MATERNAL MRNA, PROTEIN LOCALIZATIONS IN XENOPUS OOCYTES

一个单细胞，卵子，如何变成多细胞的问题 具有不同组织类型和形式的生物体是一个基本问题， 胚胎学和发育生物学。 据推测 定位的细胞质成分（细胞质决定簇）可能具有 在胚胎发育的初始阶段起着调节作用， 据推测，RNA和/或蛋白质可能是这些蛋白质的候选者。 调节分子 然而，很少有实验证明， 这一点 区域定位分子（RNA，蛋白质， 或其它成分）并不一定意味着 该组分是一种细胞质决定簇，然而，这表明， 所述组分可具有生物学上重要作用（否则， 预计将分配该部分）。 在这个提议中， 非洲爪蟾的胚胎将用于研究局部RNA， 蛋白质，因为：a）在这些卵母细胞中存在定位mRNA的空间图 (of所有现有的系统，非洲爪蟾卵母细胞最好是聚（A）+ RNA和mRNA）; B）RNA和蛋白质合成相对较好 c）卵母细胞和胚胎很容易被识别; 可获得的，并且它们的大尺寸允许容易的操作。 它们的大 大小允许将空间图用作空间分数的指导 （将单细胞卵母细胞和胚胎分成区域），然后 每个区域的RNA和蛋白质的生化分析。 空间 将通过冷冻和将卵切片在 冷冻切片机、卵子脱水和所需区域的"取出"，以及 切除部分卵皮质 在成熟卵母细胞中， 蛋白质的RNA类型）。 体外 将翻译产物或提取的区域RNA与以下进行比较： 1)该区域的稳态蛋白质; 2） 新合成的蛋白质。 这将决定一个 存在分选蛋白质的机制。 该分析将 在所有的卵子发生，减数分裂成熟，早期 胚胎发生 将针对区域特异性蛋白质制备抗体探针， a）分析蛋白质的空间分布，3）获得 蛋白质的多聚核糖体通过沉淀新生链， 挑选出特定的RNA用于未来的分析。 此外该 维持非洲爪蟾卵母细胞mRNA定位的机制 将被审查。

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 "putting-out" of the desired region, and removal of regions of the egg cortex. In the mature oocyte the amount and type of RNA for protein) in each region will be determined. The in vitro translation products or 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, 3) obtain polyribosomes for the proteins by precipitation of nascent chains in order to single out specific RNAs for future analysis. In addition, the mechanisms involved in maintaining the localized mRNA in Xenopus oocytes will be examined.