FACTORS REGULATING HPRT GENE EXPRESSION

次黄嘌呤磷酸核糖基转移酶完全缺乏 （HPRT）在人类中引起Lesch-Nyhan综合征，一种X连锁神经系统疾病， 以精神发育迟滞、舞蹈手足徐动症、自 破坏行为和高尿酸血症。局部缺乏会导致严重的 痛风型 关节炎，大约20%的患者患有 智力迟钝和痉挛。HPRT被认为是一种“管家” 基因，因为它在所有组织中以低水平组成型表达。 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 为了了解顺式作用序列在组织分化中的作用， 表达的人HPRT基因，我们已经功能分析了1600 bp 在培养的细胞和转基因小鼠中的5'侧翼区。我们 已经鉴定了一个182 bp的负调控元件（hHPRT-NE）上游 核心启动子抑制培养的成纤维细胞中的表达。 转基因小鼠研究表明，hHPRT-NE是至少一种 两个元素，指导转基因表达的前脑， 老鼠.我们发现97 bp的核心启动子显然不是 足以在转基因小鼠中组成型表达。我们有 确定了与hHPRT-NE相互作用的反式作用因子， 和大脑特异性。本申请旨在确定trans-acting 来自脑、肝和肾等组织的因子， 人HPRT基因的顺式作用调节序列， 对于维持基因的组织差异表达很重要。的 这些反式作用因子与这些序列的相互作用将是 在核苷酸水平表征。表达这些的cDNA克隆 因子将被分离并用于它们在细菌细胞中的表达 在哺乳动物细胞中，允许认证和产生 抗体和功能分析。组织- 反式作用因子的分布将在 与HPRT基因表达相关的发育。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， 顺式作用序列的组织差异表达的重要性 HPRT基因。它们还将确定“内务管理”的重要因素， 组织差异基因表达 HPRT缺陷是基因治疗的极好候选者。的 本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， 证明在设计用于这种治疗的载体中非常有价值。

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.