EXPRESSION OF CYSTATHIONINE SYNTHASE AND HUMAN DISEASE

这项提案的主要目标是确定 --胱硫醚-8-合酶(CBS)基因的表达可能起到 在人类三种疾病状态中的作用：同型半胱氨酸尿症；外周和 脑闭塞性动脉疾病；以及唐氏综合征。哥伦比亚广播公司，a 真核细胞硫代谢中心酶是硫代谢的四聚体。 63 kDa亚基它在人类21号染色体上编码；它的 遗传性缺陷是最常见的同型半胱氨酸尿症。 此外，28%的闭塞患者是试验性疾病。 据报道，他们是CBS缺乏症的杂合子。相反， CBS水平升高已在21三体中被记录在案，并可能 有助于唐氏综合症表型的产生。我们 最近分离了编码CBS的cdna克隆。有趣的是， 三种类型(I、II和III)的明显全长的cDNA是 发现，这两个词在翻译后和 未翻译的区域；它们编码63、39和61.5 kDa 分别为多肽。我们的具体目标是：1)克隆 CBS的全长基因并确定其限制性内切酶图谱和 内含子/外显子结构；2)确定三种合酶是否 在大鼠肝脏中发现的mRNAs反映了， 合成酶转录本；3)确定这些mRNAs是否 负责组织特异性合酶表达的差异； 4)实现三种不同克隆的cDNA在大肠杆菌中的表达 转基因培养的啮齿动物细胞，从而解决 S-腺苷甲硫氨酸与合成酶的相互作用机制及其作用机制 产生48 kDa亚基的特异性蛋白水解酶激活 具有更高的催化活性；5)在 一些早产儿动脉疾病患者的分子水平 CI杂合子；6)建立携带 CBS转基因大鼠作为唐氏综合征CBS剂量模型的研究 生化、细胞生物学和分子生物学方法 将被使用，包括：筛选基因组文库 (32P)标记的DNA和RNA探针；桑格DNA测序 双脱氧技术；聚合酶链式反应DNA扩增； ~(32)P标记的DNA、RNA和蛋白质印迹检测 探针或通过抗CBS抗体和(125I)蛋白G；DNA 转入培养细胞和卵母细胞显微注射。这个 这项工作的结果将补充我们对正常和 突变的CBS基因，并将阐明其意义 人类疾病中的产品。

The major goal of this proposal is to determine how levels of expression of the--cystathionine 8-synthase (CBS) gene may play a role in three human disease states: homocystinuria; peripheral and cerebral occlusive arterial disease; and Down syndrome. CBS, a central enzyme in eukaryotic sulfur metabolism, is a tetramer of 63 kDa subunit It is encoded in humans on chromosome 21; its inherited deficiency causes the most common form of homocystinuria. Further, 28% of patients with occlusive are trial disease reportedly are heterozygous for CBS deficiency. Conversely, elevated levels of CBS has been documented in trisomy 21 and may contribute to the generation of the Down syndrome phenotype. We have recently isolated cDNA clones encoding CBS. Interestingly, three types (I, II and III) of apparently full-length cDNAs were found, which differ significantly in both their translated and untranslated regions; they code for a 63, 39 and a 61.5 kDa polypeptide, respectively. Our specific aims are 1) to clone the entire gene for CBS and to determine its restriction map and intron/exon structure; 2) to determine, whether the three synthase mRNAs found in rat liver reflect alternative splicing of the, synthase transcript; 3) to ascertain whether these mRNAs are responsible for difference, in tissue-specific synthase expression; 4) to achieve expression of the three different cloned cDNAs in transfected cultured rodent cells and, thereby, address the mechanism < synthase interaction with S-adenosylmethionine and its activation by specific proteoly-sis which yields 48 kDa subunits with increased catalytic activity; 5) to provide evidence at a molecular level that some patients with premature arterial disease are CI heterozygotes; and 6) to establish transgenic mice carrying a rat CBS transgene as a model for CBS dosage in Down syndrome. Biochemical, cell biological, and molecular biological approaches will be employed, including: screening of genomic libraries with (32P) labeled DNA and RNA probes; DNA sequencing by Sanger's dideoxy technique; DNA amplification by polymerase chain reaction; DNA, RNA, and protein blotting with detection by (32p)labeled probes or by anti-CBS antibodies and (125I) protein G; DNA transfec-tion into cultured cells and oocyte microinjection. The results of this work will complement our studies of normal and mutant CBS genes and will elucidate the significance of their products in human disease.