A cDNA for a putative type III deiodinase in the trout (Oncorhynchus mykiss): influence of holding conditions and thyroid hormone treatment on its hepatic expression

Abstract

A putative rainbow trout type III deiodinase (D3) cDNA was amplified by PCR, using primers to evolutionarily conserved sequences. The RACE-derived complete cDNA was then identified by sequence comparison to that in tilapia and other vertebrates. The cDNA coded for a predicted 31,500 kDa protein of 278 amino acids, with a hydrophobic trans-membrane segment and with 80% similarity to tilapia D3 and 39% similarity to rainbow trout type II deiodinase (D2). It also showed a selenocysteine codon at position 141 and a putative SECIS element in the 3' untranslated end. In the liver, a second form of D3 was found that differed only at this 3' untranslated region; the coding region was identical in both forms. The D3 mRNA, measured by RT-PCR using primers located within the common, translated portion of the cDNA, was expressed in the brain and, depending on thyroidal status, in liver and kidney. Holding trout for 7 days in static water as opposed to flowing water caused increased plasma T4 levels, decreased hepatic D2 mRNA levels and T4 outer-ring deiodination (ORD) activity and increased D3 mRNA levels and T3 inner-ring (IRD) activity. Trout held in flowing water and fed T3 for 7 days showed increased plasma T3 levels and hepatic D3 mRNA levels and T3 IRD activity but decreased D2 mRNA levels and T4ORD activity. Trout held in static water and exposed to ambient T4 for 7 days showed increased plasma T4 levels and hepatic T3IRD activity but with no significant change in D2 or D3 mRNA levels. We conclude that hepatic D3 mRNA levels and T3IRD activity are enhanced and D2 mRNA levels and T4ORD activity are suppressed by adverse holding conditions or T3 treatment suggesting that the putative D3 cDNA and D2 cDNA represent respectively the genes determining T3IRD and T4ORD activities. However, there were changes in the ratios of mRNA levels to enzyme activity, raising the potential for post-transcriptional regulation and showing that mRNA levels alone may be unreliable indices of deiodinase activity. Post-transcriptional regulation of D3 enzyme activity may be influenced by the observed alternative 3' and 5' untranslated regions of the D3 mRNA.