Objective: To conduct clinical, genetic, and molecular diagnostics of two sisters with typical symptoms of complete androgen insensitivity syndrome. Design: Case report. Setting: Research laboratory at a university of...Objective: To conduct clinical, genetic, and molecular diagnostics of two sisters with typical symptoms of complete androgen insensitivity syndrome. Design: Case report. Setting: Research laboratory at a university of medical science. Patient(s): Two patients with 46,XY karyotype and a female phenotype were diagnosed because of primary amenorrhea. Their sister with 46,XX karyotype, her daughter, and five other family members including their mother also were examined. Intervention(s): Orchiectomy, estrogen substitution therapy. Main Outcome Measure(s): Cancer prophylaxis. Result(s): Multiple-temperature single-stranded conformation polymorphism and sequence analyses of the androgen receptor gene(AR) revealed a c.C2812T transition in exon 7 in the two sisters. Their mother and the third sister(46,XX) were carriers of the same mutation. This mutation, which previously had never been reported, resulted in Pro817Leu substitution in the ligand-binding domain of the androgen. Computer simulation of structural changes generated by Pro817Leu substitution revealed appreciable conformational changes in the region responsible for dimerization of the receptor. Conclusion(s): The novel c.C2812T transition that might impair dimerization of the receptor is responsible for the clinical symptoms of complete androgen insensitivity syndrome in the affected individuals. Molecular analysis of AR proved to be very useful for genetic counseling of the unaffected sister, who was a carrier of the same mutation.展开更多
目的系统构建雄激素受体(AR)野生型全长和4个功能域截短体的原核表达质粒,Western blot与凝胶迁移(EMSA)实验鉴定各融合蛋白及其功能。方法基于pGEX-4T-1载体,构建AR全长及各功能域的谷胱甘肽巯基转移酶(GST)融合表达重组质粒。应用异丙...目的系统构建雄激素受体(AR)野生型全长和4个功能域截短体的原核表达质粒,Western blot与凝胶迁移(EMSA)实验鉴定各融合蛋白及其功能。方法基于pGEX-4T-1载体,构建AR全长及各功能域的谷胱甘肽巯基转移酶(GST)融合表达重组质粒。应用异丙基-β-D-硫代半乳糖苷(IPTG)对重组质粒进行诱导表达,确定各重组蛋白的最佳参数。分别用AR内源性抗体和GST标签抗体进行Western blot鉴定,分析诱导前、诱导后细菌裂解液中的目的蛋白,并利用谷胱甘肽琼脂糖树脂进一步纯化。将纯化后蛋白与病毒荧光探针进行孵育,复合物于非变性凝胶中电泳检测纯化蛋白功能。结果成功构建了AR全长和3个功能域截短体的原核表达质粒。PCR和双酶切鉴定均显示在各插入片段大小相符处出现阳性条带,且测序结果与NCBI GenBank标准株比对一致。其中,96 ku GST-AR-NTD+DBD与86 ku GST-AR-NTD两个融合蛋白得以成功表达及后续纯化。纯化蛋白可与病毒基因组DNA直接结合。结论来源同一基因序列的AR截短体重组质粒原核表达条件不同,纯化后的AR蛋白可用于深入了解AR各功能域与其他分子间的直接相互作用机制。展开更多
文摘Objective: To conduct clinical, genetic, and molecular diagnostics of two sisters with typical symptoms of complete androgen insensitivity syndrome. Design: Case report. Setting: Research laboratory at a university of medical science. Patient(s): Two patients with 46,XY karyotype and a female phenotype were diagnosed because of primary amenorrhea. Their sister with 46,XX karyotype, her daughter, and five other family members including their mother also were examined. Intervention(s): Orchiectomy, estrogen substitution therapy. Main Outcome Measure(s): Cancer prophylaxis. Result(s): Multiple-temperature single-stranded conformation polymorphism and sequence analyses of the androgen receptor gene(AR) revealed a c.C2812T transition in exon 7 in the two sisters. Their mother and the third sister(46,XX) were carriers of the same mutation. This mutation, which previously had never been reported, resulted in Pro817Leu substitution in the ligand-binding domain of the androgen. Computer simulation of structural changes generated by Pro817Leu substitution revealed appreciable conformational changes in the region responsible for dimerization of the receptor. Conclusion(s): The novel c.C2812T transition that might impair dimerization of the receptor is responsible for the clinical symptoms of complete androgen insensitivity syndrome in the affected individuals. Molecular analysis of AR proved to be very useful for genetic counseling of the unaffected sister, who was a carrier of the same mutation.
文摘目的系统构建雄激素受体(AR)野生型全长和4个功能域截短体的原核表达质粒,Western blot与凝胶迁移(EMSA)实验鉴定各融合蛋白及其功能。方法基于pGEX-4T-1载体,构建AR全长及各功能域的谷胱甘肽巯基转移酶(GST)融合表达重组质粒。应用异丙基-β-D-硫代半乳糖苷(IPTG)对重组质粒进行诱导表达,确定各重组蛋白的最佳参数。分别用AR内源性抗体和GST标签抗体进行Western blot鉴定,分析诱导前、诱导后细菌裂解液中的目的蛋白,并利用谷胱甘肽琼脂糖树脂进一步纯化。将纯化后蛋白与病毒荧光探针进行孵育,复合物于非变性凝胶中电泳检测纯化蛋白功能。结果成功构建了AR全长和3个功能域截短体的原核表达质粒。PCR和双酶切鉴定均显示在各插入片段大小相符处出现阳性条带,且测序结果与NCBI GenBank标准株比对一致。其中,96 ku GST-AR-NTD+DBD与86 ku GST-AR-NTD两个融合蛋白得以成功表达及后续纯化。纯化蛋白可与病毒基因组DNA直接结合。结论来源同一基因序列的AR截短体重组质粒原核表达条件不同,纯化后的AR蛋白可用于深入了解AR各功能域与其他分子间的直接相互作用机制。
文摘目的研究六价铬对雄激素受体(AR)转录活性的影响及其与小泛素类似修饰物特异性蛋白酶(SENP)家族分子的相关性。方法(1)293T细胞经K_(2)CrO_(4)0.25,0.5,1.0,2.0,4.0,8.0和16.0μmol·L^(-1)处理24 h,LNCa P细胞经K_(2)CrO_(4)0.25,0.5,1.0,2.0,4.0,10.0和20.0μmol·L^(-1)处理24 h,用MTT法检测细胞存活率并计算半数抑制浓度(IC_(50))值;(2)将带有荧光素酶报告基因的表达雄激素反应元件的质粒和表达AR的质粒共转染293T细胞,将带有荧光素酶报告基因的表达雄激素反应元件的质粒转染LNCa P细胞,分别用K_(2)CrO_(4)0.25,0.5,1.0,2.0,4.0,8.0和16.0μmol·L^(-1)处理24 h,用双荧光素酶报告基因检测系统分析外源性和内源性AR的转录活性。(3)取对数生长期LNCa P细胞,以K_(2)CrO_(4)0.5,2.0和8.0μmol·L^(-1)处理24 h,用逆转录PCR法检测AR和SENP家族分子SENP1,SENP2,SENP3,SENP5,SENP6,SENP7和SENP8 m RNA水平,Western印迹法检测AR,SENP1和SENP3蛋白水平。结果(1)与细胞对照组相比,K_(2)CrO_(4)浓度≥1.0μmol·L^(-1)时293T细胞存活率显著降低(P<0.01),K_(2)CrO_(4)浓度≥2.0μmol·L^(-1)时LNCa P细胞存活率明显降低(P<0.01)。(2)与细胞对照组相比,K_(2)CrO_(4)浓度≥1.0μmol·L^(-1)时293T细胞中外源性AR的转录活性显著增强(P<0.05),K_(2)CrO_(4)4.0和8.0μmol·L^(-1)明显增强LNCa P细胞中内源性AR转录活性(P<0.05)。(3)与细胞对照组相比,K_(2)CrO_(4)0.5,2.0和8.0μmol·L^(-1)均不改变LNCa P细胞中AR m RNA和蛋白水平,而K_(2)CrO_(4)2.0和8.0μmol·L^(-1)组SENP1 m RNA水平显著增加(P<0.05),K_(2)CrO_(4)0.5,2.0和8.0μmol·L^(-1)组SENP3 m RNA水平显著增加(P<0.05)。与细胞对照组相比,K_(2)CrO_(4)8.0μmol·L^(-1)组SENP1蛋白水平显著增加(P<0.01),K_(2)CrO_(4)2.0和8.0μmol·L^(-1)组SENP3蛋白水平显著增加(P<0.05)。结论K_(2)CrO_(4)可能通过上调SENP1和SENP3表达而增强AR转录活性。