A GENE FAMILY EXPRESSED IN SEA URCHIN EMBRYONIC ECTODERM

海胆胚胎外胚层表达的一个基因家族

• 批准号: 2198608
• 负责人: WILLIAM H. KLEIN
• 金额: $ 21.95万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-02-01 至 1997-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2198608
• 关键词: DNA binding protein; affinity chromatography; alternatives to animals in research; antisense nucleic acid; calcium binding protein; cell cell interaction; cell type; developmental genetics; early embryonic stage; ectoderm; family genetics; gene expression; genetic enhancer element; genetic manipulation; genetic promoter element; genetic regulatory element; histochemistry /cytochemistry; in situ hybridization; invertebrate embryology; laboratory rabbit; mixed tissue /cell culture; molecular cloning; nucleic acid repetitive sequence; protein biosynthesis; protein transport; sea urchins; site directed mutagenesis; transcription factor

The goal of this project is to understand mechanisms underlying cell type specific gene activation during development using the sea urchin aboral ectoderm and the Spec/LpS1 genes as a model. Much is known about the cell lineages comprising the aboral ectoderm and the cell interactions required for differentiation. The Spec and the LpS1 genes serve as powerful markers for probing mechanisms involved in specifying aboral ectoderm. The specific aims are: (1) To characterize the Spec2a promoter. Virtually all positive control of the Spec2a gene lies within a 188-bp enhancer. This enhancer is within a 600-bp repetitive sequence element termed RSR. The RSR enhancer contains multiple DNA-elements including a key element, the A/T palindrome. Additional elements in the Spec2a promoter will be identified, along with the corresponding DNA-binding proteins. Non-Spec genes having nearby RSR elements will be cloned to ask if their presence is linked to aboral ectoderm expression. (2) To clone and characterize the A/T palindrome binding protein (A/TBP). The A/T palindrome contains a binding site for a putative bicoid-class protein. attempts will be made to purify this protein and in addition to obtain a cDNA clone using oligonucleotides with bicoid-class sequences. Once cloned, it will be determined if the A/TBP has properties consistent with a role in activation of Spec2a and aboral ectoderm specification. (3) To characterize the LpS1-beta promoter and the ectoderm and endoderm/mesoderm G-string factors. The proximal G-string is a positive Ell-regulatory element on the LpS1-beta promoter that binds to ectoderm specific and endoderm/mesoderm specific factors. Other DNA-elements will be identified on this promoter and the G-string factors will be cloned by protein purification and homology with the mammalian factor, IFI. (4) To determine the function of the Spec/LpS1 proteins. Spec/LpS1 proteins are hypothesized to function in transport of calcium across the ectoderm. This will be tested using antisense procedures. Reduced levels of calcium should result in skeletal defects in the embryo. (5) To test if suppressive cellular interactions repress Spec gene expression in non-aboral ectoderm cells. In the absence of cell interactions, blastomeres exhibit greater developmental potential than in the intact embryo. It will be determined if the Veg2 tier is capable of activating Spec genes in isolation and if this activation is suppressed upon co-culture with animal blastomeres. These experiments should lead to an understanding of Spec/LpS1 gene activation and its relationship to aboral ectoderm specification.

该项目的目的是了解细胞类型的基础机制 开发过程中使用海胆的特定基因激活 外胚层和Spec/LPS1基因作为模型。 关于 细胞谱系包含原住民和细胞相互作用 分化所需的。 规格和LPS1基因作为 用于探测指定原则的机制的强大标记 外胚层。 具体目的是：（1）表征Spec2a 发起人。 几乎所有对Spec2a基因的积极控制都在 188 bp增强剂。 该增强器在600 bp的重复序列范围内 元素称为RSR。 RSR增强子包含多个DNA元素 包括关键元素，即a/t palindrome。 在 将确定Spec2a启动子，以及相应的 DNA结合蛋白。 附近有RSR元素的非规格基因将是 克隆以询问它们的存在是否与原住民表达相关。 （2）克隆并表征A/T造成的结合蛋白（A/TBP）。 a/t palindrome包含一个假定的双子级的结合位点 蛋白质。将尝试净化这种蛋白质，除了 使用具有双核苷酸的寡核苷酸获得cDNA克隆。 克隆后，将确定A/TBP是否具有一致的属性 在激活Spec2a和Aboral外膜规范中的作用。 （3）表征LPS1-beta启动子和外胚层 内胚层/中胚层G弦因子。 近端G弦是阳性 LPS1-β启动子上与外胚层结合的ELL调节元件 特定和内胚层/中胚层特定因素。 其他DNA元素将 在该启动子上识别，G弦因子将被克隆 蛋白质纯化和与哺乳动物因子IFI的同源性。 （4）至 确定规格/LPS1蛋白的功能。 规格/LPS1蛋白是 假设在钙跨外胚层的运输中起作用。 这将使用反义程序进行测试。 降低的水平 钙应导致胚胎中的骨骼缺陷。 （5）测试是否 抑制性细胞相互作用抑制特定基因表达 非碱性外胚层细胞。 在没有细胞相互作用的情况下 与完整相比，胚泡具有更大的发育潜力 胚胎。 将确定VEG2层是否能够激活 孤立的规格基因，如果这种激活在 与动物囊泡的共同培养。 这些实验应导致 了解规格/LPS1基因激活及其与本土的关系 外胚层规范。