SPATIAL REGULATION OF DEVELOPMENTAL GENE EXPRESSION

发育基因表达的空间调控

• 批准号: 3328755
• 负责人: RONALD M EVANS
• 金额: $ 20.09万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-12 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3328755
• 关键词: DNA binding protein; Escherichia coli; X ray crystallography; beta galactosidase; bioassay; biological models; estrogens; gel filtration chromatography; gene deletion mutation; gene expression; genetic enhancer element; genetic mapping; genetic transcription; genetically modified plants; growth /development; immunocytochemistry; in situ hybridization; laboratory mouse; ligands; mammalian embryology; messenger RNA; molecular dynamics; mutagens; nuclear magnetic resonance spectroscopy; nucleic acid sequence; osteocalcin; point mutation; retinoate; retinoid binding proteins; steroid hormone receptor; transcription factor

The goal of this proposal is to establish a molecular basis for pattern formation during early vertebrate development. The discovery of the retinoic acid receptor and its homology to the steroid/thyroid receptor superfamily, offers for the first time in a vertebrate system, the hope of analyzing the mechanisms by which positional information are established and thus, how the fate and behavior of embryonic cells are controlled. Utilizing the cloned retinoic acid receptor gene products as models, we will define, in molecular terms, how retinoids influence development and physiology by controlling patterns of gene expression. We will characterize the molecular interactions of the retinoic acid receptor with DNA by identifying putative response elements (RAREs) present in the retinoic acid receptor and osteocalcin genes. In addition to biochemical dissection of the DNA-protein complex, the DNA-binding domain of the RAR and the ligand-binding domain will be overproduced in E. coli to begin NMR and X-ray crystallographic studies. Activation domains of the retinoid receptors will be identified by deletion, truncation, and point mutation analysis. Proteins interacting with the RARs will be sought by chemical approaches to identify the encoding genes. These approaches include chromatography of nuclear extracts to RAR affinity columns and the detection of receptor-DNA complexes with altered electrophoretic mobilities. In situ hybridization and immunohistochemical studies will be used to localize subtype-specific RAR mRNA and protein during mouse embryogenesis. Functional roles of the RARs during early embryonic development will be assessed by creating transgenic animals expressing a variety of mutant RAR derivatives. RAR responsive promoters linked to beta-galactosidase will be used to visualize endogenous RA gradients. In summary, retinoic acid, because of its described role in limb development and its capacity to induce differentiation in embryo carcinoma cells, is an ideal candidate for a vertebrate morphogen. In combination with the recently cloned retinoic acid receptor gene products in several species, we are now poised to examine the mechanism by which RA and the RARs specify pattern formation in vertebrates.

该提议的目的是建立模式的分子基础 早期脊椎动物发育过程中的形成。发现 视黄酸受体及其与类固醇/甲状腺受体的同源性 超家族，在脊椎动物系统中首次提供报价，希望 分析建立位置信息的机制 因此，如何控制胚胎细胞的命运和行为。 利用克隆的视黄酸受体基因产物作为模型，我们 从分子角度来定义类视黄素如何影响发展和 通过控制基因表达模式的生理学。 我们将表征视黄酸的分子相互作用 通过识别存在的假定响应元件（RAR）的受体，存在 在视黄酸受体和骨钙素基因中。此外 DNA-蛋白质复合物，DNA结合结构域的生化解剖 RAR和配体结合域的大肠杆菌将过量生产 开始NMR和X射线晶体学研究。 类视视感受体的激活结构域将通过 缺失，截断和点突变分析。蛋白质相互作用 通过化学方法，将寻求稀有 编码基因。这些方法包括核提取物的色谱法 rar亲和柱和与受体-DNA复合物的检测 电泳迁移改变。 原位杂交和免疫组织化学研究将用于 小鼠胚胎发生过程中定位亚型特异性RAR mRNA和蛋白质。 早期胚胎发育过程中ras的功能作用将是 通过创建表达各种突变体RAR的转基因动物来评估 衍生物。与β-半乳糖苷酶相关的RAR响应式启动子将是 用于可视化内源性RA梯度。 总而言之，视黄酸在肢体中所描述的作用 发育及其诱导胚胎癌分化的能力 细胞是脊椎动物形态学的理想候选者。结合 与最近克隆的视黄酸受体基因产物在几个 物种，我们现在准备检查RA和RA和 RARS在脊椎动物中指定模式形成。