MATERNAL MRNA AND PROTEIN LOCALIZATIONS IN OOCYTES

一个单一的细胞，即卵子，如何变成多细胞的问题 不同组织类型和形式的生物体是一个基本的问题 胚胎学和发育生物学。据推测， 定位的细胞质成分(细胞质决定因素)可能具有 在胚胎发育的初始阶段起着调节作用，它也一直是 推测RNA和/或蛋白质可能是这些基因的候选基因 调节分子。然而，几乎没有实验可以证明 这一点。区域性分子(RNA、蛋白质、 或其他成分)并不一定意味着 该成分是细胞质的决定因素；然而，它提示 该组件可以具有生物上重要的作用(否则为制服 预计将分发该组件)。在这个提案中，卵母细胞 非洲爪哇的胚胎将被用于研究定位的RNA和 蛋白质是因为：a)在这些卵母细胞中存在定位的mRNAs的空间图谱 (在所有现有的系统中，非洲爪哇的卵母细胞最适合 Poly(A)+RNA和mRNA)；b)RNA和蛋白质的合成相对较多 在早期发育时很容易理解；c)卵母细胞和胚胎很容易 可获得的，其大尺寸允许容易操作。他们的大 大小允许将空间地图用作空间分数的指南 (将单细胞卵母细胞和胚胎分成几个区域)，然后 对每个区域的RNA和蛋白质进行生化分析。空间上的 分离将通过冷冻和切开鸡蛋在一个 冷冻、卵子脱水和切除所需区域，以及 切除卵子皮质的区域。在成熟卵母细胞中，数量和 将确定每个区域的RNA(或蛋白质)类型。试管苗 提取的区域RNA的翻译产物将与以下内容进行比较： 1)该区域的稳态蛋白质；2)该区域的分布 每一区域新合成的蛋白质。这将确定一个 蛋白质的分离机制是存在的。这一分析将是 在整个卵子发生、减数分裂成熟和早期 胚胎发生。将对区域特异性蛋白进行抗体探针以 A)分析蛋白质的空间分布，b)获得 蛋白质的多核糖体通过新生链的有序沉淀 以挑选出特定的RNA用于未来的分析。此外， 非洲爪哇卵母细胞维持本地化mRNA的机制 将会被检查。

The question of how a single cell, the egg, becomes a multicellular organism with differing tissue types and form is a question basic to embryology and developmental biology. It has been speculated that localized cytoplasmic components (cytoplasmic determinants) may have a regulatory role in the initial stages of embryogenesis and it has also been speculated that RNA and/or protein are likely candidates for these regulatory molecules. However, few experiments exist which demonstrate this point. The existence of regionally localized molecules (RNA, protein, or other components) in the oocyte or embryo does not necessarily mean that the component is a cytoplasmic determinant; however, it is suggestive that the component may have a biologically important role (otherwise a uniform distribution of the component would be expected). In this proposal oocytes and embryos of Xenopus laevis will be used to study localized RNA and protein because: a) spatial maps of localized mRNAs exist in these oocytes (of all existing systems the Xenopus oocytes are best defined for poly(A)+RNA and mRNA); b) the synthesis of RNA and protein are relatively well understood during early development; c) oocytes and embryos are easily obtainable and their large size allows for easy manipulation. Their large size allows the spatial maps to be used as a guide for spatial fraction (division of the single-celled oocytes and embryos into regions) and then biochemical analysis of the RNA and protein in each region. The spatial fractionation will be performed by freezing, and sectioning the eggs in a cryotome, dehydration of eggs and "cutting-out" of the desired region, and removal of regions of the egg cortex. In the mature oocyte the amount and type of RNA (or protein) in each region will be determined. The in vitro translation products of the extracted regional RNA will be compared with: 1) the steady-state proteins in the region, 2) the distribution of newly-synthesized proteins in each region. This will determine if a mechanism exists for the sorting-out of proteins. This analysis will be performed during all of oogenesis, meiotic maturation, and early embryogenesis. Antibody probes will be made to region-specific proteins to a) analyze the spatial distribution of the proteins, b) obtain polyribosomes for the proteins by precipitation of nascent chains in order to single out specific RNA's for future analysis. In addition, the mechanisms involved in maintaining the localized mRNA in Xenopus oocytes will be examined.