EUKARYOTIC GENE ORGANIZATION

该项目探索LINE-1(L1)分子生物学和 它们对哺乳动物基因组的影响。L1是可转置的原型 编码逆转录酶但不包含LTR的元件 逆转录病毒特有的序列。L1占10%或更多 哺乳动物的基因组。相关元素广泛存在于真核生物中 包括昆虫和植物。L1是基因突变的一个重要来源 哺乳动物，包括人类。L1转座可以敲除基因，并具有 与肿瘤的发生有关。L1元素之间的重组也是 一个潜在的突变来源。我们的研究只集中在老鼠身上。 我们打算表征L1转座中间体。自.以来 L1的转录是其转位的第一步，一项研究将 关注L1转录的调控。小白鼠L1A-的分析 通过饱和诱变完成类型启动子的意图 来定义转录结合位点。突变体的凝胶位移分析 转录特性改变的基因将被用于研究 启动子活性所需序列与蛋白质之间的对应关系 结合部位。带有L1A型驱动报告基因的转基因小鼠将 用于研究启动子的发育规律。研究 比较重建的祖先F-F-的转录调控 现代A型启动子在组织培养细胞和凝胶中的应用 轮班实验将会延长。F型驱动的转基因小鼠 报告基因将被构建并用于其调节与 现代A型。这些实验模拟了L1在过去5-10年中的演变 在现代老鼠细胞中存活了一百万年。另一项研究将集中在L1 粒子作为可能的换位中间体。我们还将构建 过表达L1的细胞系，并利用它们产生L1颗粒。这个 这些部分IDE的结构和酶性质将是 详细地描述了。 L1的存在对其哺乳动物宿主有用吗？有两种方法将 被调查。1)表达L1的体细胞组织 学习。初步观察发现L1在卵巢癌组织中的表达 免疫系统将得到扩展。L1在免疫系统中的可能功能 系统a)L1逆转录酶在体细胞中的参与 免疫球蛋白的高度突变b)组织特异性基因的激活 胸腺中抗原提呈细胞的L1超转位。2)反- 将构建L1核酶以尝试在细胞中创造表型 品系和转基因动物。

This project explores the molecular biology of LINE-1 (L1) elements and their impact on the mammalian genome. L1 is the prototype for transposable elements which encode reverse transcriptase but do not contain the LTR sequences characteristic of retroviruses. L1 makes up 10% or more of the mammalian genome. Related elements are found throughout eukaryotes including insects and plants. L1 is a significant source of mutations in mammals, including humans. L1 transposition can knock out genes and has been implicated in tumorigenesis. Recombination between L1 elements is also a potential source of mutations. Our studies focus only in the mouse. We intend to characterize L1 transposition intermediates. Since transcription of L1 is the first step in its transposition one study will focus on the regulation of L1 transcription. Analysis of the mouse L1 A- type promoter by saturation mutagenesis will be completed with the intent of defining the transcription binding sites. Gel shift analysis of mutants with altered transcriptional properties will be used to investigate the correspondence between sequences required for promoter activity and protein binding sites. Transgenic mice with an L1 A-type driven reporter gene will be used to study the developmental regulation of the promoter. Studies comparing the transcriptional regulation of a reconstructed ancestral F- type promoter with the modern A-type in tissue culture cells and in gel shift experiments will be extended. Transgenic mice with an F-type driven reporter gene will be constructed and used to are its regulation with the modern A-type. These experiments model evolution of L1 over the last 5-10 million years in modern mouse cells. Another study will focus on the L1 particle as a likely transposition intermediate. We will also construct cell lines overexpressing L1 and use them to produce L1 particles. The structural and enzymatic properties of these part ides will be characterized in detail. Is the presence of L1 useful for its mammalian host? Two approaches will be investigated. 1) Somatic tissue in which L1 is expressed will be studied. Initial observations finding L1 expression in tissues of the immune system will be extended. Possible functions for L1 in the immune system are a) involvement of L1 reverse transcriptase in somatic hypermutation of immunoglobulins b) activation of tissue specific genes by L1 hyper-transposition in antigen presenting cells in the thymus. 2) Anti- L1 ribozymes will be constructed to attempt to create a phenotype in cell lines and transgenic animals.