To further understand the mechanisms by which protein synthesis in the exocrine pancreas is regulated by gene expression, characterization of the canine pancreas secretory system, developed in this laboratory, will be extended to include the mRNAs and genes which code for individual exocrine proteins. Full length cDNAs will be characterized by (a) hybrid selection and code analysis using our high fidelity in vitro protein synthesis and processing system coupled with two dimensional IEF/SDS gel electrophoresis and (b) nucleotide sequence analysis. Deductive methods will allow us to establish for the first time the amino acid structures of canine pancreatic exocrine proteins and to complete our analysis of transport peptide sequences associated with these proteins. A canine genomic library will be established and we will begin to isolate and characterize individual genes by R-loop analysis, restriction enzyme mapping and nucleotide sequence analysis to determine the intron/exon organization and the regulatory sequences associated with 5 feet flanking regions. An analysis will be made for multiple mRNA transcripts derived from single genes to search for alternative RNA processing mechanisms. We will correlate the organizational structure of 5 feet and 3 feet flanking sequences with (a) basal protein synthesis rates and (b) the presence of nucleotide signals which might modulate, in a coordinate manner, the synthesis of individual exocrine proteins in response to hormones and nutritional substrates. The effects of intracellular factors (calcium, calmodulin, cAMP, cGMP, and polyamines) on individual rates of gene transcription, mRNA translation and mRNA degradation will be studied. Mutagenesis studies followed by structure-function analyses will be conducted to examine in detail the transport peptide structure required for translocation of chymotrypsinogen 2 across the RER membrane and its subsequent secretion from the pancreatic acinar cell. These studies can be expected to elucidate molecular mechanisms by which genomic organization contributes to the control of pancreatic exocrine function.
