The aim of this proposal is to elucidate the molecular mechanisms which underly the differential expression of genes observed among the various cell types of multicellular organisms. Insulin and somatostatin, two key products of the endocrine pancreas, will be investigated in detail using methods of molecular genetics. Although the mechanisms which generate specificity are unknown, many studies have pinpointed transcription of DNA to messenger RNA as a key point of control. It is widely believed that cell specific gene expression results from the binding of trans-acting regulatory proteins to cis-acting DNA sequences located close to the gene. The consequence of binding is increased transcription of the gene. Presumably, the concentration of regulatory protein is substantially higher in cells which express the gene than in non- expression cells. Identification of the cis-acting elements of the somatostatin gene will be accomplished by introducing molecularly cloned DNA molecules into tissue culture cells of appropriate differentiated phenotype and comparing the level of expression with that observed following introduction to cells which do not produce somatostatin. Deletion analysis will be used to define the sequences responsible for cell specificity. Characterization of the trans-acting factor(s) responsible for selective expression of the insulin gene, will be undertaken using a genetic approach based on the presumptive ability of the trans-activator to augment expression of the transcriptional control region of the insulin gene. The approach will involve construction of a fibroblast cell line whose survival is conditionally dependent on the activity of the insulin transcriptional control region. Transfection of such cell with genomic DNA sequences under non-permissive conditions will select for cells expressing the trans-activator, whose gene can then be cloned from such cells by conventional methods. The identification of the gene coding for the insulin gene trans- activator, and the characterization of its activities would represent a substantial advance in our understanding of development in higher eukaryotes as well as possibly providing novel perspectives on the cause and treatment of diabetes, a heterogeneous disorder frequently associated with insufficient production of insulin.
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