INBORN ERRORS--MOLECULAR ANALYSIS IN CULTURED CELLS

该项目继续专注于分析培养细胞中的 具有临床意义的先天性代谢缺陷，涉及四种 氨基酸和有机酸代谢酶： 同型胱氨酸尿症，由胱硫醚β-合酶（CS）引起 甲基丙二酰辅酶A引起的甲基丙二酸血症 脱氨酶（MUT）缺乏症;高氨血症，由于 鸟氨酸转氨甲酰酶（OTC）缺乏;和丙酸 酸血症，由于丙酰辅酶A羧化酶（PCC）缺乏。 这项研究利用了两种来源的成纤维细胞系 从患有遗传缺陷的患者身上， 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 这些蛋白质。 这些研究的目的是：1）识别 MUT和PCC中天然存在的突变， 异常的线粒体摄取和处理这些核- 编码的、细胞质合成的线粒体酶; 2） 在完整的培养细胞中分析人工 OTC前导肽中的突变，并将这些突变与 已经在体外描述的效果; 3）在 分子水平的CS、MUT和PCC中的天然人类突变， 为了了解它们对线粒体转运的影响， 辅酶结合、亚基相互作用或蛋白水解活化; 4)探讨CS过度表达与 (due部分三体21）和唐氏综合征，以及CS 低表达（由于CS缺陷的杂合性）和 外周闭塞性动脉疾病;和5）发展中的分子 早期产前诊断工具和可靠的 杂合子检测这些先天性错误的每一个， 新陈代谢. 生物化学、细胞生物学和分子遗传学 将采用的方法，包括：放射性标记和 完整细胞中特异性酶的免疫化学回收; 磷酸钙介导的DNA转染; DNA、RNA和 蛋白质印迹法，用（32 P）标记的探针（DNA和 RNA）或特异性抗体和（125 I）蛋白A（蛋白质）; 噬菌体载体中的基因组克隆;突变序列 通过变性梯度（Lerman）凝胶鉴定;和DNA 通过Maxam-Gilbert或桑格双脱氧技术测序。 这些研究应提供新的信息，说明 这些先天性缺陷发病机制中的个体突变， 对人体正常代谢和体内平衡的影响。

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.