Cloning and characterization of a mRNA-encoding rat preprosomatostatin

Abstract

An undecanucleotide extended hybridization probe has been used to screen a rat medullary thyroid carcinoma cDNA library for clones which contain preprosomatostatin sequences. The nucleotide sequence encoding rat preprosomatostatin is reported. The sequence of cDNA contains 67 nucleotides in the 3'-noncoding region, 84 nucleotides in the 5'-untranslated region, and 458 bases corresponding to the coding region. The mRNA codes for a somatostatin precursor 116 amino acids in length (Mr = 12,773). The preprosomatostatin has a sequence of hydrophobic amino acids at the NH2 terminus, followed by a peptide of approximately 78 residues, which precedes somatostatin-14. The amino acid sequences of rat and human preprosomatostatin (Shen, L. P., Pictet, R. L., and Rutter, W. J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4575-4579) differ by only 4 amino acid residues. Translation of rat poly(A) RNA in a rabbit reticulocyte cell-free system followed by immunoprecipitation with antisera directed against somatostatin-14 demonstrated the synthesis of a single protein having a molecular weight of 15,000. Two proteins having molecular weights of 14,000 and 15,000 are immunoprecipitated from a wheat germ cell-free translation mixture. Northern analysis of the somatostatin mRNA indicated that it is approximately 850 nucleotides in length. Analysis of several medullary thyroid carcinomas demonstrated that one tumor, designated WF, had immunoreactive somatostatin-14 in concentrations of 350 ng of somatostatin-14/mg of protein and somatostatin mRNA that represented 10% of the cellular poly(A) RNA. Cell lines derived from this tumor may provide an attractive system to investigate the regulation of somatostatin gene expression.