ANDROGEN RECEPTORS IN THE TFM SYNDROME

本提案的目标是通过以下方式了解生化过程 并为临床提供分子基础。 操纵这些器官的异常生长。在过去三年里， 我们已经分离并生产了两个非常重要的工具[完整的 人/大鼠雄激素受体(AR)cDNA和单/多特异性抗体 为研究雄激素作用的分子机制奠定了基础。我们也 在雄激素不敏感的患者中发现AR的几个突变 睾丸女性化(TFM)综合征。基于这些发现，我们 建议检验四个假设： 假设I：AR缺失或缺陷可能导致TFM综合征。为了测试 在这个假设下，我们计划对AR基因的编码区进行测序 人和患有TFM综合征的小鼠。结果可能会让我们检测到 DNA和RNA水平的AR异常。 假设II：一些有缺陷的AR可能会阻止AR的易位 胞质到胞核。为了检验这一假设，我们计划应用 用微波固定免疫细胞学方法定位培养的AR TFM患者的成纤维细胞。假设I和II的结果可能 使我们能够确定对原子核有贡献的序列 AR的本地化流程。 假说III：TFM小鼠的前列腺腺可能被重新诱导并 将正常AR基因导入尿生殖窦(UGS)而形成 TFM小鼠。为了验证这一假设，来自TFM小鼠的UGS将 含正常AR基因的逆转录病毒转染及体内培养 以检查是否诱导了前列腺体。结果可能是 证明间充质(间质)中正常AR的必要性 前列腺发育。 假设四：受精卵可纠正小鼠的TFM综合征 显微注射(转基因实验)正常AR基因。为了测试这一点 假设正常AR基因将被克隆到表达载体中 含有非常强的启动子(延伸因子启动子)和 显微注射入TFM小鼠受精卵。这一结果可能会为 TFM综合征基因治疗的可能性。 上述研究将使评估AR在TFM中的作用成为可能 并最终可能导致新的诊断方法的发展 TFM综合征的临床治疗方法和新思路。

The goals of this proposal are to understand the biochemical procedures by which at target organs and to provide a molecular basis for the clinical manipulation of abnormal growth of these organs. For the past three years, we have isolated and produced two very important tools [full-length of human/rat androgen receptor (AR) cDNAs and mono-/poly- specific antibodies to AR] for the study of molecular mechanism of androgen action. We also found several mutations of AR in patients with androgen insensitivity or testicular feminization (Tfm) syndrome. Based on these discoveries, we propose to test four hypotheses: Hypothesis I: Absent or defective AR may cause the Tfm syndrome. To test this hypothesis, we plan to sequence the coding region of AR gene from human and mice with Tfm syndrome. The results may allow us to detect the abnormal AR at DNA and RNA levels. Hypothesis II: Some defective AR may prevent the translocation of AR from cytoplasm to nuclei. To test this hypothesis, we plan to apply the immunocytology with microwave-fixed method to locate AR in cultured fibroblasts from Tfm patients. The results of hypotheses I and II may allow us to determine the sequences contributing to the nuclear localization process for the AR. Hypothesis III: The prostatic glands from Tfm mice may be reinduced and developed by introduction of normal AR cDNA into the urogenital sinus (UGS) of Tfm mice. To test this hypothesis, the UGS from Tfm mice will be transfected with retrovirus containing normal AR cDNA and cultured in vivo to be examined whether prostatic glands are induced. The results may demonstrate the necessary of normal AR in the mesenchyme (stroma) for the prostate development. Hypothesis IV: The Tfm syndrome in mice may be corrected by zygote microinjection (transgenic experiment) of normal AR cDNA. To test this hypothesis, the normal AR cDNA will be cloned into expression vector containing a very strong promoter (elongation factor promoter) and microinjected into the zygote of Tfm mice. The results may provide the possibility of gene therapy for the Tfm syndrome. The above studies will permit an evaluation of the role of AR in Tfm syndrome and may eventually lead to the development of new diagnostic methods and new ideas in clinical treatment of Tfm syndrome.