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The revised 8307 base pair coding sequence of human thyroglobulin transiently expressed in eukaryotic cells
S A van de Graaf1, E Pauws, J J de Vijlder
1Academic Medical Centre, University of Amsterdam, The Netherlands.
European Journal of Endocrinology
|May 1, 1997
Summary
Researchers created a human thyroglobulin (TG) expression system in thyroid cells, identifying 21 sequence variations and revealing that only the N-terminal TG fragment reaches the endoplasmic reticulum.
Area of Science:
- Molecular Biology
- Cell Biology
- Endocrinology
Background:
- Human thyroglobulin (TG) is crucial for thyroid hormone synthesis.
- Accurate TG gene sequence is essential for understanding its function.
- A functional expression system is needed to study TG structure-function relationships.
Purpose of the Study:
- To develop a transient transfection system for human TG cDNA in human thyroid and COS-1 cells.
- To identify and validate variations in the human TG nucleotide sequence.
- To investigate the cellular localization and processing of expressed TG fragments.
Main Methods:
- Amplification of TG cDNA fragments using reverse transcription-PCR.
- Nucleotide sequencing and comparison with published human TG sequences.
- Transient transfection of TG constructs into HTori 3 and COS-1 cells.
- Protein expression analysis via Western blotting and immunofluorescence.
- Lectins analysis for mannose structures and cellular localization studies.
Main Results:
- Identified 30 nucleotide variations in TG cDNA, with 21 confirmed in normal human alleles, necessitating revision of the published sequence.
- Determined the mature human TG coding sequence to be 8307 nucleotides, encoding 2750 amino acids.
- Successfully expressed all four TG protein fragments in both cell lines.
- Demonstrated that only the N-terminal TG fragment, including the signal peptide, is targeted to the endoplasmic reticulum but does not reach the Golgi complex.
Conclusions:
- A novel transient transfection system for human TG was established in human thyrocytes and COS-1 cells.
- The study identified significant revisions needed for the human TG nucleotide sequence.
- The N-terminal TG fragment exhibits specific endoplasmic reticulum localization, providing insights into TG processing and potential structure-function relationships in thyroid hormonogenesis.