FIG. 6.

Inhibition of MT Mtn and Mto transcription by RNAi specific for dMTF-1. (A) dsRNA of dMTF-1 inhibits heavy metal-induced transcription of the Mtn reporter gene. The Mtn promoter-reporter gene construct was transfected into Drosophila S2 cells, and various amounts (ranging from 0.3 to 2.7 μg per 5 ml of culture medium) of dsRNA of either dMTF-1 or lacZ were included. Cytoplasmic RNA was isolated after 24 h of heavy metal induction (0.5 to 2 mM ZnCl₂ as indicated, 60 μM CdCl₂, or 500 μM CuSO₄) and the reporter gene transcripts were quantified by S1 nuclease mapping. Lanes 1 and 13, basal expression level of transfected Mtn reporter; lanes 2 to 4, 6 to 8, and 10 to 12, induction by zinc; lanes 14 to 20, induction by cadmium; lanes 21 to 27, induction by copper. LacZ dsRNA was included in lanes 5 to 8, 15 to 17, and 22 to 24 (lanes 5 to 8, 1 μg; lanes 15 to 17 and 22 to 24, 0.3 to 2.7 μg as indicated). dMTF-1 dsRNA was added in the transfections shown in lanes 9 to 12, 18 to 20, and 25 to 27 (lanes 9 to 12, 1 μg; lanes 18 to 20 and 25 to 27, 0.3 to 2.7 μg as indicated). −, control. F, free probe. (B) Inhibition of heavy metal (Cd²⁺ or Cu²⁺) induction in the presence of cotransfected (cotransf.) dMTF-1 by dsRNA of dMTF-1. The Mtn or Mto reporter construct was introduced into S2 cells together with a dMTF-1 expression vector, and results very similar to those for Mtn reporter transfection alone (described for panel A) were obtained. Lanes 1 and 10, basal transcription of the Mtn and Mto reporters, respectively. Lanes 2, 5, 8, 11, 14, and 17, cadmium induction (60 μM CdCl₂, 24 h); lanes 3, 6, 9, 12, 15, and 18, copper induction (500 μM CuSO₄, 24 h). Lanes 4 to 6 and 7 to 9, cotransfection with 10 μg of lacZ dsRNA and dMTF-1 dsRNA, respectively; lanes 13 to 15 and 16 to 18, cotransfection with 5 μg of LacZ dsRNA and dMTF-1 dsRNA, respectively. −, control. (C) dsRNA of dMTF-1 inhibits endogenous MT gene (Mtn) transcription. Various amounts of dsRNA were transfected into S2 cells. Endogenous Mtn transcripts were detected by S1 mapping after 24 h of induction with 60 μM CdCl₂ or 500 μM CuSO₄. F, free S1 probe for detecting endogenous Drosophila Mtn transcripts. Lanes: 1, basal level of endogenous Mtn transcripts; 2 and 9, after Cd²⁺ and Cu²⁺ induction, respectively; 3 to 5 and 6 to 8, effects on cadmium induction of dsRNA corresponding to lacZ and dMTF-1, respectively; 10 to 12 and 13 to 15, effects on copper induction of dsRNA corresponding to lacZ and dMTF-1, respectively. Endogenous Mtn transcripts following Cd²⁺ or Cu²⁺ induction were reduced to 20 and 47%, respectively, of the control level in the presence of dMTF-1 dsRNA, whereas lacZ dsRNA was not inhibitory.