EFFECT OF IN-VIVO ESTRADIOL TREATMENT ON CELL-FREE TRANSCRIPTION IN TROUT LIVER NUCLEAR EXTRACTS

In order to perform later studies on the transcriptional regulation of hormone-dependent genes in fish liver, we firstly examined the potent ial of trout liver nuclear extracts in a cell-free transcription syste m. As reporter genes, we used DNA sequences without G (G-free cassette s) under the control of three promoters derived from the 5' flanking s equence of the Xenopus vitellogenin B1 gene; two of them were responsi ve to the oestrogen receptor (ER) through oestrogen responsive element s (ERE). Maximal transcriptional activity was obtained within a range of 30-130 mu g protein per extract depending on the extract preparatio n. Transcription was maximal in reactions carried out at 25 degrees C. Similar transcriptional activities for the three promoters were obser ved when transcription was performed in extracts from untreated male t rout. In contrast, we observed a 4.5- to 6-fold increase in the transc ription with ERE-containing promoters in comparison with that with the minimal promoter bearing only a TATA box when extracts from oestradio l-treated male trout were used. This effect was correlated with the in crease in the nuclear ER concentration induced by in vivo hormonal tre atment. This enhanced transcription was specifically inhibited by the addition of a 25- to 100-fold excess of ERE oligonucleotide competitor . These data demonstrated, therefore, that transcription was ERE-depen dent in this system and suggest strongly that it was mediated by the t rout ER. Addition of oestradiol or the anti-oestrogens hydroxytamoxife n or ICI 164384 had no effect on the transcriptional activity of the t wo ERE-containing promoters, indicating that transcription was hormone -independent in trout liver nuclear extracts.