| Abstract: | 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. |
