INBORN ERRORS--MOLECULAR ANALYSIS IN CULTURED CELLS

该项目继续专注于对培养的细胞进行分析 临床上有意义的先天性代谢错误涉及四个 氨基酸和有机酸代谢的酶： 由胱硫醚β-合酶(CS)引起的同型半胱氨酸尿症 缺乏；甲基丙二酸血症，由甲基丙二酰辅酶A引起 变位酶(MUT)缺乏症；高氨血症 鸟氨酸转氨甲基酶(OTC)缺乏症；丙酸 酸血症，由于丙酰辅酶A羧基酶(PCC)缺乏。 这项拟议的研究利用了两种来源的成纤维细胞系 来自每一个基因决定缺陷的患者 这些酶和表达突变蛋白的细胞系 通过体外诱变分离的编码cDNA而产生的 这些蛋白质。这些研究的目的是：1)确定 MUT和PCC中自然发生的突变导致 线粒体异常摄取和处理这些核- 编码，细胞质合成，线粒体酶；2) 在完整培养细胞中分析人工合成的影响 OTC前导肽的突变，并将其与 已经在体外描述的效果；3)在 中国人CS、MUT和PCC的分子水平自然突变 为了了解它们对线粒体运输的影响， 辅酶结合、亚基相互作用或蛋白水解性激活； 4)解决CS过度表达之间可能存在的关系 (由于部分21三体)和唐氏综合征，以及CS之间 表达不足(由于CS缺乏的杂合性)和 外周闭塞性动脉疾病；5)发展中的分子 将允许早期产前诊断和可靠的工具 这些先天缺陷中每一个的杂合子检测 新陈代谢。生化、细胞生物学和分子遗传学 将采用各种方法，包括：放射性标记和 免疫化学恢复完整细胞中的特定酶； 磷酸钙介导的DNA转染法；DNA，RNA和 用(32P)标记的探针(DNA和 RNA)或特定抗体和(125I)蛋白A(蛋白质)； 噬菌体载体中的基因组克隆；突变序列 变性梯度(勒曼)凝胶和DNA鉴定 用Maxam-Gilbert或Sanger双脱氧技术测序。 这些研究应该提供关于两个角色的新信息 这些先天错误的发病机制中的个体突变和 论人类正常的代谢和动态平衡机制。

This project continues to focus on an analysis in cultured cells of clinically significant inborn errors of metabolism involving four enzymes of amino acid and organic acid metabolism: homocystinuria, due to cystathionine beta-synthase (CS) deficiency; methylmalonic acidemia, due to methylmalonyl CoA mutase apoenzyme (MUT) deficiency; hyperammonemia, due to ornithine transcarbamylase (OTC) deficiency; and propionic acidemia, due to propionyl CoA carboxylase (PCC) deficiency. The proposed research makes use both of fibroblast lines derived from patients with genetically determined defects in each of these enzymes and of cell lines expressing mutant proteins generated by in vitro mutagenesis of isolated cDNAs encoding these proteins. These studies are aimed at: 1) identifying naturally occurring mutations in MUT and PCC which result in abnormal mitochondrial uptake and processing of these nuclear- coded, cytoplasmically synthesized, mitochondrial enzymes; 2) analyzing in intact cultured cells the effects of artificial mutations in the OTC leader peptide and comparing these to the effects already described in vitro; 3) characterizing at the molecular level natural human mutations in CS, MUT, and PCC in order to understand their effects on mitochondrial transport, coenzyme binding, subunit interaction, or proteolytic activation; 4) addressing the possible relationship between CS overexpression (due to partial trisomy 21) and Down's syndrome, and between CS underexpression (due to heterozygosity for CS deficiency) and peripheral occlusive arterial disease; and 5) developing molecular tools which will permit early prenatal diagnosis and reliable heterozygote detection for each of these inborn errors of metabolism. Biochemical, cell biological, and molecular genetic approaches will be employed, including: radiolabeling and immunochemical recovery of specific enzymes in intact cells; calcium phosphate-mediated DNA transfection; DNA, RNA, and protein blotting with detection by (32P) labeled probes (DNA and RNA) or by specific antibodies and (125I) protein A (proteins); genomic cloning in bacteriophage vectors; mutant sequence identification by denaturing gradient (Lerman) gels; and DNA sequencing by Maxam-Gilbert or Sanger dideoxy techniques. These studies should provide new information on both the role of individual mutations in the pathogenesis of these inborn errors and on normal mechanisms of metabolism and homeostasis in man.