BIOCHEMICAL CHARACTERIZATION OF DROSOPHILA COLLAGEN

果蝇胶原蛋白的生化特性

• 批准号: 3284112
• 负责人: LOUANN W MURRAY
• 金额: $ 6.84万
• 依托单位: LOS ANGELES COUNTY HARBOR-UCLA MED CTR
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1988-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3284112
• 关键词: basement membrane; diapause; electron microscopy; gel electrophoresis; gene expression; high performance liquid chromatography; ion exchange chromatography; larva; mature animal

The purpose of the proposed research is to biochemically characterize the collagens from larval and adult Drosophila melanogaster. Preliminary studies have shown that (1) treatment of larvae with -amino-propionitrile allows extraction of collagen in quantities sufficient for further characterization, (2) there are biochemical differences between the larval and adult collagens, suggesting a developmental shift in collagen gene expression, and (3) the Drosophila collagens are more similar to the vertebrate collagen types IV and VII than to the interstitial collagens. The specific goals are to (1) purify native Drosophila collagens, (2) determine the number of collagen types in Drosophila, (3) determine the subunit composition of each type, (4) characterize the denatured subunits, (5) compare larval and adult Drosophila collagen, and (6) compare Drosophila collagens to human collagen, particularly Types IV and VII, looking for structural and functional analogies. Type IV collagen molecules form a mesh-like network that is visable ultrastructurally as the lamina densa of basement membranes. Type VII collagen molecules can be reconstituted into a fiber form that closely resembles anchoring fibrils, which are morphologically identifiable structures that are associated with basement membranes (Murray it al., submitted). Comparison of Drosophila to human collagens will identify conserved structural features that are essential to proper function of collagen in basement membranes and connective tissues. Native collagen molecules will be separated and characterized by a combination of salt fractionation, ion exchange and reverse phase HPLC, electrophoresis and electron microscopy. Isolated molecules and molecular aggregates will be examined by rotary shadowing and negative staining. Denatured subunits will be separated and characterized by ion exchange and reverse phase HPLC, peptide mapping and amino acid analysis. Elucidation of the molecular diversity of the Drosophila collagen family would provide a foundation for future studies in a genetically and developmentally well characterized organism.

拟议研究的目的是从生化角度表征 来自幼虫和成年果蝇的胶原蛋白。 初步研究表明（1）对幼虫进行处理 -氨基丙腈可以提取足够量的胶原蛋白 为了进一步表征，（2）之间存在生化差异 幼虫和成虫胶原蛋白，表明发育发生转变 胶原蛋白基因表达，（3）果蝇胶原蛋白更相似 与间质胶原蛋白相比，对脊椎动物 IV 型和 VII 型胶原蛋白的影响更大 胶原蛋白。 具体目标是 (1) 纯化天然果蝇胶原蛋白，(2) 确定果蝇中胶原蛋白类型的数量，（3）确定 每种类型的亚基组成，(4) 表征变性亚基， (5) 比较幼虫和成虫果蝇胶原蛋白，以及 (6) 比较 果蝇胶原蛋白到人类胶原蛋白，特别是 IV 型和 VII 型， 寻找结构和功能的类比。 IV型胶原蛋白 分子形成网状网络，其超微结构可见 基底膜致密层。 VII型胶原蛋白分子可以 重组成与锚定原纤维非常相似的纤维形式， 它们是与相关的形态学上可识别的结构 基底膜（Murray等人提交）。 果蝇与 人类胶原蛋白将识别保守的结构特征 对于基底膜中胶原蛋白的正常功能至关重要， 结缔组织。 天然胶原蛋白分子将被分离并通过 盐分馏、离子交换和反相 HPLC 的组合， 电泳和电子显微镜。 孤立的分子和分子 将通过旋转阴影和负染色检查聚集体。 变性的亚基将被分离并通过离子交换和表征 反相 HPLC、肽图谱和氨基酸分析。 阐明果蝇胶原蛋白家族的分子多样性 将为未来的遗传学和遗传学研究奠定基础 发育良好的有机体。