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 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 approximately 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.
Linard
B
B
Endocrinologie Moléculaire de la Reproduction, UPRES-A CNRS 6026, Rennes, France.
Pakdel
F
F
Marmignon
M H
MH
Saligaut
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D019556:COS Cells; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005260:Female; D007668:Kidney / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; D014446:Tyrosine 3-Monooxygenase