MOLECULAR GENETICS OF LOW RENIN HYPERTENSION

描述（改编自申请人摘要）：研究者 实验室继续研究生物化学，调节和遗传学， 11 β-羟类固醇脱氢酶肾（K型或2型）同工酶 （11-HSD）及其基因（HSD 11 K）。 这种酶在 维持盐皮质激素受体的特异性。 突变 这种基因会导致孟德尔式的高血压， 盐皮质激素过多 在生物化学研究中，它们会表达 野生型E.大肠杆菌中，并比较其动力学性质 与胎盘中的天然酶和重组酶的那些相比 在培养的哺乳动物细胞中表达。 然后，他们将修改酶， 增加其溶解性和活性，并使用亲和层析， 以活性形式将酶纯化至均质。 调查人员将 表征跨膜结构域之间的关系， 酶活性，并将表征的核苷酸结合位点的酶活性， 酵素 他们将重新创造突变， 盐皮质激素过量，以检测低水平的残留活性。 研究人员将确定和表征转录调控 在HSD 11 K中的元素。 研究人员将使用瞬时转染技术， 报道构建体、电泳迁移率变动分析和DNA酶I 足迹分析研究增强子和沉默子元件，并将 通过寻找组织特异性DNA酶鉴定可能的基因座控制区 I染色质超敏反应。 调查人员将确认 通过产生合适的菌株， 转基因小鼠 在遗传学研究中，他们将确定额外的 突变导致明显的盐皮质激素过量综合征， 确定它们在哺乳动物和/或细菌表达中的功能作用 系统. 研究人员将确定11-HSD K在 原发性高血压的发展，使用联系和关联 问题研究

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.