CHARACTERIZATION OF LOCALIZED MATERNAL MRNA

胚胎分化在一定程度上受决定因素的影响 不对称分布在卵子的细胞质中。植物区 似乎包含至少四组决定因素，那些 形态：中胚层、内胚层、背轴结构和胚芽。 细胞谱系。本地化母体RNA是一类罕见的可以 行列式的代码。空间信息传递的机制 建立和维护RNA的分布是未知的 代表了细胞生物学和发育生物学的中心问题。 现代和经典研究表明，细胞骨架起着 重要的角色。最近，我们分离到了两个新的非洲爪哇细胞骨架 定位于植物极的卵母细胞中的相关转录本 (xCAT-2和xCAT-3)。值得注意的是，XCAT-2与Manos同源，即 果蝇的后部决定因素。 在该方案中，实验被概括为：(1)研究空间和 XCAT-2和XCAT-3在正常发育过程中的时间表达(2) 鉴定XCAT-2和XCAT-3蛋白产物并检测其 在早期发育中起作用。(3)确定RNA定位信号 以及参与XCAT-2和XCAT-3定位的蛋白结合成分。 总体策略将是确定如何继承XCAT 通过RNA印迹和原位杂交研究胚胎发育过程中的变化。错失- XCAT-2和XCAT-3蛋白在卵母细胞中的表达和不表达， 胚胎和动物的帽子将被用来描述它们可能的 在发展中的作用。XCAT-2和XCAT-3的显微注射研究 突变结构将使我们能够识别所需的RNA序列 定位并评估相关蛋白的功能。 通过研究本地化的母体RNA及其与 细胞骨架，我们希望了解分子极性是如何建立的 在卵母细胞中的作用以及它如何指导早期 胚胎。

Embryonic differentiation is mediated in part by determinants asymmetrically distributed in the cytoplasm of the egg. The vegetal region of frog eggs appears to contain at least four sets of determinants, those specifying: mesoderm, endoderm, the dorsal axial structures and the germ cell lineage. Localized maternal mRNAs are a rare class of RNAs that can code for determinants. The mechanism through which the spatial distribution of RNAs is established and maintained is unknown and represents a central problem in both cell and developmental biology. Modern and classical studies have suggested that the cytoskeleton plays an important role. Recently, we have isolated two novel Xenopus cytoskeletal associated transcripts in oocytes that are localized to the vegetal pole (XCAT-2 and XCAT-3). Remarkably, XCAT-2 is homologous to manos, the posterior determinant in Drosophila. In this proposal experiments are outlined to: (1) study the spatial and temporal expression of XCAT-2 and XCAT-3 during normal development. (2) characterize the XCAT-2 and XCAT-3 protein products and examine their function in early development. (3) determine the RNA localization signal and protein binding components involved in XCAT-2 and XCAT-3 localization. The general strategy will be to establish how the XCATs are inherited during embryogenesis by RNA blot and in situ hybridization studies. Mis- expression and non-expression of XCAT-2 and XCAT-3 proteins in the oocyte, embryo, and animal caps will be used to characterize their possible functions in development. Microinjection studies of XCAT-2 and XCAT-3 mutant constructs will allow us to identify the RNA sequences required for localization and to assess the function of associated proteins. By studying localized maternal RNAs and their relationship to the cytoskeleton, we hope to understand how molecular polarity is established in the oocyte and how it directs regional specification of the early embryo.