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EGG CYTOPLASMIC INFORMATION IN CELL DIFFERENTIATION

细胞分化中的卵细胞质信息

基本信息

批准号：
3320982
负责人：
J RICHARD WHITTAKER
金额：
$ 29.13万
依托单位：
MARINE BIOLOGICAL LABORATORY
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-09-01 至 1993-02-28
项目状态：
已结题

项目摘要

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。决定性因素已在操作上加以界定在早期细胞分裂过程中，限制性细胞谱系：分离的卵裂期卵裂球发展和自我分化只根据已经确定的命运决定因素可能与早期生产有关，明显的组织特异性mRNA和最终的表达，组织特异性蛋白和超微结构特征。主要本项目将审查的假设是：（a）概念卵细胞质决定因素，如实验所定义的，离散的信息大分子，可以从根据种类和类别进行化学鉴定， (b)这些决定子要么是特定基因的启动子，功能或本身是组织特异性mRNA分子，参与表型表达的蛋白质，和（c）所述试剂导致新分化的表达（transdetermination）当人工转移到其他谱系细胞时，可用于设计用于因子的生物测定，以帮助进一步净化。这一建议集中在两个方面对比决定系统，肌肉表达，依赖于母体来源的基因激活剂，和内胚层碱性磷酸酶的发育，包括一种酶的母体mRNA。翻译测定（非洲爪蟾卵母细胞注射，免疫沉淀，酶活性翻译产物）正在开发中并用于鉴定乙酰胆碱酯酶和碱性磷酸酶的mRNA，磷酸酶。其他研究方法包括分离海鞘肌肉乙酰胆碱酯酶的基因序列，原肌球蛋白基因和碱性磷酸酶基因。 cDNA 探针将被用于在特定时间监测基因表达和胚胎中的位置。蛋白质本身作为在卵裂球中分化的优异表达标志物隔离实验继续检查决定因素分离模式和免疫细胞化学方法，光和电子显微镜水平正在应用于他们的侦测该项目还使用了显微注射技术，谱系特异性细胞质的电融合转移和分离材料（如聚A RNA）作为设计生物制品的基础决定因素分析。