MOLECULAR GENETICS OF LOW RENIN HYPERTENSION

描述(改编自申请人的摘要)：调查人员的 实验室继续研究生物化学、调节和遗传学。 11β-羟基类固醇脱氢酶肾脏(K或2型)同工酶的研究 (11-HSD)及其基因(HSD11K)。这种酶在体内起着关键作用 维持盐皮质激素受体的特异性。基因突变 该基因导致孟德尔式高血压，即表观高血压综合征 盐皮质激素过多。在生化研究中，他们将表达 大肠杆菌中野生型11-HSD K同工酶及其动力学性质的比较 与胎盘中天然酶和重组酶的同源性 在培养的哺乳动物细胞中表达。然后，他们会将这种酶改造成 增加其溶解度和活性，并使用亲和层析 以活性形式提纯酶，使其均一。调查人员将 表征跨膜结构域和膜跨结构域之间的关系 酶活性，并将表征核苷酸结合部位 酵素。他们将重新创造突变，导致表观综合症 过量的矿物质皮质激素，以检测低水平的残留活性。 调查人员将识别和描述转录调控 HSD11K中的元素。调查人员将使用瞬时转染法 报告基因构建、凝胶迁移率改变分析和DNA酶I 足迹分析研究增强剂和消音器元件，并将 通过寻找组织特异性DNA酶来确定可能的基因座控制区 I染色质过敏。调查人员将确认其功能正常 通过产生适当的菌株来确定可能的基因控制区 转基因小鼠。在基因研究中，他们将识别出更多 突变导致明显的盐皮质激素过多综合征和 确定它们在哺乳动物和/或细菌表达中的功能效应 系统。调查人员将确定11-HSD K在 高血压病的发展--连锁与关联并用 学习。

DESCRIPTION (Adapted from applicant's abstract): The investigators' laboratory continues to study the biochemistry, regulation and genetics of the kidney (K or type 2) isozyme of 11 Beta-hydroxysteroid dehydrogenase (11-HSD) and its gene (HSD11K). This enzyme plays a crucial role in maintaining the specificity of the mineralocorticoid receptor. Mutations in the gene cause a Mendelian form of hypertension, the syndrome of apparent mineralocorticoid excess. In biochemical studies, they will express the wild type 11-HSD K isozyme in E. coli and compare its kinetic properties with those of the native enzyme in placenta and the recombinant enzyme expressed in cultured mammalian cells. They will then modify the enzyme to increase its solubility and activity, and use affinity chromatography to purify the enzyme to homogeneity in an active form. The investigators will characterize the relationship between the membrane spanning domains and enzymatic activity, and will characterize the nucleotide binding site of the enzyme. They will recreate mutations causing the syndrome of apparent mineralocorticoid excess in order to detect low levels of residual activity. The investigators will identify and characterize transcriptional regulatory elements in HSD11K. The investigators will use transient transfection of reporter constructs, electrophoretic mobility shift assays and DNAse I footprinting assays to study enhancer and silencer elements, and will identify possible locus control regions by looking for tissue-specific DNAse I hypersensitivity in chromatin. The investigators will confirm functioning of putative locus control regions by producing appropriate strains of transgenic mice. In genetic studies, they will identify additional mutations causing the syndrome of apparent mineralocorticoid excess and determine their functional effects in mammalian and/or bacterial expression systems. The investigators will determine the role of 11-HSD K in the development of essential hypertension, using both linkage and association studies.