FACTORS REGULATING HPRT GENE EXPRESSION

次黄嘌呤磷酸核糖转移酶完全缺乏症 人类(HPRT)导致X连锁神经病学Lesch-Nyhan综合征 精神障碍的特征是智力低下、舞蹈动作障碍、自我 肢解行为和高尿酸血症。部分缺乏症导致严重的 痛风性关节炎的形式，其中约20%的患者患有 智力低下和痉挛。HPRT被认为是一种“内务” 基因，因为它在所有组织中都有低水平的结构性表达。 然而，在某些地方，它的表达水平要高出五到七倍 中枢神经系统。 了解顺式作用序列在组织分化中的作用 人HPRT基因的表达，我们对1600个碱基进行了功能分析 在培养细胞和转基因小鼠中的5‘侧翼区。我们 已确定上游有182个碱基的负调控元件(hHPRT-NE) 抑制培养成纤维细胞表达的核心启动子。 转基因小鼠的研究表明，hHPRT-NE至少是 两个直接在小鼠前脑中表达转基因的元件 老鼠。我们发现97个碱基的核心启动子显然不是 足以在转基因小鼠中进行结构性表达。我们有 已确定的与hHPRT-NE相互作用的反式作用因子，两者都普遍存在 而且是大脑特有的。这个应用程序建议识别交易行为 来自大脑、肝脏和肾脏等组织的因子，它们相互作用 人HPRT基因的顺式作用调控序列和 对维持该基因的组织差异表达很重要。这个 这些反式作用因子与这些序列的相互作用将是 以核苷酸水平为特征的。表达这些基因的cDNA克隆 因子将被分离并用于在细菌细胞中表达 在哺乳动物细胞中，允许认证和产生 分别用于抗体和功能分析。组织- 反式作用因子的分布将在 HPRT基因表达与发育的关系。这些研究 将支持和确认我们正在进行的关于功能划分的研究 顺式作用序列对组织差异表达非常重要 HPRT基因。他们还将界定对“内务管理”和 组织差异基因表达。 HPRT缺乏症是一种很好的基因治疗方法。这个 这项研究提供的信息将通过定义序列和 HPRT基因适当表达模式所需的因子， 事实证明，在设计这种疗法的载体方面非常有价值。

Complete deficiency of the enzyme hypoxanthine phosphoribosyltransferase (HPRT) in humans causes Lesch-Nyhan syndrome, an X-linked neurological disorder characterized by mental retardation, choreoathetosis, self- mutilative behavior and hyperuricemia. Partial deficiency causes a severe form of gouty arthritis and approximately 20% of these patients have mental retardation and spasticity. HPRT is considered a "housekeeping" gene as it is constitutively expressed at low levels in all tissues. However, it is expressed at five- to seven-fold higher levels in parts of the central nervous system. To understand the role of cis-acting sequences in the tissue-differential expression of the human HPRT gene, we have functionally analyzed 1600 bp of the 5' flanking region in cultured cells and in transgenic mice. We have identified a 182 bp negative regulatory element (hHPRT-NE) upstream of the core promoter which represses expression in cultured fibroblasts. Transgenic mice studies have shown that the hHPRT-NE is one of at least two elements that direct transgene expression in the forebrain of the mouse. We have found that the core promoter of 97 bp is apparently not sufficient for constitutive expression in transgenic mice. We have identified trans-acting factors interacting with hHPRT-NE, both ubiquitous and brain-specific. This application proposes to identify trans-acting factors from tissues such as brain, liver and kidney, that interact with the cis-acting regulatory sequences of the human HPRT gene and are important for maintaining tissue-differential expression of the gene. The interaction of these trans-acting factors with these sequences will be characterized at the nucleotide level. cDNA clones expressing these factors will be isolated and used for their expression in bacterial cells and in mammalian cells, to allow authentication and the generation of antibodies, and for functional analysis, respectively. The tissue- distribution of the trans-acting factors will be studied during development in relation to that of HPRT gene expression. These studies will support and confirm our ongoing studies on functional delineation of cis-acting sequences important for tissue-differential expression of the HPRT gene. They will also define factors important for "housekeeping" and tissue-differential gene expression. HPRT deficiency is an excellent candidate for gene therapy. The information provided by this study will, by defining the sequences and factors required for appropriate patterns of expression of the HPRT gene, prove very valuable in designing vectors for such therapy.