Molecular cloning of the cDNAs for pituitary glycoprotein hormone alpha subunits of two species of duck and their gene regulation

The cDNAs encoding pituitary glycoprotein hormone a subunits (PGHas) of two species of duck (Muscovy duck, Cairina moschata and Pekin duck, Anas platy rhynchos domesticus) were cloned and sequenced to better understand the phy logenic diversity and evolution of PGH alpha molecules in vertebrates. Olig onucleotide primers were designed and used for reverse transcription PCR (R T-PCR) amplification of PGH alpha cDNA fragments from total cellular RNA of pituitary glands. The remaining sequences were completed by rapid amplific ation of the cDNA ends. The nucleotide sequence of isolated PGH alpha cDNA of both ducks are identical, including 81 bp of 5' untranslated region (UTR ), 360 bp of coding region, and 272 bp of 3'-UTR followed by a 13 bp poly(A )(+) tract. The total number of amino acids deduced from the cDNA of the du ck PGHa is 120 with a signal peptide of 24 amino acids and a mature protein of 96 amino acids. PGH alphas of the ducks (order Anseriformes) share 96% homology of amino acid sequence in signal peptide, and 100% homology in mat ure proteins with chicken, quail and turkey (order Galliformes). Our data t hus demonstrate identical inter-order homology of PGH alpha mature protein in birds. Ten cysteine residues, presumably forming five disulfide bonds wi thin the a subunit, and four proline residues, presumably responsible for f olding of the molecule, are conserved in the alpha subunit of ducks. Northe rn blot analysis revealed that PGHa mRNA is expressed only in the pituitary . In order to study factors regulating the gene expression of PGH alpha mRN A, duck pituitary fragments were incubated with GnRH, TRH, testosterone, or triiodothyronine (T-3). GnRH and TRH increased, while testosterone and T-3 decreased, PGH alpha mRNA levels. This is the first report in birds of TRH up-regulation and down-regulation by testosterone and T-3 under in vitro c onditions. The present study demonstrates both ducks have the same cDNA nuc leotide and deduced amino acid sequences in the PGH alpha subunit, exhibiti ng identical inter-genus homology within the family of Anatidae. The findin gs from mRNA expression work suggest that hypothalamic GnRH and TRH up-regu late, while testosterone and T-3 down-regulate, PGH alpha gene expression i n ducks.