The objectives of this research are to characterize the developmental expression of gastrin and to characterize the mechanisms regulating gastrin gene expression during transcriptional elongation and termination. The first objective is to understand the mechanism by which the human gastrin gene is selectively expressed during development. These studies will be based on the novel discovery of transient expression of a gastrin promoter/T antigen construct in the hepatobiliary tract of transgenic mice. This preliminary finding will be extended to determine the developmental stage-specific, cell lineage-specific, and regional patterns of expression of the gastrin/SV40 T antigen fusion gene. The question of the role of gastrin development will be addressed by a gene knock-out strategy in which the gastrin gene will be disrupted by insertional mutagenesis involving homologous recombination. The chimeric mice will be examined for any abnormal phenotype by histochemical, molecular biological and tissue transplantation techniques. It is hoped that these studies will illuminate the fundamental questions of whether gastrin plays a central role in the development of the hepatobiliary tract, pancreatic islets, and gastric antral mucosa. This information could potentially be important in understanding certain diseases of the liver, pancreas and stomach. The second objective of this proposal is to study the mechanisms of gastrin gene regulation during transcriptional elongation and termination. These studies will be extended with the goal of dissecting the biochemical steps at the molecular level using an in vitro system that includes the gastrin gene terminator, the human elongation factor TFIIS, the putative transcription termination factor (TTF), and RNA polymerase II. The nature of the interaction of the TFIIS zinc finger with nucleic acids will be defined at the atomic level by three dimensional NMR. The mechanisms of RNA cleavage by TFIIS during pausing and termination will be explored in depth. Identification of a putative TTF gene will be attempted by a novel approach involving yeast genetics. The protein-protein interactions between TFIIS and RNA pol II will be examined by chemical (site-directed cross-linking), biochemical and physical methods (NMR analysis of the TFIIS polymerase binding domain). In addition to producing information on the transcriptional regulation of the gastrin gene, these studies will help elucidate the basic biological architecture of the eukaryotic transcription elongation complex.
