Genomic organization of the human thyroglobulin gene: the complete intron-exon structure

Objective: In order to complete the knowledge of the genomic organization o f the human thyroglobulin gene, the present work was designed to establish the intron-exon organization from exon 24 to exon 35 and to construct a mor e complete physical map of the gene. Design: Screening of two genomic libraries, and subsequent restriction mapp ing, hybridization and sequencing were used to characterize the recombinant phages. Methods: Two human genomic DNA libraries were screened by in situ hybridiza tion. Southern blotting experiments were performed to characterize the phag e inserts. The Long PCR method was used to amplify the genomic DNA region c ontaining exon 24. Intron-exon junction sequences were determined by using the Taq polymerase-based chain termination method. Results: We isolated and characterized five lambda phage clones that includ e nucleotides 4933 to 6262 of the thyroglobulin mRNA, encompassing exons 25 -35 of the gene. The remaining exon 24 (nucleotides 4817-4932) was sequence d from the amplified fragment. In total, 8010 intronic bases were analyzed. Conclusions: The present study shows that the five phages isolated and the amplified fragment include 59.4 kb genomic DNA, covering 1446 nucleotides o f exonic sequence distributed over 12 exons, from exon 24 to exon 35. Using previous studies and our current data, 220 kb of the human thyroglobulin g ene was analyzed, a physical map was constructed, and all exon-intron junct ions were sequenced and correlated with the different domains of the protei n. In summary, the thyroglobulin gene contains 48 exons ranging in size fro m 63 nucleotides to 1101 nucleotides.