OBJECTIVE: In order to complete the knowledge of the genomic organization of 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 more complete physical map of the gene. DESIGN: Screening of two genomic libraries, and subsequent restriction mapping, hybridization and sequencing were used to characterize the recombinant phages. METHODS: Two human genomic DNA libraries were screened by in situ hybridization. Southern blotting experiments were performed to characterize the phage inserts. The Long PCR method was used to amplify the genomic DNA region containing 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 include nucleotides 4933 to 6262 of the thyroglobulin mRNA, encompassing exons 25-35 of the gene. The remaining exon 24 (nucleotides 4817-4932) was sequenced 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 of 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 gene was analyzed, a physical map was constructed, and all exon-intron junctions were sequenced and correlated with the different domains of the protein. In summary, the thyroglobulin gene contains 48 exons ranging in size from 63 nucleotides to 1101 nucleotides.
Site-specific gene cassette insertion by combining artificial restriction DNA cutter and single-stranded DNA specific endonuclease