EXPRESSION OF CYSTATHIONINE SYNTHASE AND HUMAN DISEASE

该提案的主要目标是确定 β-胱硫醚8-合酶（CBS）基因的表达可能起作用， 在三种人类疾病状态中的作用：高胱氨酸尿症;外周和 脑动脉闭塞性疾病和唐氏综合征。 哥伦比亚广播公司a 真核生物硫代谢的中心酶，是一种四聚体， 63 kDa亚单位它在人类21号染色体上编码;其 遗传性缺陷导致最常见形式的同型胱氨酸尿症。 此外，28%的闭塞性患者为试验疾病 据报道是CBS缺陷的杂合子。 相反地， CBS水平升高在21三体中有记录， 有助于唐氏综合症表型的产生。 我们 最近已经分离出编码CBS的cDNA克隆。 有趣的是， 三种类型（I、II和III）的明显全长cDNA被 这两种语言在翻译和翻译上都有很大的不同， 非翻译区;它们编码63，39和61.5 kDa的 多肽，分别。 我们的具体目标是：1）克隆 CBS的整个基因，并确定其限制性酶切图谱， 内含子/外显子结构; 2）确定，是否三个合酶 在大鼠肝脏中发现的mRNA反映了选择性剪接， 合成酶转录物; 3）确定这些mRNA是否 负责差异，在组织特异性合成酶表达; 4)为了实现三种不同克隆的cDNA在 转染培养的啮齿动物细胞，从而解决了 合成酶与S-腺苷甲硫氨酸的相互作用及其 通过产生48 kDa亚基的特异性蛋白水解激活 具有增加的催化活性; 5）提供证据， 分子水平，一些患有早发性动脉疾病的患者 是CI杂合子;和6）建立携带 大鼠CBS转基因作为唐氏综合征中CBS剂量的模型。 生物化学、细胞生物学和分子生物学方法 将采用，包括：筛选基因组文库， (32P)标记的DNA和RNA探针;桑格法DNA测序 双脱氧技术;聚合酶链反应; DNA、RNA和蛋白质印迹法，通过（32 p）标记的检测 探针或通过抗CBS抗体和（125 I）蛋白G; DNA 转染培养细胞和卵母细胞显微注射。 的 这项工作的结果将补充我们对正常和 突变CBS基因，并将阐明其意义， 人类疾病的产品。

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.