title: The revised 8307 base pair coding sequence of human thyroglobulin transiently expressed in eukaryotic cells.
abstract: We developed a transient transfection system for human thyroglobulin (TG) cDNA in both human thyroid cells and in COS-1 cells. Four overlapping TG cDNA fragments were amplified by reverse transcription-PCR from RNA of normal thyroid tissue. The most 5' fragment includes the natural translation initiation site and the sequence encoding the signal peptide (SP). After subcloning, the nucleotide sequence was determined and compared with the published human sequence, resulting in the detection of 30 nucleotide variations. For validation purposes, all variations were screened in 6-12 normal human alleles. Twenty-one were present in all screened alleles and have to be revised in the published nucleotide 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 transfected in HTori 3 and COS-1 cells and protein expression was detected 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 mannose structures detected on the proteins by lectins and localization after expression in the cells suggest that only the N-terminal TG fragment (containing the SP) is directed to the endoplasmatic reticulum but is unable to reach the Golgi complex. The described expression system in human thyrocytes will be a helpful tool in studying the structure-function relationship of human TG in thyroid hormonogenesis.
authors.affiliation: Academic Medical Centre, University of Amsterdam, The Netherlands.
authors.forename: S A
authors.initials: SA
authors.lastname: van de Graaf
authors.forename: E
authors.initials: E
authors.lastname: Pauws
authors.forename: J J
authors.initials: JJ
authors.lastname: de Vijlder
authors.forename: C R
authors.initials: CR
authors.lastname: Ris-Stalpers
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D004251:DNA Transposable Elements; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005057:Eukaryotic Cells / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012016:Reference Values; D013954:Thyroglobulin / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D004251:DNA Transposable Elements; D018076:DNA, Complementary; D010446:Peptide Fragments; D013954:Thyroglobulin