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The DNA segment corresponding to nucleotides 661 to 932 or 661 to 797 (Fig. 1) as subcloned downstream of the rat type 1 deiodinase coding region in the mammalian expression vector pUHD10 [M. Gossen and H. Bujard, Proc. Natl. Acad. Sci. U.S.A. 89, 5547 (1992)]. Plasmids containing the wild-type deiodinase SECIS element or a nonfunctional mutant of this element downstream of the deiodinase coding region served as positive and negative controls, respectively. The plasmids with deiodinase sequences were transfected, together with a control plasmid that expresses human growth hormone, into human embryonic kidney 293 cells. Deiodinase activity, in counts per minute of iodine released per microliter of transfected cell sonicate, was normalized to units of human growth hormone in 100 μl of medium, as described [G. W. Martin III, J. W. Harney, M. J. Berry, RNA 2, 171 (1996)].
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The DNA segment corresponding to nucleotides 661 to 932 or 661 to 797 (Fig. 1) as subcloned downstream of the rat type 1 deiodinase coding region in the mammalian expression vector pUHD10 [M. Gossen and H. Bujard, Proc. Natl. Acad. Sci. U.S.A. 89, 5547 (1992)]. Plasmids containing the wild-type deiodinase SECIS element or a nonfunctional mutant of this element downstream of the deiodinase coding region served as positive and negative controls, respectively. The plasmids with deiodinase sequences were transfected, together with a control plasmid that expresses human growth hormone, into human embryonic kidney 293 cells. Deiodinase activity, in counts per minute of iodine released per microliter of transfected cell sonicate, was normalized to units of human growth hormone in 100 μl of medium, as described [G. W. Martin III, J. W. Harney, M. J. Berry, RNA 2, 171 (1996)].
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Transfection procedures: HeLa cells were co-transfected with 1 μg of cytomegalovirus (CMV)-β-Gal (Stratagene) and 5 μg of pCl vector (Promega), pCl-P35 (18), or pCl-MC066L-A mixed with lipofectin. Thirty hours after transfection, the cells were treated with UV (312 nm) light for 15 s at room temperature or continuously with 100 μM hydrogen peroxide or cycloheximide (1 μg/ml; Sigma) and TNF-α (10 ng/ml; Boehringer Mannheim) or cycloheximide and anti-Fas (1.25 μg/ml; CH-11 antibody. Kamiya Biomedical, Seattle, WA). Twelve hours later, cells were fixed, stained with X-Gal (Promega), and examined microscopically. The control wells contained 100 to 200 flat, blue cells per microscopic field. The number of flat, blue cells in six fields of the untreated well of each transfectant was compared to the number of flat, blue cells in six fields of the treated well, and the percent viability was determined. The HaCaT cells were co-transfected with 0.33 μg of CMV-β-Gal and 1.67 μg of pCl, pCl-P35, or pCl-MC066L-A mixed with lipofectamine reagent (Life Technologies). Thirty hours after transfection, the cells were treated for 30 s with UV (312 nm) light or 10 μM hydrogen peroxide. Cells were fixed, stained, and scored for viability as described for HeLa cells.
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We thank D. Hatfield for suggestions regarding selenium labeling of proteins and J. Bertin and J. Cohen for critically reading the manuscript, providing pCl-P35, and offering advice regarding transfections and viability assays. Supported in part by NIH grant DK47320 (M.J.B.).
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