Fig. 1. Chicken TRα (c-ErbA) and v-ErbA can be expressed in the Xenopus oocyte and can repress transcription driven by the TRβA gene promoter. (A) In vivo synthesis of chicken TRα and v-ErbA. Oocytes were left uninjected (lane 1), or injected with 5 ng of Xenopus RXRα mRNA along with 5 ng of mRNA for Xenopus TRβ (lane 2), chicken TRα (lane 3) or v-ErbA (lane 4) mRNA. The oocytes were cultured in the presence of [³⁵S]methionine for 10 h, and oocyte protein extract was prepared as described in Materials and methods. Proteins synthesized in the oocyte during the incubation period were visualized by SDS–PAGE and autoradiography. The positions of the proteins derived from the injected mRNAs are indicated to the right of the autoradiograph; molecular weight marker size is indicated to the left. (B) Transcriptional regulation by chicken TRα and v-ErbA. Groups of 20 oocytes were injected into the cytoplasm with 5 ng of Xenopus RXRα mRNA (lanes 5–10) along with 5 ng of Xenopus TRβ (lanes 5 and 6), chicken TRα (lanes 7 and 8) or v-ErbA (lanes 9 and 10) mRNA; the oocytes in lanes 1–4 were not injected with mRNA. After a 12 h incubation, the oocytes were left uninjected (lanes 1 and 2), or injected into the nucleus with 1 ng (lanes 3–10) of double-stranded reporter plasmid DNA (pTRβA); the oocytes were then cultured for an additional 16 h in the absence (–) or presence (+) of 100 nM thyroid hormone (T₃), following which total RNA was extracted and levels of H4 and TRβA mRNA were measured by primer extension as described in Materials and methods. The primer against histone H4 mRNA that is stored in the oocyte was included in all reactions to serve as a loading control. Transcriptional activity (txn) was quantitated by normalizing the signal for TRβA in each sample against that for H4 in that sample, and expressing the result in arbitrary units where 1 unit = transcriptional activity in the absence of injected mRNA (the average of signals from lanes 3 and 4).