Magnuson M A, Dozin B, Nikodem V M
J Biol Chem. 1985 May 25;260(10):5906-12.
A plasmid cDNA library was constructed using poly(A+) RNA isolated from the livers of rats treated with 3,5,3'-triiodothyronine (T3) and fed a high carbohydrate diet. This library was screened by differential colony hybridization with [32P]cDNA probes made from hypothyroid and hyperthyroid rat liver poly(A+) RNA to obtain clones representing T3-inducible mRNAs. Using plasmid cDNAs to 4 different T3-inducible mRNAs, we have studied by hybridization assay the responses of these mRNAs to different thyroidal steady states and to a high carbohydrate diet. The fold of induction (hypothyroid to hyperthyroid) varied from about 4.0 (mRNA 5-8D) to 13.2 (mRNA 4-12B). The linearity of response with regard to nuclear receptor occupancy was estimated by assessing the relative mRNA levels in a euthyroid state. Three of the mRNAs demonstrated nonlinear responses with the largest portion of the induction occurring in the euthyroid to hyperthyroid transition. An induction by the high carbohydrate diet was clearly seen for only one mRNA (5-8D) suggesting that these two pathways of induction are independent. In a study of the response kinetics of each mRNA to a nuclear receptor saturating dose of T3 in hypothyroid animals, an increase was seen within 4 h (the earliest time point examined) for one of the mRNAs. The other 3 mRNAs did not increase significantly until 8 h after the T3 dose. Northern analysis showed a single mRNA corresponding to each of these 4 clones with sizes ranging from about 1375 to 7600 bases. Two mRNAs (5-9E and 4-12B) were shown by hybrid-selected translation to code for proteins of molecular mass of about 27 and 46 kDa, respectively. The availability of several different cDNA probes to T3 responsive liver mRNAs should facilitate future studies on the mechanism of action of this hormone.
使用从用3,5,3'-三碘甲状腺原氨酸（T3）处理并饲喂高碳水化合物饮食的大鼠肝脏中分离的聚腺苷酸（poly(A+)）RNA构建了质粒cDNA文库。该文库通过与由甲状腺功能减退和甲状腺功能亢进大鼠肝脏聚腺苷酸（poly(A+)）RNA制备的[32P]cDNA探针进行差异菌落杂交来筛选，以获得代表T3诱导型mRNA的克隆。使用针对4种不同T3诱导型mRNA的质粒cDNA，我们通过杂交测定研究了这些mRNA对不同甲状腺稳态和高碳水化合物饮食的反应。诱导倍数（甲状腺功能减退到甲状腺功能亢进）从约4.0（mRNA 5-8D）到13.2（mRNA 4-12B）不等。通过评估甲状腺功能正常状态下的相对mRNA水平来估计与核受体占有率相关的反应线性。其中三种mRNA表现出非线性反应，最大部分的诱导发生在甲状腺功能正常到甲状腺功能亢进的转变过程中。仅一种mRNA（5-8D）明显可见高碳水化合物饮食诱导，这表明这两种诱导途径是独立的。在对甲状腺功能减退动物中每种mRNA对核受体饱和剂量T3的反应动力学研究中，其中一种mRNA在4小时内（检查的最早时间点）出现增加。其他3种mRNA直到T3给药后8小时才显著增加。Northern分析显示与这4个克隆中的每一个相对应的单个mRNA，大小范围约为1375至7600个碱基。通过杂交选择翻译显示两种mRNA（5-9E和4-12B）分别编码分子量约为27和46 kDa的蛋白质。几种不同的针对T3反应性肝脏mRNA的cDNA探针的可用性应有助于未来对该激素作用机制的研究。
