Figure 1.

Identification of cis-regions for Cu responsiveness. A, Schematic diagram of constructs. A plasmid construct containing the 5′ flanking region (solid line), 5′ untranslated region (black box), and the coding region (gray box; three amino acids) of FeSOD connected to the GUS gene was introduced into P. patens subsp. patens by polyethylene glycol-mediated transformation. Nucleotide positions are indicated in base pairs from the transcription initiation site. Tnos, Nopaline synthase terminator. B, GUS activities of the transgenic moss plants treated with or without CuSO₄. Left, The repression of the GUS activities by Cu is indicated as the relative value. The bars represent the GUS activities of transgenic moss plants cultured in the presence of 2.0 μm CuSO₄. The GUS activities of the transgenic plants cultured in the absence of CuSO₄ were arbitrarily set to 1.0 (dotted line). For each construct, at least three independent transgenic plants were examined. The bars indicate ± sd. Right, The average GUS activities are expressed in nanomoles of 4-methylumbelliferone (4-MU) produced per milligram of extract protein per minute. The GUS activities of transgenic moss plants containing the promoterless-GUS construct were below 2 × 10⁻² nmol 4-MU min⁻¹ mg⁻¹ and were not affected by Cu (data not shown). C, RT-PCR analysis of GUS gene expression in transgenic moss plants cultured with (+) or without (−) CuSO₄. After amplification with specific primers, the products were detected by DNA gel-blot hybridization. GAPDH was used as an internal control of RT-PCR.