MECHANISMS OF THYROID HORMONE ACTION IN DEVELOPMENT

描述(改编自申请人的摘要)：长期目标 这项研究项目是为了阐明细胞过程中涉及的 甲状腺激素的作用。具体地说，这项建议涉及 脊椎动物发育过程中调节TH代谢的分子机制 物种。在胎儿中，就像在成人中一样，特定的碘甲状腺原氨酸脱碘酶 将甲状腺激素和其他甲状腺激素转化为活性和非活性 代谢物。最近，一个编码I型大鼠肝脏5‘- 分离并测定了脱碘酶(5‘D)的序列。因此，研究 在分子水平上，旨在研究 在发育过程中受到调节的脱碘酶现在可以进行了。二 将使用动物模型：老鼠，它传统上被用作 研究哺乳动物TH经济的实验模型系统，以及青蛙， 它的主要优势是其幼虫形式是自由游泳和 因此很容易受到实验操作的影响。具体目标1是 分离和鉴定蝌蚪中存在的5‘D和5D的cDNA。在……里面 特定目标2和#4这些蝌蚪和大鼠的cDNA将被用作分子 表征脱碘酶翻译前调控的几种方法 不同发育阶段的酶通过量化mRNA水平和通过 原位杂交。TH等因素在5‘-D调节中的作用 将在大鼠胚胎和蝌蚪中研究5D mRNA的水平 (特异性靶点3)和培养的胎鼠肝细胞和蝌蚪尾巴 提示(具体目标5)。TH对5‘-D和5D基因转录的影响 然后使用转录分析(特定目标6)来确定。 具体目标#7是从青蛙和大鼠基因组文库中分离基因 它们编码5‘D和5D及其相关的DNA侧翼区。

DESCRIPTION (Adapted from the applicant's abstract): The long term goal of this research project is to elucidate the cellular processes involved in thyroid hormone (TH) action. Specifically, this proposal addresses the molecular mechanisms which regulate TH metabolism in developing vertebrate species. In the fetus, as in the adult, specific iodothyronine deiodinases convert thyroxine and other thyroid hormones into active and inactive metabolites. Recently, a cDNA which encodes the type I rat liver 5'- deiodinase (5'D) was isolated and its sequence determined. Thus, studies at a molecular level aimed at investigating the mechanisms by which deiodinases are regulated during development can now be undertaken. Two animal models will be employed: the rat, which is traditionally used as an experimental model system for studying mammalian TH economy, and the frog, which offers the major advantage that its larval form is free swimming and thus readily amenable to experimental manipulation. Specific Aim 1 is to isolate and characterize cDNAs for the 5'D and 5D present in tadpoles. In Specific Aim 2 and #4 these tadpole and rat cDNAs will be used as molecular probes to characterize the pretranslational regulation of the deiodinase enzymes at various stages of development by quantitating mRNA levels and by in situ hybridization. The role of TH and other factors in regulating 5'D and 5D mRNA levels will be investigated in rat fetuses and tadpoles (Specific Aim 3) and in cultured fetal rat hepatocytes and tadpole tail tips (Specific Aim 5). The effect of TH on 5'D and 5D gene transcription will then be determined using a transcription assay (Specific Aim 6). Specific Aim #7 is to isolate from frog and rat genomic libraries the genes which encode the 5'D and 5D and their associated DNA flanking regions.