The complete sequence of human preprocalcitonin

Abstract

DNA complementary to mRNA extracted from the thyroid glands of patients suffering from medullary carcinoma of the thyroid (MCT), a calcitonin-producing tumour, was inserted in the Pst site of pBR 322 by G-C tailing. The recombinant plasmids were used to transform Escherichia coli DP 50. Ampicillin-resistant clones were screened using a ³²P-labelled cDNA to mRNA extracted from a case of MCT particularly rich in calcitonin (CT) mRNA. Positive clones were subsequently rescreened using a ³²P poly(T) probe. Eighty clones were thus purified, and the inserts obtained by digestion with PstI were subjected to positive hybridization selection with subsequent translation in vitro. An insert stimulating synthesis of the protein and containing restriction sites compatible with the previously published complete sequence of calcitonin mRNA from rat was sequenced. This cDNA insert contained the entire coding region of 426 bp, 70 bp at the 5′-end, and 295 bp upstream from the poly(A) tail. The complete amino acid sequence of human preprocalcitonin could thus be deduced.