TRACE METAL METABOLISM IN DEVELOPING ARTEMIA SALINA

本项目的目标是向学生介绍 早期胚胎发育中的痕量金属和金属蛋白。在……里面 具体地说，研究重点将放在分离和表征上 卤虫卤虫金属硫蛋白(MT)样蛋白的研究 以及在发育过程中影响其表达的因素。 金属硫蛋白是一类低分子金属结合蛋白 多种生物中的蛋白质，包括甲壳类动物。他们是 具有高度保守的一级结构和高度的 对铜和锌等必需痕量金属的亲和力 它们是正常细胞生长和分化所必需的。这个 金属硫蛋白参与金属离子动态平衡与人体健康 有记录在案。金属硫蛋白可被重金属和一些 其他细胞外应激因素也使这一点具有吸引力 用于研究真核基因表达的系统。 一类低分子量金属结合蛋白，称为 锌结合蛋白(锌结合蛋白，II组)在胞浆中被检测到 卤虫提取物。这种混合物至少由四种物质组成 离散的MT样锌金属蛋白，其中更多的是 发育调节的。这些分子将被提纯到均一状态 使用凝胶过滤、离子交换层析和C18反相 高效液相色谱。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法检测产品纯度。 将分析每种蛋白质的氨基酸组成，以 确定它们与哺乳动物金属硫蛋白的相似性。 这四种蛋白质各自表达的发育概况 将会被确定。随后，胚胎将暴露在非致命性环境中 铜和镉的浓度，以确定金属对 单个蛋白质的表达。 最后，将从卤虫中筛选出细菌cdna文库。 来分离这种蛋白质的潜在基因。Southern杂交分析 利用人类MT基因探针对卤虫基因组DNA的研究揭示了 卤虫中存在MT-LIME基因序列。阳性克隆将是 DNA的分离和插入片段的桑格测序 测序。 最初，学生将接触到基本的生化和 分子生物学技术将会给他们留下深刻的印象 基本化学原理在生命研究中的应用 有机体。此后，将鼓励他们设计和实施 回答关于以下特定问题的原始实验方案 胚胎发育中的金属离子动态平衡。

The objective of this project is to introduce students to the role of trace metals and metalloproteins in early embryonic development. In particular, the research will focus on the isolation and characterization of metallothionein(MT)-like proteins from the brine shrimp Artemia salina and the factors which influence its expression during development. Metallothioneins are a class of low molecular weight metal binding proteins found in a variety of organisms, including crustacean. They are characterized by their highly conserved primary structure and high affinity for essential trace metals such as copper and zinc, both of which are required for normal cell growth and differentiation. The involvement of MT in metal ion homeostasis and human health is well documented. The fact that MT is inducible by heavy metals and a number of other extracellular stress factors also makes this an attractive system for the study of eukaryotic gene expression. A class of low molecular weight metal binding proteins, referred to as ZnBPII (zinc binding proteins, group II), has been detected in cytosolic extracts from Artemia. The mixture is composed of at least four discrete MT-like zinc metalloproteins one of more of which are developmentally regulated. The molecules will be purified to homogeneity using gel filtration, ion-exchange chromatography, and C18 reverse phase HPLC chromatography. Purity of the product will be checked by SDS-Page. The amino acid composition will be analyzed for each of the proteins to determine their similarity to mammalian metallothioneins. A developmental profile for the expression of each of the four proteins will be determined. Subsequently, embryos will be exposed to non-lethal concentrations of copper and cadmium to determine the effect of metal on the expression of the individual proteins. Finally, a bacterial cDNA library generated from Artemia will be screened to isolate the potential gene for this protein. Southern blot analysis of Artemia genomic DNA using a human MT gene probe has revealed the presence of MT-lime gene sequences in Artemia. Positive clones will be isolated and the inserts sequenced by the Sanger method for DNA sequencing. Initially, students will be exposed to the basic biochemical and molecular biology techniques will be to impress on them the importance of fundamental chemical principles as they apply to the study of living organisms. Thereafter, they will be encouraged to design and implement original experimental protocols to answer a specific question regarding metal ion homeostasis in embryonic development.