GENETICS OF NOTOCHORD DEVELOPMENT

描述：(改编自调查人员摘要)这是第一次 竞争续订需要五年支持的RO1应用程序 通过鉴定SD患者中突变的基因研究脊索发育 入口点。在拨款首次发放后的三年内， 申请者使用胚胎嵌合体研究来确定SD和TC (另一个影响脊索发育的突变)在细胞中发挥作用--自主 方法，并开始了一系列实验，以分离SD的基础上 地图位置。当前的应用程序建议完成这些实验 并开始在基因和基因水平上表征SD基因产物 蛋白质表达。目前，SD定位于0.3厘米的间隔；一侧 的间隔与650 kb的BAC重叠群(其中600 kb 是非重组的)，另一侧由分子标记D2UCl3定义， 这意味着2919个重组体中有7个远离SD。在第一个具体目标中， 申请者将通过使用BAC和染色体行走来完成物理地图 YAC，并试图通过进行BAC转基因来缩小临界区 救援实验，并通过在新的 回交。(申请人在拨款审查之前提供了最新情况，即 另外两步染色体行走已经弥合了身体上的差距， 将临界区域缩小到&lt；750 kb)。在第二个具体目标中 在SD的分子鉴定中，对候选基因的搜索将通过 1-2个BAC克隆的序列采样，如果关键区域可以缩小到 &lt；150 kb；如果区域较大，则使用 来自E9.5-10.5全胚胎的组织。候选基因的初步验证 SD中的突变将基于表达和序列结构分析 比较突变组织和正常组织，然后是转基因方法 挽救或复制突变的表型。最后，在第三个具体目标中， SD基因产物的机制研究将通过分离出一株 全长cdna，产生抗体，并进行详细分析 RNA和蛋白质的表达。

DESCRIPTION: (Adapted from investigator's abstract) This is the first competitive renewal of an RO1 application that requests five years of support to study notochord development by identifying the gene mutated in Sd as an entry point. In the three years since the grant was first awarded, the applicant used embryonic chimera studies to determine whether or not Sd and tc (another mutation that affects notochord development) act in a cell-autonomous manner, and initiated a series of experiments to isolate Sd on the basis of its map position. The current application proposes to complete these experiments and to begin characterizing the Sd gene product at the level of gene and protein expression. At present, Sd is localized to a 0.3 cM interval; one side of the interval is closed physically with a 650 kb BAC contig (of which 600 kb is non-recombinant), the other side is defined by a molecular marker, D2Ucl3, that lies 7/2919 recombinants away from Sd. In the first specific aim the applicant will complete the physical map by chromosome walking using BACs and YACs, and try to narrow the critical region by carrying out BAC transgenic rescue experiments and by generating additional recombinant animals in a new backcross. (The applicant provided an update prior to the grant review that an additional two steps of chromosome walking had closed the physical gap, narrowing the critical region to <750 kb). In the second specific aim directed at molecular identification of Sd, a search for candidate genes will proceed by sequence sampling of 1-2 BAC clones if the critical region can be narrowed to <150 kb; if the region is larger, cDNA selection will be carried out using tissue from E9.5-10.5 whole embryos. Initial verification of a candidate gene as mutated in Sd will be based on expression and sequence structure analyses that compare mutant and normal tissues, followed by transgenic approaches to rescue or reproduce the mutant phenotype. Finally, in a third specific aim, mechanistic studies of the Sd gene product will be initiated by isolating a full-length cDNA, generating antibodies, and carrying out a detailed analysis of RNA and protein expression.