EGG CYTOPLASMIC INFORMATION IN CELL DIFFERENTIATION

细胞分化中的卵细胞质信息

• 批准号: 3320981
• 负责人: J RICHARD WHITTAKER
• 金额: $ 27.96万
• 依托单位: MARINE BIOLOGICAL LABORATORY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-01 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3320981
• 关键词: RNA biosynthesis; Tunicata; acetylcholinesterase; alkaline phosphatase; cell differentiation; complementary DNA; cytochalasins; cytoplasm; dissection; early embryonic stage; egg /ovum; embryogenic cleavage; enzyme induction /repression; enzyme inhibitors; enzyme mechanism; extrachromosomal DNA; fertilization; gel electrophoresis; gene expression; genetic transcription; histogenesis; immunoelectrophoresis; messenger RNA; microinjections; molecular cloning; monophenol monooxygenase; myogenesis; nonmammalian vertebrate embryology; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; phase contrast microscopy; protein biosynthesis; saltwater environment; videotape /videodisc

This project examines the nature and properties of the egg cytoplasmic determinants of tissue differentiation that occur in Ascidian embryos. Determinants have been defined operationally by their differential segregation during early cell divisions into restricted cell lineages: cleavage stage blastomeres in isolation develop and self-differentiate only according to already fixed fates. Determinants can be related to early production of apparently histospecific mRNAs and the eventual expression of histospecific proteins and ultrastructural features. The major hypotheses to be examined by this project are (a) the conception that egg cytoplasmic determinants, as defined experimentally, are discrete informational macromolecules that can be isolated from the embryo and identified chemically according to kind and class, (b) that the determinants are either initiators of specific gene functions or are themselves histospecific mRNA molecules for proteins involved in phenotypic expression, and (c) the agents cause expression of new differentiation (transdeterminations) when moved artificially to other lineage cells and this property can be used in devising a biological assay for the factors to aid in their further purification. This proposal is focused on two contrasting determinant systems, muscle expression which seems to depend on maternally-derived gene activator(s), and endodermal alkaline phosphatase development which apparently involves a maternal mRNA for the enzyme. Translational assays (Xenopus oocyte injections, and immunoprecipitation of enzymatically active translation products) are being developed and used to identify mRNAs for acetylcholinesterase and alkaline phosphatase. Other methods of study involve isolating ascidian gene sequences for the muscle acetylcholinesterase and tropomyosin genes and for the alkaline phosphatase gene. cDNA probes will be used to monitor gene expression at certain times and locations in the embryos. The proteins themselves serve as excellent expression markers of differentiation in blastomere isolation experiments that continue to examine determinant segregation patterns, and immunocytochemical methods at the light and electron microscopic levels are being applied to their detection. The project also uses techniques of microinjection and electrofusion transfer of lineage-specific cytoplasm and isolated materials (e.g. poly A RNA) as a basis for devising biological assays of determinants.

这个项目研究了鸡蛋的性质和特性。 组织分化的细胞质决定因素 海鞘胚胎。决定因素已经在操作上定义了 通过它们在早期细胞分裂中的不同分离 限制性细胞谱系：分离的卵裂期卵裂球 只有按照已定的标准发展和自我区分 命运。决定因素可能与早期生产 明显具有组织特异性的mRNAs及其最终表达 组织特异性蛋白和超微结构特征。少校 这个项目要检验的假设是：(A)概念 根据实验定义，卵子细胞质的决定因素是 离散的信息性大分子，可以从 胚胎，并根据种类和类别进行化学鉴定， (B)决定因素是特定基因的启动子 功能或本身是组织特异性的mRNAs分子 参与表型表达的蛋白质，以及(C)试剂 新分化的原因表达(转译) 当人工转移到其他谱系细胞时，这一特性 可以用来设计一种生物测定法，以帮助 他们的进一步净化。这项建议主要集中在两个方面 截然不同的决定系统，肌肉表情看起来 依赖母源基因激活剂(S)，以及 内皮型碱性磷酸酶的发育明显 涉及母体的酶的信使核糖核酸。翻译分析 (非洲爪哇卵母细胞注射和免疫沉淀 酶活性翻译产品)正在开发中 并用于鉴定乙酰胆碱酯酶和碱性的mRNAs 磷酸酶。研究的其他方法包括分离海鞘。 肌乙酰胆碱酯酶基因序列和 原肌球蛋白基因和碱性磷酸酶基因。Cdna 探针将被用来监测特定时间的基因表达 以及胚胎中的位置。这些蛋白质本身就是 卵裂球分化的优秀表达标志 继续检查行列式的分离实验 分离模式和免疫细胞化学方法 光学和电子显微镜水平正在被应用于他们的 侦测。该项目还使用了微注射和 谱系特异性细胞质和分离株的电融合转移 材料(例如，聚A RNA)作为设计生物的基础 决定因素的分析。