RECEPTOR MUTANTS IN VITAMIN D RESISTANT RICKETS OF BONE

维生素 D 抗性骨佝偻病中的受体突变体

• 批准号: 3242176
• 负责人: MARK R HUGHES
• 金额: $ 14.63万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3242176
• 关键词: Epstein Barr virus; RNA; RNA splicing; acetylcholine; alleles; androgens; cholesterol; complementary DNA; developmental genetics; estrogens; fibroblasts; gene frequency; genetic library; genome; glucocorticoids; hormone receptor; human subject; insulin; ligase; low density lipoprotein; lymphoblast; molecular cloning; mutant; neurotransmitter receptor; pancreatic ribonuclease; plasmids; progesterone; protein structure function; renal rickets; sterols; tissue /cell culture; western blottings

The proposed research focuses on the molecular mechanism of action of the sterol 1,25 dihydroxyvitamin D with major emphasis in understanding the structure and func-tion of the i intracellular receptor protein for this hormone. Despite very low abun-dance of the cellular receptor protein (0.001%), human expression cDNA libraries have been used to clone the full coding resign of the protein. Sequence analysis confirm regions of the gene similar to other cloned receptors thus for (estrogen, progesterone, glucocorticoid, and androgen). The natural gene for the human receptor has recently been isolated and the intron-exon junctions identified. It is proposed to use this information to analyze defective receptor proteins in families with the genetic disease, vitamin D- resistant rickets - type II. Patients with this autosomal recessive disorder display hypocalcemic, hyperparathyroidism and severe osteomalacia/rickets of bone. Cells from these patients will be analyzed for functionality as assessed by hormone binding, nuclear translocation, DNA binding and induction of alternate enzyme (24-hydroxylase) pathways. Genomic libraries will be; constructed from the patient DNA. Screened for receptor containing sequences and sequenced for mutation identification. Restriction fragment length polymorphism (RFlP), polymerase chain reaction (POR) amplification, and ribonuclease A digestion will be employed to identify the patient mutation(s). Subsequently, the identified gene defect (point mutation, deletion, insertion, splice-junction error, promoter defect) will be introduced into the normal clone to recreate the patients mutation. Transfection into COS-l receptor negative cells would be expected to reproduce the identified mutant protein with the same phenotype as isolated from the patient. The in vitro produced defective protein will be tested for functionality (hormone binding, DNA binding, 24- hydroxylase activation) to gain insights into the critically important amino acids of the receptor. Protein DNA contact points will be determined by alkylation interference experiments using dimethylsulfate and ethylnitrosourea. Similarly, mutations involving hormone binding affinity will be studied to define in molecular terms the steroid binding pocket and the critical amino acids involved in ligand interaction on with the receptor.

拟议的研究重点是作用的分子机制 甾醇 1,25 二羟基维生素 D 的主要重点是 了解细胞内的结构和功能 这种激素的受体蛋白。 尽管丰度很低 细胞受体蛋白 (0.001%)，人类表达 cDNA 库已被用来克隆完整的编码设计 蛋白质。 序列分析确认该基因的区域类似于 其他克隆受体（雌激素、孕激素、 糖皮质激素和雄激素）。 人类的天然基因 最近已分离出受体，内含子-外显子连接 确定。 建议使用此信息来分析缺陷 患有遗传病的家族中的受体蛋白，维生素D- 抵抗性佝偻病 - II 型。 患有这种常染色体的患者 隐性病症表现为低钙血症、甲状旁腺功能亢进和 严重的骨软化症/骨佝偻病。 来自这些患者的细胞 将通过激素结合评估进行功能分析， 核易位、DNA 结合和交替诱导 酶（24-羟化酶）途径。 基因组文库将是； 由患者 DNA 构建而成。 筛选含有受体 序列并测序以进行突变鉴定。 限制 片段长度多态性 (RFIP)、聚合酶链式反应 (POR) 扩增，并采用核糖核酸酶 A 消化 识别患者突变。 随后，确定的 基因缺陷（点突变、缺失、插入、剪接点 错误、启动子缺陷）将被引入到正常克隆中 重现患者突变。 转染至COS-1 受体阴性细胞有望复制 鉴定出具有与分离的相同表型的突变蛋白 病人。 体外产生的缺陷蛋白将是 测试功能（激素结合、DNA 结合、24- 羟化酶激活）以深入了解关键 受体的重要氨基酸。 蛋白质 DNA 接触点 将通过烷基化干扰实验确定 硫酸二甲酯和乙基亚硝基脲。 同样，突变 将研究涉及激素结合亲和力的定义 分子术语类固醇结合口袋和关键氨基 参与配体与受体相互作用的酸。