METALLOTHIONEIN FUNCTION AND GENE REGULATION

对金属硫蛋白(MT)在正常人中作用的认识 基因表达和细胞分化的过程是必不可少的 以评估其在致癌过程中的作用。我们对此采取的方法 一个作用是解决MT基因的调节和MT基因的功能 MT蛋白作为相互关联的过程，因为两者都受到 金属离子的结合和稳态。赤眼莲 紫癜性金属硫蛋白基因SpMTA是一种合适的模型。 测试这些关系，主要是因为它的表达式是 仅限于海洋的特定细胞谱系(败育的外胚层) 海胆胚胎。要利用单元格类型中的此成员资格 特定的一组基因、SpMTA功能的不同方面和SpMTA基因 将研究监管，包括SPMTA是否可能 蛋白质可能参与调节其他基因的表达。 SET，以及在影响细胞分化方面。其效果 将通过监测MT的表达来检验MT表达的增加 这些基因在细胞内MT水平一直是 通过转基因表达提高。为此，SpMTA mRNA或SpMTA CDNAs，后者受全局表达或 败育外胚层细胞型特异性启动子，将显微注射入 用于在发育中的胚胎中表达的蛋。对细胞的影响 分化将在形态上以及通过 标记物mRNAs水平。 该项目的第二个和补充目标将是分析 金属诱导SpMTA转录的组成和机制 吉恩。来自正常胚胎和慢性暴露于 An(II)离子(已知其SpMTA基因转录在 非常高的水平)将用作转录来源 各种因素。随后将克隆金属反应因子(MRF)。 还将从这些细胞中产生体外转录系统 细胞核，为了分析(A)蛋白质因子的功能， 特别是克隆的MRF，以及(B)特定DNA的参与 SpMTA基因转录调控的元件。功能界别 SpMTA基因中MRF等金属相关因子的特性 转录，以及推导出的克隆的MRF的结构特性，将 与MT和含金属转录物的性质进行比较 因素，特别是了解金属监管的动态，在 MT基因调控与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 用于评估人类MT在癌症中的潜在作用，以及 未来诊断和诊断的假设和方法的发展 治疗。

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.