THE REVISED 8307-BASE-PAIR CODING SEQUENCE OF HUMAN THYROGLOBULIN TRANSIENTLY EXPRESSED IN EUKARYOTIC CELLS

We developed a transient transfection system for human thyroglobulin ( TG) cDNA in both human thyroid cells and in COS-1 cells. Four overlapp ing TG cDNA fragments were amplified by reverse transcription-PCR from RNA of normal thyroid tissue. The most 5' fragment includes the natur al translation initiation site and the sequence encoding the signal pe ptide (SP), After subcloning, the nucleotide sequence was determined a nd compared with the published human sequence, resulting in the detect ion of 30 nucleotide variations. For validation purposes, all variatio ns were screened in 6-12 normal human alleles, Twenty-one were present in all screened alleles and have to be revised in the published nucle otide sequence. Since one variation concerns a triplet insertion, the coding sequence of the mature human thyroglobulin is 8307 nucleotides encoding 2750 amino acids. The TG cDNA constructs were transiently tra nsfected in HTori 3 and COS-1 cells and protein expression was detecte d using a polyclonal anti-human-TG on fixed cells and after SDS-PAGE. In both cell-lines all four TG protein fragments were expressed, The m annose structures detected on the proteins by lectins and localization after expression in the cells suggest that only the N-terminal TG fra gment (containing the SP) is directed to the endoplasmatic reticulum b ut is unable to reach the Golgi complex. The described expression syst em in human thyrocytes will be a helpful tool in studying the structur e-function relationship of human TG in thyroid hormonogenesis.