FUNCTIONAL ANALYSIS OF THE TAN1 GENE

描述：（改编自研究者摘要） 格兰特是分析人类TAN-1基因的功能，现在被称为 NOTCH 1，其编码一种跨膜受体，涉及许多正常的 发展过程和病理状态。本实验室以前的工作 表明该基因表达为350 kD前体多肽， 裂解产生异二聚体，其中细胞外230 kD亚基 通过非共价力保持与120 kD跨膜亚基结合。 配体的结合似乎通过解离230 kD的 亚基，否则抑制跨膜的内在活性 亚单位计划进行实验以调查与以下有关的许多问题： 在激活过程中受体的变化，包括是否 230 kD亚基的释放涉及120 kD亚基的蛋白水解裂解， 亚基以去除230 kD亚基结合的位点。的部分 负责抑制120 kD亚基的230 kD亚基将被 通过在230 kD亚基内产生突变来研究。的命运 在配体诱导的从受体复合物解离后，230 kD亚基将 进行研究，以解决实验观察中明显的不一致性 关于释放后游离亚基的目的地。为了获得更多 准确评估NOTCH 1的解剖部位和发育时间 激活后，转基因小鼠将产生报告转基因， NOTCH 1敏感性控制下的海蜇绿色荧光蛋白 启动子为了解决与不同的监管有关的问题， NOTCH 1 -3基因，特别是关于反馈调节 转录，这三个基因的启动子将被分析并用于 研究与之结合的调节蛋白。更好地了解 NOTCH 1在胸腺细胞发育中的作用，将进行实验，其中 NOTCH 1信号在T细胞前体中被破坏或修饰，因为它们 在来源于恒河猴的胸腺培养系统中分化。最后， 将研究由NOTCH 1激活产生的基因表达的改变 通过总细胞RNA探针与cDNA微阵列的杂交。除了 提供了对由NOTCH 1直接和间接调控的基因的深入了解， 利用微阵列的研究可能揭示出基因的身份， 与截短的NOTCH 1在具有以下特征的细胞的恶性转化中的作用 染色体易位，分裂NOTCH 1基因。

DESCRIPTION: (Adapted from the investigator's abstract) The purpose of this grant is to analyze the function of the human TAN-1 gene, now referred to as NOTCH1, which encodes a transmembrane receptor implicated in a number of normal developmental processes and pathologic states. Previous work by this laboratory indicates that the gene is expressed as a 350 kD precursor polypeptide that is cleaved to produce a heterodimer in which an extracellular 230 kD subunit remains bound to a 120 kD transmembrane subunit by non-covalent forces. Engagement of ligand seems to activate NOTCH1 by dissociating the 230 kD subunit, which otherwise represses the intrinsic activity of the transmembrane subunit. Experiments are planned to investigate a number of issues related to changes in the receptor during the process of activation, including whether the release of the 230 kD subunit involves a proteolytic cleavage of the 120 kD subunit to remove the site at which the 230 kD subunit is bound. The portion the 230 kD subunit responsible for the repression of the 120kD subunit will be investigated by generating mutations within the 230 kD subunit. The fate of the 230 kD subunit after ligand-induced dissociation from the receptor complex will be studied to resolve apparent inconsistencies in experimental observations about the destination of free subunit after its release. To gain a more accurate assessment about the anatomic sites and developmental timing of NOTCH1 activation, transgenic mice will be generated with reporter transgenes encoding the jelly fish green fluorescent protein under control of a NOTCH1-sensitive promoter. To address questions related to the differential regulation of the NOTCH1-3 genes, particularly with regard to feedback regulation of transcription, the promoters for these three genes will be analyzed and used to investigate regulatory proteins that bind to them. To better understand the role of NOTCH1 in thymocyte development, experiments will be performed in which NOTCH1 signaling is disrupted or modified in T cell precursors as they differentiate in a thymic culture system derived from Rhesus macaques. Finally, alterations of gene expression produced by activation of NOTCH1 will be studied by hybridization of probes for total cellular RNA to cDNA microarrays. Besides providing insights into genes regulated directly and indirectly by NOTCH1, studies with microarrays may reveal the identity of genes which collaborate with truncated NOTCH1 in the malignant transformation of cells having chromosomal translocations that divide the NOTCH1 gene.