CLONING AND EXPRESSION OF HUMAN TSH

STUDIES OF THE BIOCHEMISTRY AND PHYSIOLOGY OF HUMAN THYROID-STIMULATING HORMONE (HTSH) HAVE BEEN HAMPERED BY THERECENT TERMINATION OF DISTRIBUTION OF PITUITARY-DERIVED REAGENTS DUE TO POTENTIAL CONTAMINATION WITH THE INFECTIOUS AGENTS OF CREUTZFELD-JAKOB DISEASE AND OTHERS. CONTINUED ACQUISITION OF KNOWLEDGE ABOUT THE COMPLEX INTERACTION BETWEEN THE ENDOCRINE MILIEU, TSH, AND THE THYROID GLAND REQUIRES A CONSTANT AND REPRODUCIBLE SUPPLY OF A HIGHLY PURIFIED HTSH PREPARATION. IT IS POSSIBLE TO EMPLOY RECOMBINANT DNA TECHNOLOGY TO PRODUCE THIS GLYCOPROTEIN. INTEGRATED GENETICS, INC. RECENTLY SYNTHESIZED THE CDNA ENCODING FOR THE HUMAN ALPHA SUBUNIT. A GENOMIC FRAGMENT THAT CONTAINS THE ENTIRE CODING SEQUENCE FOR THE HTSH BETA SUBUNIT IS AVAILABLE FOR USE. WITH THESE CLONES, IT WILL BE POSSIBLE TO GENETICALLY ENGINEER THEM INTO THE EUKARYOTIC EXPRESSION VECTOR, BOVINE PAPILLOMA VIRUS (BPV). THE MOUSE EPITHELIAL CELLS (C127) WILL BE TRANSFECTED WITH THE BPV VECTORS THAT CONTAIN THE GENES FOR THE ALPHA AND BETA SUBUNITS. CELLS THAT TRANSCRIBE THESE GENES AND SECRETE BIOLOGICALLY ACTIVE HTSH WILL BE IDENTIFIED BY RADIOIMMUNOASSAY AND BIOASSAY. THIS PROPOSAL DESCRIBES THE METHOD THAT WILL BE EMPLOYED TO ACHIEVE THESE GOALS.