METALLOTHIONEIN FUNCTION AND GENE REGULATION

金属硫蛋白的功能和基因调控

• 批准号: 2102070
• 负责人: MARTIN J NEMER
• 金额: $ 12.44万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-15 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2102070
• 关键词: alternatives to animals in research; cell differentiation; complementary DNA; ectoderm; embryo /fetus; gene expression; genetic promoter element; genetic regulation; genetic transcription; genetically modified animals; metallothionein; microinjections; molecular cloning; sea urchins

An understanding of the role of metallothionein (MT) in the normal processes of gene expression and cellular differentiation is essential for evaluating its involvement in carcinogenesis. Our approach to such a role is to address the regulation of the MT gene and the function of the MT protein as interrelated processes, since both are influenced by the binding and homeostasis of metal ions. The Strongylocentrotus purpuratus metallothionein gene, SpMTA, is an appropriate model for testing these relationships, principally because its expression is restricted to specific cell lineages (the aboral ectoderm) of the sea urchin embryo. To take advantages of this membership in a cell-type specific set of genes, diverse aspects of SpMTA function and SpMTA gene regulation will be studied, including the possibility that the SPMTA protein may be involved in regulating the expression of other genes in the set, as well as in influencing cellular differentiation. The effect of increasing MT expression will be tested by monitoring the expression of these genes in embryos whose intracellular levels of MT have been elevated through transgenic expression. For this, SpMTA mRNA or SpMTA cDNA, the latter under the regulation of either a globally expressed or aboral ectoderm cell-type specific promoter, will be microinjected into eggs for expression in developing embryos. Effects on cellular differentiation will be monitored morphologically as well as by the levels of the marker mRNAs. A second and complementary aim of this project will be to analyze the components and mechanisms of metal-induced transcription of the SpMTA gene. Nuclei from normal embryos and embryos chronically exposed to An(II) ions (known to have their SpMTA genes transcribed at extraordinarily high levels) will be used as sources of transcription factors. The metal-response factors (MRFs) will be subsequently cloned. An in vitro transcription system will also be generated from these nuclei, in order to analyze (a) the functions of protein factors, especially of cloned MRFs, and (b) the involvement of specific DNA elements in the regulation of SpMTA gene transcription. The functional properties of MRFs and other metal-associated factors in SpMTA gene transcription, and the deduced structural properties of cloned MRFs, will be compared with the properties of MT and metal-containing transcription factors, especially to understand the dynamics of metal regulation, in the interrelated processes of MT gene regulation and MT functionality. The results of these studies will provide a basic conceptual framework for evaluation of the potential role of human MT in cancer, and for development of future hypotheses and approaches to diagnosis and therapies.

金属硫蛋白（MT）在正常人肝组织中的作用 基因表达和细胞分化的过程是必不可少的 用于评估其在致癌作用中的作用。 我们对这种 一个作用是解决MT基因的调节和 MT蛋白是相互关联的过程，因为两者都受到 金属离子的结合和体内平衡。 球海胆 purpuratus金属硫蛋白基因，SpMTA，是一个合适的模型， 测试这些关系，主要是因为它的表达是 仅限于海洋的特定细胞谱系（反口外胚层） 海胆胚胎 要利用单元类型中的成员身份， 特定的基因组，SpMTA功能的不同方面和SpMTA基因 监管将被研究，包括SPMTA的可能性， 蛋白质可能参与调节其他基因的表达， 以及影响细胞分化。 效果 增加MT表达的可能性将通过监测 这些基因在胚胎中的细胞内MT水平已经被 通过转基因表达提高。 为此，SpMTA mRNA或SpMTA cDNA，后者受全局表达或 反口外胚层细胞类型特异性启动子，将被显微注射到 在发育中的胚胎中表达。 对细胞的影响 分化将在形态学上以及通过 标记物mRNA的水平。 该项目的第二个补充目标是分析 金属诱导SpMTA转录的组分和机制 基因 正常胚胎和长期暴露于 An（II）离子（已知其SpMTA基因在 非常高的水平）将被用作转录的来源 因素 随后将克隆金属响应因子（MRF）。 一个体外转录系统也将产生这些 细胞核，为了分析（a）蛋白质因子的功能， 特别是克隆的MRF，和（B）特异性DNA的参与 调控SpMTA基因转录的元件。 功能 SpMTA基因中MRF和其他金属相关因子的性质 转录，以及克隆MRF的推断结构特性，将 与MT和含金属转录的性质相比， 因素，特别是了解金属调节的动态， MT基因调控和MT功能的相关过程。 这些研究的结果将提供一个基本的概念框架 用于评估人MT在癌症中的潜在作用，以及用于 发展未来的假设和诊断方法， 治疗