REGULATION AND FUNCTION OF METALLOTHIONEIN GENES

金属硫蛋白（MT）是小的、富含Cys的金属结合蛋白， 在真核生物中普遍存在。 MT被认为是辅助金属 体内平衡和解毒，最近，已经涉及到 应激反应，胚胎发生/发育，镉致癌，顺铂 抗性和转录因子活性的调节。 然而，MT功能的尖锐测试，基于消融或 MT在特定组织中的过表达，尚未进行。 同样地，目前对控制癌症的分子机制的认识， 金属诱导的、细胞特异性的和/或发育调节的 MT在多细胞真核生物中的表达/转录是有限的。 本研究旨在研究MT基因在线虫中的调控和功能 秀丽隐杆线虫（C. elegans）。 蛋白免疫细胞化学和 将使用原位mRNA杂交来确定时间、细胞和组织中的mRNA水平。 以及2种异源MT（命名为CeMT 1）的亚细胞表达模式 和CeMT 2，在连续的发育阶段。 体内 MT基因（mtl-1和mtl-1）启动子的转录活性 MTL-2）将通过创建和 鉴定转基因C.表达报告基因驱动的 由TL发起人。 位于mtl基因5'端侧翼的DNA将被 通过诱变进行修饰，以鉴定和表征金属 调控元件（MRE）和控制细胞内 转录的特异性和发育时间。 的功能 每个顺式元件将在生理环境中确定 完整（转基因）动物。 镉/锌活化的发生 MRE结合蛋白（MRE-BP）将在凝胶迁移中确定， 足迹分析。 MRE-BP将（a）通过以下方法纯化至均匀 常规方法和亲和层析，（B）表征 （c）在体外转录系统中进行生物化学测试，以及（d） 部分排序。 通过以下方法获得编码MRE-BP的cDNA克隆： cDNA文库的常规和表达筛选 酵母系统中的互补。 最后将层压CeMT， 在特定的细胞和组织中过度表达或异位表达， 转基因长优雅 我们将确定CEMT是否是必不可少的， 正常发育，应激反应，广谱解毒， 锌的细胞内分布。 新概念源自 拟议的研究将适用于镉诱导的致癌作用，因为 金属/胁迫对MT基因调控的保守性， MT功能。 分析了C.雅利安人对环境的反应 致癌物镉可能提示以下疾病的机械原因和治疗方法：（a） 金属促进的恶性肿瘤和（B）变得交叉耐药的肿瘤 到顺式DDP和烷基化剂。

Metallothionein (MTs) are small, Cys-rich metal-binding proteins that are ubiquitous among eukaryotes. MTs are thought to subserve metal homeostasis and detoxification and recently, have been implicated in stress responses, embryogenesis/development, Cd-induced cancer, cis-DDP resistance and the regulation of transcription factor activities. However, incisive tests of MT functions, based on the ablation or overexpression of MTs in specific tissues, have not been performed. Likewise, current knowledge of the molecular mechanisms that govern metal-inducible, cell-specific and/or developmentally-modulated expression/transcription of MTs in multicellular eukaryotes is limited. We will study the regulations and function of MT genes in the nematode Caenorhabdities elegans (C. elegans). Protein immunocytochemistry and in situ mRNA hybridization will be used to determine temporal, cellular and subcellular patterns of expression for 2 iso-MTs, designated CeMT1 and CeMT2, during successive developmental stages. In vivo transcriptional activities of the promoters of the MT genes (mtl-1 and mtl-2) will be monitored in individual cells by creating and characterizing transgenic C. elegans that express reporter genes driven by tl promoters. DNA flanking the 5' ends of the mtl genes will be modified by mutagenesis in order to identify and characterize metal regulatory elements (MREs) and DNA sequences that control the cellular specificity and developmental timing of transcription. The function of each cis element will be determined in the physiological environment of intact (transgenic) animals. The occurrence of cadmium/zinc-activated MRE binding proteins (MRE-BP) will be established in gel shift and footprinting assays. The MRE-BP will be (a) purified to homogeneity via conventual procedures and affinity chromatography, (b) characterized biochemically (c) tested in an in vitro transcription system and (d) partially sequenced. cDNA clones encoding MRE-BPs will be obtained by conventional and expression screening of cDNA libraries of complementation in a yeast system. Finally CeMTs will be laminated, overexpressed or ectopically expressed in specific cells and tissues of transgenic C. elegans. We will determine whether CeMTs are essential for normal development, a stress response, broad spectrum detoxification and the intracellular distribution of zinc. New concepts derived from the proposed studies will be applicable to Cd-induced carcinogenesis because of the conserved nature of both MT gene regulation by metals/stress and MT functions. Analysis of C. elegans' response to the environmental carcinogen cadmium may suggest mechanistic causes and therapies for (a) metal-promoted malignancies and (b) tumors that become cross-resistant to cis-DDP and alkylating agents.