Molecular cloning of Pst I fragments from rat double stranded thyroglobulin complementary DNA

Thyroglobulin mRNA was prepared from thyroid glands of rats chronically treated with propylthiouracil. The double stranded complementary DNA was synthesized using Avian Myeloblastosis Virus reverse transcriptase and subjected to restriction with the endonuclease Pst I. The restriction fragments were ligated into the unique Pst I site of the plasmid pBR 322 and the resulting recombinant DNA was used to transform E.coli to tetracyclin resistance. The colonies harboring recombinant plasmids (42 out of 1852) could be classified into three categories containing 320, 550 and 640 base pair inserts. One clone from each size class was selected and the ability of their plasmid DNA to bind functionaly active rat thyroglobulin mRNA was demonstrated in a positive translation assay. Altogether the three cloned DNA fragments represent about 20% of thyroglobulin structural gene sequence.