REGULATION OF TRANSLATION DURING EARLY DEVELOPMENT

这一建议解决了翻译调控的问题， 在基因表达控制的基本要素，在早期 发展 具体来说，我们要问的是蛋白质合成是如何 受精后激活，以及在受精过程中如何调节 胚胎发生 本研究选择的系统是海胆卵， 胚胎发育 提出的一般方法是纯化和 描述翻译的关键组成部分，并开发工具， 监测这些组成部分的水平、形式和活动的变化， 最终发现控制它们相互作用的机制。 第一、 我们将纯化和表征蛋白质的起始因子， 从海胆卵和胚胎中合成并产生抗体， 纯化的因素。 用免疫化学和凝胶电泳 技术，我们将确定水平和分子形式（例如， 磷酸化）的因素在未受精卵和胚胎。 第二、 在蛋白质合成中具有活性的海胆卵无细胞裂解物将 按照公布的程序制备。 我们还将构建一个 系统完全是高度纯化的海胆成分，并使用这两个 用于详细分析纯化的翻译蛋白的作用的测定 在蛋白质合成的激活起始途径的组成部分。 第三，我们将研究受精过程中核糖体蛋白的变化 和早期发展，并建立如何改变是联系在一起的， 受精时的翻译激活。 细胞骨架的作用 在早期发育过程中调节蛋白质速率的变化 还将研究合成。 最后，我们将净化和表征 大量和特异性信使核糖核蛋白mRNA和蛋白质 颗粒使用免疫学和凝胶电泳技术，并试图 识别掩蔽因子。 开发的工具和信息 从这些实验中获得的信息将使我们能够阐明 mRNP进入多聚核糖体的分子机制。 这些研究 将阐明不仅重要的控制机制，在早期 发展，而且重要的一般机制的翻译控制 在所有真核细胞中。

This proposal addresses the question of translational regulation as a fundamental element in the control of gene expression during early development. Specifically, we are asking how protein synthesis is activated following fertilization and how it is regulated during embryogenesis. The system chosen for this study is the sea urchin egg and developing embryo. The general approach proposed is to purify and characterize the key components of translation, and develop tools which can monitor changes in the levels, forms and activities of these components, to ultimately discover the mechanisms which govern their interactions. First, we shall purify and characterize the initiation factors of protein synthesis from sea urchin eggs and embryos and produce antibodies against the purified factors. Using immunochemical and gel electrophoretic techniques, we will determine levels and molecular forms (e.g., phosphorylation) of the factors in unfertilized eggs and embryos. Second, sea urchin egg cell-free lysates active in protein synthesis will be prepared following published procedures. We will also construct an assay system entirely of highly purified sea urchin components, and use these two assays for detailed analysis of the role of the purified translational components of the initiation pathway in activation of protein synthesis. Third, we will examine changes in ribosomal proteins during fertilization and early development, and establish how changes are linked to the translational activation at fertilization. The role of cytoskeletal changes during early development in modulating the rate of protein synthesis will also be studied. Finally, we shall purify and characterize the mRNA and proteins of bulk and specific messenger rebonucleoprotein particles using immunological and gel electrophoretic techniques and try to identify a masking factor. The tools developed and the information obtained from these experiments will then allow us to elucidate the molecular mechanisms of mobilization of mRNP into polysomes. These studies will elucidate not only important controlling mechanisms during early development, but also important general mechanisms of translational control in all eukaryotic cells.