Amplification and hormone-regulated expression of a mouse mammary tumor virus-Eco gpt fusion plasmid in mouse 3T6 cells.
Mouse 3T6 cells were transformed with a chimeric DNA plasmid, pSVMgpt, in which the mouse mammary tumor virus (MMTV) promoter was fused to the Escherichia coli gene encoding xanthine-guanine phosphoribosyl transferase (Eco gpt). The transformants exhibited glucocorticoid-inducible expression of Eco gpt. With limiting xanthine concentrations, conditions were established in which cell growth became hormone dependent. Cells selected for their ability to grow in limiting concentrations of both xanthine and glucocorticoids contained amplified levels of Eco gpt DNA, and expression of Eco gpt remained glucocorticoid inducible in these amplified cells. Thus, amplification of the MMTV promoter region in itself did not abolish hormonal responsiveness of a gene. In addition to increased levels of Eco gpt DNA, some of the selected cells also exhibited increased levels (two- to threefold) of glucocorticoid receptors. Lastly, we found that excessive expression of Eco gpt is toxic to 3T6 cells; by maintaining low hormone levels and, therefore, low levels of expression, we were able to select cells with amplified Eco gpt. Thus, the MMTV promoter may be of general utility in expressing genes whose products may be lethal if they are produced in excessive quantities.
D000818:Animals; D002478:Cells, Cultured; D005784:Gene Amplification; D005786:Gene Expression Regulation / Q000187:drug effects*; D005938:Glucocorticoids / Q000494:pharmacology*; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D051379:Mice; D010430:Pentosyltransferases / Q000235:genetics; D010957:Plasmids; D012333:RNA, Messenger / Q000096:biosynthesis; D011965:Receptors, Glucocorticoid / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid
D005938:Glucocorticoids; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase