CLONING OF A CDNA CODING FOR ACTIVE TYROSINE-HYDROXYLASE IN THE RAINBOW-TROUT (ONCORHYNCHUS-MYKISS) - COMPARISON WITH OTHER HYDROXYLASES AND ENZYMATIC EXPRESSION

Although catecholamines are of critical importance for neuroendocrine function in teleost fishes, there has been no tool to give access to p retranslational regulation of their synthesis enzymes. In this study, we undertook cloning of the cDNA coding for the tyrosine hydroxylase ( TH) of the rainbow trout (Oncorhynchus mykiss). First, we looked for a tissue sufficiently rich in TH to make an expression library, The cDN A coding for the rainbow trout TH (rtTH) was then cloned and sequenced . The rtTH sequence encodes a protein of 489 amino acids. Several doma ins and amino acids required for enzyme activity, like cysteines or ph osphorylation sites, are highly conserved between species. Northern bl ot analysis showed a single rtTH messenger RNA of 4;2 kb expressed in the anteroventral brain. The ability of rtTH to hydroxylate L-tyrosine was analyzed by transient expression of the rtTH cDNA in COS-1 cells. In vitro TH activity, using COS-1 cell extracts, demonstrated that TH activity in transfected cells was 40-fold higher than in untransfecte d cells. Western blot analysis revealed a single protein of similar to 65 kDa in both COS-I cells and in trout brain. This rtTH cDNA pro vid es us with a tool for further studies on pretranslational regulation o f the enzyme in salmonids.