Environmental Toxicology Using Transgenic Models

描述(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来研究与环境健康有关的问题。目前的研究主要集中在有毒金属Cd和必需金属锌调控基因表达的机制上。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。镉中毒会对许多主要器官系统造成损害，导致伊泰病、生长迟缓、不孕和癌症。相比之下，锌是一种必需的金属，是数百种蛋白质活动所必需的，但在高浓度时是有毒的。具体地说，我们的研究集中在金属反应元件结合转录因子-1(MTF-1)上，它协调镉和锌诱导的几个保护性基因的转录(例如，金属螯合剂金属硫蛋白、金属输出体锌转运体-1和谷氨酰半胱氨酸合成酶重链，谷胱甘肽合成的限速步骤)。MTF-1具有细胞金属传感器的功能，但其感知不同金属的分子机制尚不清楚。我们的研究表明，镉和锌利用重叠但不同的机制来激活MTF-1的基因表达。值得注意的是，小鼠MTF-1基因是必不可少的，纯合子敲除胚胎在怀孕中期死亡，因此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，该蛋白在发育过程中的功能也是如此。该因子的六个锌指结构域从昆虫到哺乳动物都高度保守，这表明它具有保守的功能。因此，这项建议的具体目的是：1)确定在镉和锌诱导过程中与MTF-1特异相互作用的蛋白质，并研究它们在MTF-1基因表达调控中的作用；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)发展斑马鱼作为模型系统，以揭示MTF-1在胚胎发育中的基本功能。与MTF-1相互作用的蛋白质将使用Superose-6高效液相色谱、免疫沉淀、结合部位层析和蛋白水解肽的质谱学来研究。组蛋白乙酰化的作用将通过染色质免疫沉淀、与HAT表达载体共转染、检测MTF-L复合体中特定的HAT蛋白以及分析HAT基因突变的酵母中MTF-L的功能来研究。利用吗啉反义寡核苷酸、整体原位杂交和发育中胚胎的形态计量学分析，研究mtf-L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.