REGULATION OF PURINE METABOLISM IN HUMAN CELLS

嘌呤代谢紊乱发生在约5%的人群中， 最终导致1%的临床疾病。 的临床表现 这些疾病从痛风到严重的联合免疫缺陷 疾病 尽管在这条通路中的紊乱对 人类疾病的发病机制，现在看来，更好地了解 在最基本的层面上， 对生物医学科学的进步有更广泛的影响。 本 调查，我们将集中在a）定义的性质和后果， 在人类中自发发生的特定突变， 表达次黄嘌呤鸟嘌呤磷酸核糖基转移酶（HPRT）， 腺嘌呤磷酸核糖基转移酶（APRT），B）开发一种方法， 将HPRT互补DNA插入培养的神经元细胞中， 修饰的嗜神经病毒作为载体，和c）在脑中阐明 HPRT的正常作用以及由HPRT引起的代谢畸变 其不足。 在这项赠款的任期内，我们建议利用 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， 本领域技术人员使用的复杂但非常强大的仪器 临床医生 具体来说，我们的方法将利用a）重组DNA 技术包括cDNA克隆和测序，构建有用的 嵌合质粒和定点诱变，B）高度复杂的 蛋白质水平的方法学，包括X射线晶体学， 免疫细胞化学定位，必要时进行微测序， 单克隆抗体技术，和c）在床边，正电子发射 断层扫描（PET）。 我们在人体嘌呤代谢调节方面的工作 多年来，细胞从床边转移到细胞，再到蛋白质 现在又回到了基因和床边。 我们期望 这些研究不仅会大大扩展我们的信息库， 与HPRT缺乏相关的特定疾病有关 （Lesch-Nyhan综合征;痛风）和APRT（2，8二羟腺嘌呤结石病）， 也将为人类基因和蛋白质的研究提供重要信息 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.