REGULATION OF PURINE METABOLISM IN HUMAN CELLS

大约5%的人会出现嘌呤代谢障碍，并可能 最终导致临床疾病的比例为1%。多发性硬化症的临床表现 这些疾病从痛风到严重的联合免疫缺陷。 疾病。尽管这条途径中的错位对 人类疾病的发病机制，现在看来有了更好的理解 其中一些最基本的障碍甚至可能 对生物医学科学进步的更广泛的影响。在现在 调查，我们将重点放在a)界定性质和后果 在人类中自发发生的特定突变会改变 次黄嘌呤鸟嘌呤磷酸核糖基转移酶(HPRT)的表达及 腺嘌呤磷酸核糖基转移酶(APRT)，b)发展一种方法 将HPRT互补DNA插入体外培养的神经细胞 以修饰的嗜神经病毒为载体，以及c)在脑内阐明 HPRT的正常作用及其引起的代谢异常 它的不足之处。在这笔赠款的任期内，我们建议利用 现有最先进的科学技术和实验方法 从基础生物化学家的高度复杂的工具到 技术人员使用的复杂但功能强大的仪器 临床医生。具体地说，我们的方法将利用)重组DNA 涉及cDNA克隆和测序的技术，构建实用的 嵌合质粒和定点突变，b)高度复杂 蛋白质水平的方法学，包括X射线结晶学， 免疫细胞化学定位，必要时进行显微测序和 单抗技术，以及c)在床边，正电子发射 断层扫描(PET)。我们在人体嘌呤代谢调控方面的工作 多年来，细胞从床边移动到细胞，再到蛋白质 现在是关于基因的问题，也回到了床边。这是我们的期望 这些研究不仅将大大扩展我们的信息库 与HPRT缺乏相关的特定疾病相关的 (Lesch-Nyhan综合征；痛风)和APRT(2，8二羟基腺嘌呤结石病)， 还将提供有关人类基因和蛋白质的重要信息 结构和功能以及基因转移，特别是在 神经系统疾病。

Disorders of purine metabolism occur in about 5% of the population and may eventually lead to clinical disease in 1%. The clinical manifestations of these disorders range from gout to severe combined immunodeficiency disease. Despite the importance of derangements in this pathway to the pathogenesis of human disease, it now appears that a better understanding of some of those disorders at the most fundamental levels may have even broader implications to the progress of biomedical science. In the present investigation, we will focus on a) defining the nature and consequences of the specific mutations occurring spontaneously in humans which alter the expression of hypoxanthine guanine phosphoribosyltransferase (HPRT) and adenine phosphoribosyltransferase (APRT), b) developing an approach to inserting the HPRT complementary DNA into neuronal cells in culture using a modified neurotropic virus as the vector, and c) elucidating in the brain the normal role of HPRT as well as the metabolic aberrations resulting from its deficiency. During the tenure of this grant we propose to exploit the most advanced scientific techniques and experimental approaches available ranging from the highly sophisticated tools of the basic biochemist to the complex but highly powerful instrumentation utilized by the skilled clinician. Specifically, our approach will utilize a) recombinant DNA techniques involving cDNA cloning and sequencing, construction of useful chimeric plasmids and site directed mutagenesis, b) highly sophisticated methodology at the level of the protein including X-ray crystallography, immunocytochemical localization, and, as necessary, microsequencing and monoclonal antibody techniques, and c) at the bedside, Positron Emission Tomography (PET). Our work on the Regulation of Purine Metabolism in Human Cells has moved over the years from the bedside to the cell to the protein and now to the gene as well as back to the bedside. It is our expectation that these studies will not only substantially expand our information base related to the specific diseases associated with a deficiency of HPRT (Lesch-Nyhan syndrome; Gout) and APRT (2,8 dihydroxyadenine stone disease), but also will provide important information on human gene and protein structure and function as well as on gene transfer particularly in the neurologic diseases.