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Cloning of a cDNA Coding for Active Tyrosine Hydroxylase in the Rainbow Trout ( Oncorhynchus mykiss ): Comparison with Other Hydroxylases and Enzymatic Expression
Author(s) -
Linard B.,
Pakdel F.,
Marmig M. H.,
Saligaut C.
Publication year - 1998
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1998.71030920.x
Subject(s) - complementary dna , biology , northern blot , rainbow trout , microbiology and biotechnology , cdna library , tyrosine hydroxylase , tyrosine , trout , messenger rna , amino acid , cloning (programming) , coding region , biochemistry , enzyme , gene , fishery , fish <actinopterygii> , computer science , programming language
Although catecholamines are of critical importance for neuroendocrine function in teleost fishes, there has been no tool to give access to pretranslational 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 cDNA coding for the rainbow trout TH (rtTH) was then cloned and sequenced. The rtTH sequence encodes a protein of 489 amino acids. Several domains and amino acids required for enzyme activity, like cysteines or phosphorylation sites, are highly conserved between species. Northern blot 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 untransfected cells. Western blot analysis revealed a single protein of ∼65 kDa in both COS‐1 cells and in trout brain. This rtTH cDNA provides us with a tool for further studies on pretranslational regulation of the enzyme in salmonids.