Detection and partial sequence analysis of gastrin mRNA by using an oligodeoxynucleotide probe.

Abstract

A specific deoxyoligonucleotide probe was used to detect gastrin mRNA in poly(A)-enriched RNA preparatiaons from hog antrum. The nucleotide sequence of the oligonucleotide, d(C-T-C-C-T-C-C-A-T-C-C-A), was deduced from the unique amino acid sequence Trp-Met-Glu-Glu of gastrin. When used with hog antral RNA, the dodecanucleotide is an effective primer for the synthesis of gastrin-specific c[complementary]DNA as judged by nucleotide sequence analysis of cDNA isolated by polyacrylamide gel electrophoresis. An 81-nucleotide sequence corresponding to the region of the gastrin mRNA that codes for the known amino acid sequence of the G34 progastrin intermediate species was determined, and the presence of 2 consecutive basic residues preceding the G34 sequence in the prohormone was demonstrated. Hybridization of gastrin cDNA or synthetic dodecanuleotide to hog antral RNA separated by gel electrophoresis on agarose gels in the presence of methylmercuric hydroxide indicates that the mRNA coding for gastrin is 620 nucleotides long. The gastrin precursor peptide apparently contains 110-140 amino acids. This method should be of general application for detection and characterization of mRNA corresponding to proteins of known amino acid sequence.