Cloning and sequencing of feline thyrotropin (fTSH): heterodimeric and yoked constructs.
The genes encoding the mature common glycoprotein alpha (CGA) and hormone-specific beta subunits of feline thyroid stimulating hormone (fTSH) were cloned and sequenced. The feline CGA gene was cloned from RNA extracted from the feline pituitary gland by the reverse transcription polymerase chain reaction (RT-PCR). The gene fragment that encodes mature TSHbeta was cloned from feline genomic DNA after direct polymerase chain reaction (PCR). In both cases, primers were based on the consensus sequences from TSH in other species. The resulting 510 bp PCR product for the CGA-subunit included the full coding sequence for the 96 amino acid mature subunit preceded by a 24 amino acid signal peptide. The 850 bp sequence of fTSHbeta genomic DNA consisted of two coding exons, an intron of 418 bp, and a 60 bp signal sequence. The octapeptide immunoaffinity tag FLAG was added to 3' end of the alpha gene to facilitate detection and purification. Both genes were cloned independently downstream from the EF1alpha promoter of the PEAK transfer vector to facilitate co-expression studies in PEAK cells (modified human embryonic kidney (HEK) cells). A single-chain analogue of fTSH termed yoked fTSH (yfTSH) was developed by fusing the nucleotides encoding the C-terminus of the beta-subunit fused to the N-terminus of the alpha-subunit with DNA encoding the C-terminal peptide (CTP) of human chorionic gonadotropin beta-subunit as a linker peptide. The resulting single-chain analogue encoded from N-terminus to C-terminus: beta-CTP-alpha-FLAG. The resulting DNA sequence was cloned, sequenced, ligated and recloned into expression vector PEAK. This report constitutes the first cloning and sequencing of the genes encoding the subunits of feline thyrotropin.
Rayalam
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Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA.
Eizenstat
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Hoenig
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Ferguson
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002415:Cats / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D013972:Thyrotropin / Q000235:genetics*
D015292:Glycoprotein Hormones, alpha Subunit; D012313:RNA; D013972:Thyrotropin