MOLECULAR GENETICS OF FACTOR IX AND OTHER BLOOD PROTEINS

因子IX在外源性和内源性免疫中起着关键作用， 凝血途径。 经常委员会的设立 因此，因子IX（FIX）生物合成的潜在机制是 不仅对理解血友病B的分子基础至关重要， 还探讨了FIX在血液凝固及其稳态中的作用。 本提案的长期目标是建立全面的 负责FIX基因转录的控制机制，第一步 在FIX生物合成中。 这将通过实现三个 具体目标：（一）建立准确的基本机制 负责FIX基因的总体转录控制;（ii） 建立具体机制， 改善血友病B Leyden表型;和（iii） 建立年龄相关调节的分子基础 FIX基因的表达。 目标1将通过建立详细的 基本结构要素、其功能和相互关系图 FIX基因的整体调控的关系，从而提供了一个 为其余的研究奠定了坚实的基础。 体外系统性 顺式作用DNA序列的鉴定和功能分析 元素和与它们相互作用的反作用因子将被携带， 通过定点诱变，核酸酶超敏反应测定，DNA- 蛋白质和蛋白质-蛋白质相互作用分析以及 关键的反式作用因子的表征，如果它们是新的。在 体内，这些元件在FIX小基因结构背景下的功能 将在与研究结合设计的转基因小鼠中进行测试 目标2和3。 目的2，血友病的发病机制 B莱顿表型，这是由于一组单碱基突变 包含在一个小的5'端区域，将通过扩展我们的研究， 新的发现包括生长激素的可能参与 途径，进行了大量的体外和体内分析，包括 用转基因小鼠结合垂体切除术进行测试。 我们有 最近发现了存在于5'端的1号线序列的新作用 与年龄相关的FIX基因调控。 目标3将通过以下方式实现： 建立序列的作用机制， 其余的基因结构。 在这项研究中，转基因小鼠将作为 重要体内分析系统。拟议的研究将大大 有助于我们理解FIX生物合成的具体， 血液凝固的整体调节及其体内平衡， 一般来说，这对改进或新颖的发展， 治疗人类出血和血栓性疾病。

Factor IX plays a pivotal role in both the extrinsic and intrinsic pathways of bolld coagulation. Establishment of the regualtory mechanisms underlying factor IX (FIX) biosynthesis, therefore, is critical for understanding not only the molecular basis of hemophilia B, but also the role of FIX in the blood coagulation and its homeostasis. The long-term goal of this proposal is to establish the comprehensive control mechanism responsible for FIX gene transcription, the first step in the FIX biosynthesis. This will be accomplished by achieving three specific aims, (I) to establish the precise fundamental mechanism responsible for the overall transcriptional control of the FIX gene; (ii) to establish specific mechanisms responsible for the puberty-onset amelioration of the hemophilia B Leyden phenotype; and (iii) to establish the molecular basis underlying the age-associated regulation of FIX gene expression. Aim 1 will achieved by establishing a detailed map of essential structural elements, their functions and mutual relationship n the overall regulation of the FIX gene, thus providing a solid base for the rest of the proposed studies. Systematic in vitro identification and functional analysis of cis-acting DNA sequence elements and trans-acting factors interacting with them will be carried out by site-directed mutagenesis, nuclease hypersensitivity assay, DNA- protein and protein-protein interaction analyses as well as characterization of crucial trans-acting factors, if they are novel. In vivo, functions of these elements in context of FIX minigene structures will tested in transgenic mice designed in conjunction with the studies for aims 2 and 3. Aim 2, mechanisms responsible for the hemophilia B Leyden phenotype, which are due to a set of single-base mutations contained in a small 5' end region, will be studied by extending our new findings including possible involvement of growth hormone pathway, with intensive in vitro as well as in vivo analyses including testing with transgenic mice combined with hypophysectomy. We have recently found a novel role of the Line 1 sequence present at the 5' end in the age-associated FIX gene regulation. Aim 3 will achieved by establishing the mechanism of action of the sequence in context of the rest of gene structure. In this study, transgenic mice will serve as an important in vivo assay system. Proposed studies will significantly contribute to our understanding of the FIX biosynthesis in specific, and the overall regulation of blood coagulation and its homeostasis in general, and this to the development of improved or perhaps novel teratments for bleeding and thrombotic disorders in humans.