Environmental Toxicology Using Transgenic Models

描述（由申请人提供）：这些研究的总体长期目标是应用分子、生物化学和转基因方法研究环境健康相关问题。目前的研究主要集中在有毒金属镉和必需金属锌调节基因表达的机制。镉是一种广泛存在的环境毒素，对公众健康构成越来越大的威胁。它是一种常见的工业污染物，也存在于香烟烟雾中。镉中毒会损害许多主要器官系统，导致Itai-Itai病，生长迟缓，不育和癌症。相比之下，锌是一种必需的金属，它是数百种蛋白质活性所必需的，但在高浓度时是有毒的。具体来说，我们的研究集中在金属反应元件结合转录因子-1（MTF-1）上，该转录因子协调Cd和Zn诱导的几种保护性基因（例如，金属螯合剂金属硫蛋白、金属输出体锌转运蛋白-1和γ-谷氨酰半胱氨酸合成酶重链、谷胱甘肽合成中的限速步骤）。MTF-1作为细胞金属传感器发挥作用，但它感测不同金属的分子机制还不清楚。我们的研究表明，镉和锌利用重叠但不同的机制激活基因表达使用MTF-1。值得注意的是，小鼠MTF-1基因是必不可少的，纯合敲除胚胎在妊娠中期死亡，从而强调了这种转录因子的重要性。MTF-1是否是其他物种的必需基因仍有待确定，因为这种蛋白质在发育过程中的功能。该因子的六个锌指结构域在昆虫和哺乳动物中高度保守，表明其功能保守。因此，本提案的具体目标是：1）鉴定在Cd与Zn诱导期间与MTF-1特异性相互作用的蛋白质，并检查它们在MTF-1基因表达调节中的作用; 2）确定组蛋白乙酰化在MTF-1基因表达激活中的作用;以及3）开发斑马鱼作为模型系统以揭示MTF-1在胚胎发育期间的基本功能。与MTF-1相互作用的蛋白质将使用Superose-6 HPLC、免疫沉淀、结合位点色谱和蛋白水解肽的质谱进行研究。将使用染色质免疫沉淀、与HAT表达载体共转染、检测MTF-1复合物中的特异性HAT蛋白以及分析具有HAT基因突变的酵母中的MTF-1功能来检查组蛋白乙酰化的作用。本研究将利用吗啉代反义寡核苷酸、整片原位杂交和胚胎发育的形态计量学分析来研究MTF-1在斑马鱼发育过程中的功能。

DESCRIPTION (provided by applicant): The overall long-term objective of these studies is to apply molecular, biochemical and transgenic approaches to study environmental health-related issues. Current studies focus on the mechanisms by which the toxic metal Cd, and the essential metal Zn regulate gene expression. Cd is a widespread environmental toxin that poses an increasing threat to public health. It is a common industrial pollutant that is also present in cigarette smoke. Cd poisoning causes damage to many major organ systems, leading to Itai-Itai disease, retardation of growth, sterility and cancer. In contrast, Zn is an essential metal, which is required for the activity of hundreds of proteins, but is toxic when in high concentration. Specifically, our studies concentrate on metal-response element-binding transcription factor-1 (MTF-1), which coordinates the Cd- and Zn-induced transcription of several protective genes (e.g., the metal chelator metallothionein, the metal exporter zinc-transporter-1, and gamma-glutamylcysteine synthetase heavy chain, the rate limiting step in glutathione synthesis). MTF-1 functions as a cellular metal-sensor, but the molecular mechanisms by which it senses different metals are not well understood. Our studies suggest that Cd and Zn utilize overlapping yet distinct mechanisms for activation of gene expression using MTF-1. Remarkably, the mouse MTF-1 gene is essential and homozygous knockout embryos die at midgestation, thus underscoring the importance of this transcription factor. Whether MTF-1 is an essential gene in other species remains to be determined, as does the function of this protein during development. The six zinc-finger domain of this factor is highly conserved from insects to mammals, which indicates a conserved function. Therefore, the specific aims of this proposal are to: 1) Identify proteins which interact with MTF-1 specifically during Cd versus Zn induction, and examine their roles in MTF-1 -regulation of gene expression; 2) Determine the roles of histone acetylation in the MTF-1 activation of gene expression; and 3) Develop the Zebrafish as a model system to reveal essential functions of MTF-1 during embryonic development. Proteins which interact with MTF-1 will be studied using Superose-6 HPLC, immunoprecipitation, binding-site chromatography and mass spectrometry of proteolytic peptides. Roles of histone acetylation will be examined using chromatin immunoprecipitation, co-transfection with HAT expression vectors, detection of specific HAT proteins in the MTF-l complex, and analysis of MTF-l functions in yeast with mutations in HAT genes. Functions of MTF-l during development will be examined in Zebrafish using morpholino antisense oligonucleotides, whole mount in situ hybridization and morphometric analyses of developing embryos.