d) = 3. 4 nM), similar to that previously reported for Medium-ERalpha but lower than reported for catfish ERbeta. Short-ERalpha cDNA encodes a protein that lacks most of the receptor protein and does not bind estrogen. Northern hybridization confirmed the existence of multiple hepatic ERalpha RNAs that include the size range of the ERalpha 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 ERalpha 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, ERalpha 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 ERalpha antisense RNA in a tissue-specific manner. In conclusion, channel catfish seemingly have three broad classes of ERalpha 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 ERalpha or related proteins that modulate ERalpha or ERbeta 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.
Patiño
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Texas Cooperative Fish & Wildlife Research Unit, Texas Tech University, Lubbock, Texas 79409-2120, USA. r.patino@ttu.edu
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Gale
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Wu
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Maule
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Chang
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D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D019556:COS Cells; D018076:DNA, Complementary / Q000032:analysis; D047628:Estrogen Receptor alpha; D005260:Female; D015723:Gene Library; D007059:Ictaluridae; D017403:In Situ Hybridization; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D010053:Ovary / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis*; D011960:Receptors, Estrogen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017421:Sequence Analysis; D014018:Tissue Distribution; D014162:Transfection
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D018076:DNA, Complementary; D047628:Estrogen Receptor alpha; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D011994:Recombinant Proteins