Molecular Cloning and Sequence Analysis of the cDNA for Thyroid-Stimulating Hormone β Subunit of Muscovy Duck

Abstract

The cDNA-encoding thyroid-stimulating hormone β subunit (TSHβ) of the Muscovy duck (Cairina moschata) was cloned and sequenced to investigate the phylogenic diversity and evolution of TSH molecules. Oligonucleotide primers were designed and used for reverse transcription and polymerase chain reaction amplification of a TSHβ cDNA fragment from the total cellular RNA of pituitary glands. The remaining sequence was completed by rapid amplification of cDNA ends. The nucleotide sequence of Muscovy duck TSHβ cDNA includes 66 bp of 5′-untranslated region (UTR), 402 bp of coding region, and 82 bp of 3′-UTR, followed by an 18-bp poly(A)+ tract. The total number of amino acids deduced from the cDNA of duck TSHβ is 134, with a signal peptide of 19 amino acids and a mature protein of 115 amino acids. The homologies of the amino acid sequence of Muscovy duck TSHβ compared with those of chicken, quail, mammals, amphibian (frog), and teleosts are 97, 98, 68–69, 56, and 42–47%, respectively. To test for tissue specificity of the duck TSHβ cDNA, total cellular RNA samples from brain, liver, pituitary gland, testis, and thyroid gland were analyzed by Northern blotting. A band, approximately 700 bases, was found in the pituitary gland alone. The pituitary tissue fragments were treated for 24 h in serum-free medium containing thyrotropin-releasing hormone (TRH), triiodothyronine (T3), or thyroxine (T4). TRH increased and T3 and T4 decreased TSHβ mRNA levels. This study demonstrated that the amino acid sequence of TSHβ subunits is highly conserved among birds, exhibiting a high degree of inter-order homology, and that hypothalamic TRH upregulates the TSHβ mRNA expression in duck.