Molecular cloning and sequence analysis of multiple cDNA variants for thyroid-stimulating hormone β subunit (TSHβ) in the fathead minnow ( Pimephales promelas)

Abstract

We cloned and sequenced full-length cDNAs encoding the β subunit of thyroid-stimulating hormone (TSHβ) from the pituitary of fathead minnow ( Pimephales promelas) using 5′- and 3′-rapid amplification of cDNA ends (RACE). Three cDNA variants for TSHβ with lengths of 1184-, 1093-, and 818-bp were identified. The cDNA variant of 1184-bp included 453-bp of open-reading frame and 610-bp of 3′ UTR followed by a poly(A)site. This cDNA encodes 150 amino acids including a 19 residue signal peptide and a mature TSHβ protein of 131 residues with sequence identities of 97–53% to other fishes and 42–39% to mammals. The 1093-bp cDNA variant was identical to the 1184-bp variant in the open-reading frame, but contained a deletion of 40-bp in the 3′ UTR. The 818-bp cDNA variant, however, contained 498-bp of open-reading frame followed by 227-bp of 3′ UTR and a poly(A)site. The deduced amino acid sequence for this cDNA variant showed 99.2% homology with the 1184- and 1093-bp variants of TSHβ, but a single deletion of 332-bp nucleotides spanning the predicted stop codon and 3′ UTR resulted in a deduced amino acid sequence with 15 additional residues on the C terminus. The presence of this 818-bp cDNA variant in the pituitary was further confirmed by PCR using primers developed to the 5′ and 3′ UTR. PCR and Southern blot analyses of genomic DNA suggested only one gene for TSHβ. Sequencing of this gene revealed a hairpin loop structure of approximately 300-bp located in the 3′ UTR and corresponding to the region of the 332-bp deletion in the 818-bp transcript.