MOLECULAR BASIS OF SECRETION IN EXOCRINE PANCREAS

外分泌胰腺分泌的分子基础

• 批准号: 3226077
• 负责人: GEORGE A SCHEELE
• 金额: $ 29.27万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-05-01 至 1992-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3226077
• 关键词: SDS polyacrylamide gel electrophoresis; autoradiography; bacteriophage lambda; calcium; calmodulin; cyclic nucleoside monophosphate; diet; dogs; electrofocusing; enzyme inhibitors; enzyme mechanism; fluorimetry; gastrointestinal absorption /transport; gel electrophoresis; gene expression; genetic library; genetic manipulation; genetic recombination; genetic translation; hormone regulation /control mechanism; immunochemistry; laboratory rabbit; laboratory rat; membrane permeability; messenger RNA; microsomes; nucleic acid probes; nucleic acid sequence; nutrition related tag; pancreas enzyme; pancreatic juice; peptidases; polyamines; protein biosynthesis; protein engineering; protein sequence; protein transport; proteins; radiotracer; secretion; simian virus 40; tissue /cell culture; zymogens; SDS polyacrylamide gel electrophoresis; autoradiography; bacteriophage lambda; calcium; calmodulin; cyclic nucleoside monophosphate; diet; dogs; electrofocusing; enzyme inhibitors; enzyme mechanism; fluorimetry; gastrointestinal absorption /transport; gel electrophoresis; gene expression; genetic library; genetic manipulation; genetic recombination; genetic translation; hormone regulation /control mechanism; immunochemistry; laboratory rabbit; laboratory rat; membrane permeability; messenger RNA; microsomes; nucleic acid probes; nucleic acid sequence; nutrition related tag; pancreas enzyme; pancreatic juice; peptidases; polyamines; protein biosynthesis; protein engineering; protein sequence; protein transport; proteins; radiotracer; secretion; simian virus 40; tissue /cell culture; zymogens

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 prime 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 prime and 3 prime 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.

进一步了解蛋白质合成的机制 外分泌胰腺受基因表达，表征的调节 在本实验室开发的犬胰腺分泌系统将是 扩展到包括针对单个外分泌的mRNA和基因 蛋白质。 全长cDNA的特征是（a）混合选择 使用我们的高保真体体外蛋白质合成和代码分析 加工系统与二维IEF/SDS凝胶电泳相结合 （b）核苷酸序列分析。 演绎方法将使我们能够 首次建立犬胰腺的氨基酸结构 外分泌蛋白并完成我们对运输肽的分析 与这些蛋白质相关的序列。 犬基因组库将是 建立，我们将开始隔离和表征单个基因 通过R环分析，限制酶图和核苷酸序列 分析以确定内含子/外显子组织和监管 与5个主要侧翼区域相关的序列。 分析将是 为从单基因衍生而来的多个mRNA转录本来搜索 替代RNA处理机制。 我们将关联 （a）的5个质子和3个侧翼序列的组织结构 基础蛋白质合成速率和（b）存在核苷酸信号 可能以坐标方式调节个体的合成 响应激素和营养底物的外分泌蛋白。 这 细胞内因子的影响（钙，钙调蛋白，营地，CGMP和 多胺）关于基因转录，mRNA翻译和 将研究mRNA降解。 诱变研究，然后 结构功能分析将进行详细检查 胰胆蛋白酶原易位所需的转运肽结构 2跨膜及其随后的胰腺分泌物 腺泡细胞。 这些研究可以预期阐明分子 基因组组织有助于控制的机制 胰腺外分泌功能。