CHARACTERIZATION OF LOCALIZED MATERNAL MRNA

本地化母体 mRNA 的特征

• 批准号: 3284140
• 负责人: MARY LOU KING
• 金额: $ 23.74万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3284140
• 关键词: RNA binding protein; Xenopus; Xenopus oocyte; bioassay; cytoskeletal proteins; developmental genetics; early embryonic stage; embryogenesis; gene expression; genetic manipulation; genetic transcription; genetic translation; immunochemistry; immunoprecipitation; in situ hybridization; laboratory mouse; messenger RNA; microinjections; molecular cloning; molecular polarity; nonmammalian vertebrate embryology; nucleic acid sequence; oogenesis; protein structure function; western blottings

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.

胚胎分化部分由决定因子介导 不对称地分布在卵子的细胞质中。 植物区 青蛙卵的数量似乎包含至少四组决定因素， 说明：中胚层、内胚层、背轴结构和胚芽 细胞谱系 局部母体mRNA是一类罕见的RNA， 行列式的代码。 空间的机制 RNA的分布是建立和维持是未知的， 代表了细胞和发育生物学的中心问题。 现代和经典的研究表明，细胞骨架起着重要的作用， 重要的作用 最近，我们分离出两种新的爪蟾细胞骨架， 定位于植物极的卵母细胞中的相关转录物 （XCAT-2和XCAT-3）。 值得注意的是，XCAT-2与马诺斯同源， 后决定簇 在这个建议中，实验概述了：（1）研究空间和 正常发育期间XCAT-2和XCAT-3的时间表达。 （二） 表征XCAT-2和XCAT-3蛋白质产物并检查它们的 在早期发展中发挥作用。 (3)确定RNA定位信号 以及参与XCAT-2和XCAT-3定位的蛋白结合组分。 一般的策略是确定XCAT是如何遗传的 通过RNA印迹和原位杂交研究， 错了 卵母细胞中XCAT-2和XCAT-3蛋白的表达和不表达， 胚胎和动物帽将被用来表征他们可能的 在发展中的作用。 XCAT-2和XCAT-3的显微注射研究 突变体构建体将使我们能够确定所需的RNA序列， 定位和评估相关蛋白的功能。 通过研究定位的母体RNA及其与 细胞骨架，我们希望了解分子极性是如何建立的 以及它如何指导早期卵母细胞的区域特化。 胚胎