CHARACTERIZATION OF LOCALIZED MATERNAL MRNA

本地化母体 mRNA 的特征

• 批准号: 3284139
• 负责人: MARY LOU KING
• 金额: $ 10.35万
• 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 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3284139
• 关键词: Xenopus; cytoskeleton; developmental genetics; early embryonic stage; egg /ovum; embryogenic cleavage; gene expression; genetic library; genetic manipulation; genetic recombination; genetic transcription; genetic translation; immunochemistry; messenger RNA; microinjections; molecular cloning; nonmammalian vertebrate embryology; nucleic acid sequence

Embryonic determination is mediated in part by morphogenetic substances specifically localized in the cytoplasm of the egg. The vegetal region of eggs in general and frog eggs in particular is of special interest because two known determinants, those specifying the germ cell lineages and those inducing the dorsal axial structures, are known to reside in this region. Recent evidence suggests that mRNAs could function as determinants. This laboratory has found that a small group of mRNAs are localized to the vegetal pole of Xenoupus oocytes and has cloned one such RNA (Veg2). Veg2 RNA is specifically associated with the oocyte's cytoskeleton. In this proposal experiments are outlined to (1) determine when Veg2 RNA is expressed during Xenopus development and to characterize its protein product; (2) examine the pattern of inheritance of Veg2 RNA and its protein product during normal and perturbed embryogenesis; (3) establish the requirements for correct localization of Veg2 RNA to the vegetal pole; (4) explore the relationship of Veg2 RNA to the oocyte's cytoskeletion; (5) initiate studies designed to assay for the developmental function of Veg2 RNA; (6) determine if other vegetally localized mRNAs are associated with the cytoskeleton. The general strategy involves using the Veg2 cDNA clone in blot and in situ hybridization procedures to follow its developmental fate at the cellular level. Microinjection of Veg2 RNA into the animal pole of normal and perturbed eggs will yield information concerning the mechanisms of message localization. Antibodies will be raised to Veg2 protein produced in an expression vector system and used to characterize the Veg2 protin as to its biochemical nature and its possible function in development.

胚胎决定部分是由形态发生介导的 特别定位于卵子细胞质中的物质。 的 一般来说，卵的生长区，特别是青蛙卵， 特别有趣的是，因为两个已知的决定因素，那些指定 生殖细胞谱系和那些诱导背轴 已知结构存在于该区域中。 最近的证据 表明mRNA可能起决定性作用。 这 一个实验室发现，一小部分mRNA定位于 异种卵母细胞的植物极，并克隆了这样一个 RNA（Veg2）。 Veg 2 RNA特异性地与 卵母细胞的细胞骨架。 在本提案中，实验概述了（1）确定何时 Veg 2 RNA在非洲爪蟾发育过程中表达， 表征其蛋白质产物;（2）检查 正常人和哺乳动物中Veg 2 RNA及其蛋白产物遗传 （3）建立胚胎发育的要求; Veg 2 RNA在植物极的正确定位;（4）探索 Veg 2 RNA与卵母细胞骨架的关系;（5） 启动旨在分析发育功能的研究 （6）确定其他植物定位的mRNA 与细胞骨架有关。 一般策略包括在印迹中使用Veg 2 cDNA克隆 和原位杂交程序来跟踪它的发育 在细胞水平上的命运。 将Veg 2 RNA显微注射到 正常和扰动卵的动物极将产生信息 关于消息本地化的机制。 抗体 将在表达载体中产生Veg 2蛋白 系统，并用于表征Veg 2蛋白， 生物化学性质及其在发育中的可能作用。