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THYROID HORMONE RECEPTORS IN DEVELOPMENT

发育中的甲状腺激素受体

基本信息

批准号：
2889018
负责人：
VALERIE Anne GALTON
金额：
$ 21.5万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-05-01 至 2001-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2889018
关键词：
Rana alternatives to animals in research developmental genetics enzyme inhibitors gene expression genetic transcription hormone receptor hormone regulation /control mechanism metamorphosis molecular cloning nucleic acid probes polymerase chain reaction thyroid hormone binding protein thyroid hormones

项目摘要

The long range goal of this research program is to elucidate the mechanisms of action of thyroid hormone (TH) during development using the amphibian Rana catesbeiana (RC) as the animal model. TH is essential for all aspects of development in this species. The proposed research will determine 1) the role that TH receptors (TRs) and deiodinases play in the coordination of metamorphosis and 2) why the TRbeta gene is responsive to TH in the tadpole but not in the adult frog.. The first three SAs will test the following hypothesis: an important feature in the coordination of metamorphosis is the control by TH per se of the expression of four genes whose products are involved in determining the level of TH action in the cell; t he genes for the TRalpha, TRbeta, 5-Deiodinase (5D) and 5'- Deiodinase (5'D). Thus the relationships between the level of expression of these genes and the ability of a tissue to exhibit its TH-dependent developmental characteristics will be investigated. Studies will determine, in two tissues (leg and tail) which metamorphose at different stages of the life-cycle an thus at markedly different plasma TH levels, 1) whether these genes are expressed in tadpoles in the absence of TH and 2) the effects of thyroid status on the expression of these genes during pre- and prometamorphosis and at climax. Next, the role of 5D and 5'D in regulating tissue sensitivity to TH will be examined by inhibiting the activity of these enzymes in tadpoles undergoing TH-induced metamorphosis. Then the cell-specific patterns of expression and regulation of the TR, 5D and 5'D genes will be defined using an RT in situ PCR technique. SA #5 will test a second hypothesis: the loss of TH-responsiveness of the TRbeta gene following metamorphosis is due to either the absence of a factor(s) essential for the response or the presence of a factor)s) that interferes with the response. The presence of such a factor in frog or tadpole nuclear extracts will first be demonstrated using constructs containing the TRbeta promoter and a X. laevis oocyte nuclei expression system. Then, electromobility shift assays, DNA footprinting and Far-western blotting techniques will be used to ascertain if more than one factor is involved, the location of the DNA/protein binding sites and whether binding to DNA is direct or indirect. Time permitting we will clone and characterize the cDNAs for the protein(s). Thyroid dysfunction during human development results in brain damage, the most severe manifestation of which is cretinism. Yet the mechanisms and regulation of TH action during this period have received relatively little attention, due in part to the difficulties of conducting studies in fetal mammals. Thr proposed studies will utilize an eminently suitable animal model to investigate these important biological processes.

这项研究计划的长期目标是阐明其机制。两栖动物发育过程中甲状腺激素(TH)的作用以卡氏林蛙(Rana catesbeiana，RC)为动物模型。这对所有方面都是必不可少的在这个物种中的发育。拟议的研究将确定1) TH受体和脱碘酶在协调中的作用和2)为什么TRbeta基因对TH有反应蝌蚪，但不在成年青蛙身上..前三个SA将测试下面的假设：协调的一个重要特征变态本身是由四个基因的表达控制的谁的产品参与确定该行动的级别细胞；TRAlpha、Trbeta、5-脱碘酶(5D)和5‘-脱碘酶基因脱碘酶(5‘D)。因此，表达水平之间的关系这些基因和组织表现出其TH依赖的能力将调查其发育特点。研究将会确定在两个组织(腿和尾巴)中哪个组织在不同的变态生命周期的不同阶段因此具有明显不同的血浆TH水平，1) 这些基因是否在缺乏TH和2的情况下在蝌蚪中表达) 甲状腺状况对上述基因表达的影响和变态前，并达到高潮。接下来，5D和5‘D在调节组织对TH的敏感性将通过抑制 TH诱导变态的蝌蚪体内这些酶的活性。然后是tr，5D的细胞特异性表达和调控模式 5‘D基因的定义将使用RT原位聚合酶链式反应技术。SA#5 将检验第二个假设：TRbeta的TH反应性的丧失变形后的基因要么是由于缺乏一个因素(S) 对某一因素的反应或存在至关重要S)干扰的你的回应。这种因子在青蛙或蝌蚪身上的存在首先将使用包含 Trbeta启动子和乳突X.laevis卵母细胞核表达系统。然后, 电迁移率改变分析、DNA足迹和远蛋白质印迹将使用技术来确定是否涉及多个因素， DNA/蛋白质结合位点的位置以及与DNA的结合是否直接或间接。如果时间允许，我们将克隆和描述蛋白质的DNA(S)。人类发育过程中的甲状腺功能障碍会导致大脑损伤，它最严重的表现是克汀病。然而，这些机制和在此期间，对此行动的监管收到的相对较少注意，部分原因是对胎儿进行研究的困难哺乳动物。建议的研究将利用一种非常合适的动物模型来研究这些重要的生物过程。