CHARACTERIZATION OF THE CHOLECYSTOKININ AND GASTRIN GENES FROM THE BULLFROG, RANA-CATESBEIANA - EVOLUTIONARY CONSERVATION OF PRIMARY AND SECONDARY SITES OF GENE-EXPRESSION

The gastrin and cholecystokinin (CCK) genes, and the complementary DNA s they encode, have been isolated and sequenced from the bullfrog, Ran a catesbeiana. The CCK gene promoter region possess the same four well characterized transcriptional control elements as the human CCK gene, namely an E-box, AP-1 binding site, Spl site, and TATA box. In contra st, no obvious regulatory motifs are conserved in the gastrin gene. Al ignment of the bullfrog preprohormone sequences with other members of the CCK/gastrin peptide family showed that preproCCK has been conserve d to a greater degree during evolution than preprogastrin. In mammalia n species, gastrin gene expression is typically associated with the an trum, and CCK with the small intestine and brain. However numerous sec ondary sites of CCK/gastrin gene expression have also been found. RT-P CR showed a high degree of conservation of both primary and secondary sites Of CCK/gastrin production between mammals and the bullfrog, with gastrin messenger RNA being detected in the antrum, duodenum, colon, pancreas, brain, and testes, whereas CCK mRNA was observed in the brai n, lung, testes, and throughout the length of the small intestine. In situ hybridization using radiolabeled gene specific antisense oligonuc leotides uncovered CCK and gastrin messenger RNA in distinct areas of the bullfrog central nervous system and pituitary gland. Notably, the gastrin gene was expressed in the pituitary gland and hypothalamus of the bullfrog, as previously seen in mammals. This highly preserved tis sue expression pattern suggests that gastrin plays specific roles in t he hypothalamus and pituitary gland that are distinct from those of CC K. Our findings show that in spite of the structural resemblance, bull frog CCK and gastrin constitute independent neuroendocrine peptide sys tems.