The somatostatins constitute a family of structurally related small peptides synthesized as large precursors within multiple organs, including the islets, intestine and central and peripheral nervous systems. They serve both hormonal and paracrine functions in the regulation of secretions and probably as neurotransmitters and/or neuromodulators; their actions are essential for metabolic homeostasis. The production of the somatostatins is, in turn, regulated by hormonal and paracrine influences. Importantly, transcription of the rat somatostatin gene is powerfully stimulated by cAMP, A cAMP response element of the gene, TGACGTCA resides in the sequence at -48 to -41. We plan to investigate at a molecular level the cellular mechanisms involved in the expression of the somatostatin gene. The hypothesis to be tested is that the cell- specific and cAMP-regulated expression of the somatostatin gene is mediated by the complex coordinate interactions of several transcriptional factors, some of which bind to the cAMP response element and others bind to more upstream and downstream elements.
Aims of this proposal are 1) to characterize the cis-acting nucleotide sequences in the 5'-flanking region of the rat somatostatin gene that specify islet cell-specific gene expression using fusion genes of the regulatory region of the somatostatin gene and the bacterial reporter gene chlormaphenicol acetyl transferase (CAT). 2) To identify the DNA binding proteins in islet cells that initiate cell-specific transcription of the somatostatin gene using mutational analyses DNA footprinting, photoaffinity cross-linking, and gel mobility shift-assays. These DNA sequences will be used in affinity purification of the DNA binding proteins. Protein isolation will be followed by protein sequencing and gene cloning. Additional experiments will test the hypothesis that similar but distinctly different cis-trans factors mediate cAMP stimulated gene transcription in other non-islet cells using chimeric fusions between the hCG alpha and somatostatin genes expressed in placental cells. The results of these studies may provide information relevant to the pathogenesis of diabetes mellitus and other disorders of hormone deficiencies, as well as insights into the functions of the somatostatins in the central and peripheral nervous system.
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