FUNCTIONAL ANALYSIS OF THE TAN-1 GENE

这项资助的目的是分析人类TAN-1的功能， 基因在肿瘤和正常组织中的表达。这种基因最初是在 跨越人类急性T细胞白血病复发染色体断裂点的位点 淋巴母细胞肿瘤正常全长TAN-1 cDNA的分析 揭示了与果蝇Notch基因的强序列同源性， 编码一种跨膜蛋白，控制细胞命运的选择。 发育中的果蝇的许多组织。初步数据显示 将TAN-1 cDNA片段插入骨髓干细胞， 设计来模拟易位所破坏的基因， 小鼠淋巴母细胞肿瘤。研究TAN-1蛋白（tan-1）在 正常细胞显示它在一个位点被蛋白水解切割， 在跨膜区的外部， 末端多肽片段与截短的多肽非常相似 由肿瘤细胞中易位的染色体产生。两者 全长和加工的tan-1多肽定位于 核周细胞质，而肿瘤细胞中的截短产物是 主要是核内的。这种多肽的核定位 似乎是由一个强大的核定位信号序列决定的 在蛋白质的胞内区域内。此外，该产品 BCL-3原癌基因（bcl-3），最近发现， 参与转录因子NF κ B的调节，结合到 TAN-1的细胞内区域含有六个所谓的锚蛋白重复序列。 本申请中提出的研究将调查以下四个方面： TAN-1功能，包括 发育阶段特异性T淋巴细胞转化与 截短的tan-1多肽对Notch样基因的自动调节。的 核定位对于恶性转化重要性将是 将开发一种有效的体外测定系统来研究 tan-1不同区域在转化能力中的作用 蛋白质。与tan-1的细胞外结构域结合的配体将 寻找并克隆他们的基因。可能参与的 将使用体外结合研究NF κ B途径中的tan-1 用tan-1、bcl-3和NF κ B以及通过细胞共转染进行的研究 用TAN-1、BCL-3和NF κ B cDNA沿着报告基因， 通常由NF κ B调控的启动子。

The purpose of this grant is to analyze the function of the human TAN-1 gene in neoplasia and normal tissues. This gene was originally discovered spanning the site of a recurrent chromosomal breakpoint in human acute T lymphoblastic neoplasms. Analysis of normal, full length TAN-1 cDNA revealed strong sequence homology to the Drosophila gene Notch, which encodes a transmembrane protein that controls the choice of cell fates m numerous tissues of the developing fruit fly. Preliminary data indicate that insertion into bone marrow stem cells of TAN-1 cDNA fragments designed to mimic the gene broken by the translocation induces T lymphoblastic neoplasms in mice. Studies of the TAN-1 protein (tan-1) in normal cells show that it is proteolytically cleaved at a site just external to the transmembrane region and that the residual carboxy terminal polypeptide fragment closely resembles the truncated polypeptides produced from the translocated chromosome in neoplastic cells. Both the full length and processed tan-1 polypeptides are localized to the perinuclear cytoplasm, while the truncated product in neoplastic cells is predominantly intranuclear. The nuclear location of this polypeptide appears to be determined by a strong nuclear localization signal sequence within the intracellular domain of the protein. Additionally, the product of the BCL-3 proto-oncogene (bcl-3), which has recently been found to be involved in regulation of the transcription factor NFkappaB, binds to an intracellular region of tan-1 containing six so-called ankyrin repeats. Research proposed in this application will investigate four aspects of TAN-1 function, including the alternative possibilities of the developmental stage-specific transformation of T lymphocytes versus the autoregulation of Notch-like genes by truncated tan-1 polypeptides. The importance of nuclear localization for malignant transformation will be examined and an efficient in vitro assay system will be developed to study the role of different regions within tan-1 in the transforming ability of the protein. Ligands that bind to the extracellular domain of tan-1 will be sought and their genes molecularly cloned. The possible involvement of tan-1 in the NFkappaB pathway will be investigated using in vitro binding studies with tan-1, bcl-3, and NFkappaB and by a co-transfection of cells with TAN-1, BCL-3, and NFkappaB cDNAs along with a reporter gene behind a promoter normally regulated by NFkappaB.