Wan M, Heuchel R, Radtke F, Hunziker P E, Kägi J H
Biochemisches Institut der Universität Zürich, Switzerland.
Experientia. 1995 Jun 14;51(6):606-11. doi: 10.1007/BF02128753.
We explored the molecular genetics underlying the massive induction of isoMTs by Zn2+ or Cd2+ in metal tolerant rabbit kidney (RK-13) sub-line cells, using band shift assays and Southern blotting analysis. In sub-line cells accommodated to intermediate metal concentrations (100 microM Zn2+; 1-20 microM Cd2+) evidence suggested that the increase in the capacity for isoMT synthesis is brought about by an increased binding activity of the nuclear transcription factors MTF-1 and Sp1. Using quantitative band shift analysis with a mouse MRE-d oligonucleotide probe, the binding of both transcription factors was found to be enhanced two to three times over the binding activity measured in the unexposed parental RK-13 cells. Their increase in binding activity is probably the cause of the overexpression of MT genes and the development of metal tolerance in these cells. In cells tolerant to the highest concentrations of metal the analysis of Southern blot signals revealed MT gene amplification to be the most probable cause of the increased MT production. Thus, in cells of sub-lines growing in the presence of 350 microM Zn2+, two of the isoMT genes were coordinately triplicated and in cells tolerant to 150 microM Cd2+ one isoMT gene was amplified two-fold.
我们利用凝胶迁移试验和Southern印迹分析，探究了金属耐受兔肾（RK - 13）亚系细胞中锌离子（Zn2+）或镉离子（Cd2+）大量诱导金属硫蛋白异构体（isoMTs）的分子遗传学机制。在适应中等金属浓度（100微摩尔/升Zn2+；1 - 20微摩尔/升Cd2+）的亚系细胞中，有证据表明，isoMT合成能力的增强是由核转录因子MTF - 1和Sp1的结合活性增加所致。使用小鼠金属反应元件（MRE）- d寡核苷酸探针进行定量凝胶迁移分析发现，与未接触金属的亲代RK - 13细胞相比，这两种转录因子的结合活性增强了两到三倍。它们结合活性的增加可能是这些细胞中MT基因过度表达和产生金属耐受性的原因。在耐受最高金属浓度的细胞中，Southern印迹信号分析表明，MT基因扩增最有可能是MT产量增加的原因。因此，在含有350微摩尔/升Zn2+的环境中生长的亚系细胞中，两个isoMT基因协同三倍复制；在耐受150微摩尔/升Cd2+的细胞中，一个isoMT基因扩增了两倍。
