Axis Determination and Germ Plasm Assembly in Drosophila

果蝇轴测定和胚芽质组装

• 批准号: 6897532
• 负责人: ROBERT E BOSWELL
• 金额: $ 32.83万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6897532
• 关键词: Drosophilidae; RNA binding protein; cell cycle; cell differentiation; egg /ovum; gene interaction; germ cells; immunoprecipitation; laboratory mouse; molecular assembly /self assembly; molecular asymmetry; monoclonal antibody; nuclear matrix; nucleic acid purification; nucleic acid sequence; oogenesis; polymerase chain reaction; protein localization; protein protein interaction

DESCRIPTION (provided by applicant): Cell-cell interactions are important in establishing cellular diversity in the development of multicellular organisms. In Drosophila melanogaster, it has become apparent that inductive interactions between the germline-derived oocyte and the overlying somatic posterior follicle cells establish the major body axes of the egg chamber and embryo. Posterior follicle cell-to-oocyte signaling also establishes a specialized region of cytoplasm within the posterior of the oocyte that is indispensable for germ cell determination and abdominal segmentation of embryos (the germ plasm). Although these inductive interactions are critical for defining the axes and determination of the germline, thus far, few components that function within the oocyte to regulate this important signal transduction pathway have been identified. Investigating these processes will haw important implications for understanding human developmental defects and cancers. Our long-tern objectives are to elucidate the molecular mechanisms that define the major body axes and assembly of germ plasm components in Drosophila. The mago nashi (mago) gene has been demonstrated to encode a protein that localizes within the posterior pole of the oocyte to regulate the response of the oocyte to the posterior follicle cell-to-oocyte signal. Utilizing a yeast two-hybrid screen the Tsunagi protein, an RNA-binding protein, was identified as a Mago interactor. The Mago:Tsunagi complex is required to define the major body axes and is indispensable for localizing components of the germ plasm. The objectives in this proposal are as follows: (1) Germline clonal analysis, immunological techniques and cytological methods will be used to investigate whether mago nashi and tsunagi interact for the determination and/or differentiation the oocyte. (2) To identify factors that interact with Mago protein localized within the posterior pole, immunoprecipitation and biochemical methods will be employed. (3) Two approaches will enable identification and purification of RNAs bound in vivo to Mago:Tsunagi complexes. In the first approach, RNAs transcribed from a germarial and/or ovarian cDNA iterative affinity selection library will be co-immunoprecipitated with Mago:Tsunagi complexes from ovarian extracts. In the second approach, immunoprecipitations of ovarian extracts will be used to purify endogenous RNAs that form a complex with Mago and Tsunagi.

描述（由申请人提供）：在多细胞生物的发育过程中，细胞-细胞相互作用对建立细胞多样性很重要。在黑腹果蝇中，生殖系衍生的卵母细胞与上覆的体细胞后卵泡细胞之间的诱导相互作用建立了卵室和胚胎的主要体轴。后卵泡细胞到卵母细胞的信号传导也在卵母细胞的后部建立了一个特殊的细胞质区域，这对于生殖细胞的测定和胚胎的腹部分割（胚质）是必不可少的。虽然这些诱导相互作用对于确定轴和确定种系至关重要，但迄今为止，在卵母细胞内调节这一重要信号转导途径的成分很少被确定。研究这些过程将对理解人类发育缺陷和癌症具有重要意义。我们的长期目标是阐明确定果蝇主要体轴和种质成分组装的分子机制。mago nashi （mago）基因编码一种蛋白，该蛋白位于卵母细胞的后极，以调节卵母细胞对后卵泡细胞-卵母细胞信号的反应。利用酵母双杂交筛选，将Tsunagi蛋白（一种rna结合蛋白）鉴定为Mago相互作用物。Mago:Tsunagi复合体用于确定主要的身体轴，并且对于定位种质成分是必不可少的。本研究的目的如下：(1)利用生殖系克隆分析、免疫学技术和细胞学方法，探讨日本巨尾和日本巨尾是否相互作用，以确定和/或分化卵母细胞。(2)通过免疫沉淀法和生化法确定与后极定位的Mago蛋白相互作用的因子。(3)有两种方法可以鉴定和纯化体内结合在Mago:Tsunagi复合物上的rna。在第一种方法中，从生殖和/或卵巢cDNA迭代亲和选择文库中转录的rna将与卵巢提取物中的Mago:Tsunagi复合物共同免疫沉淀。在第二种方法中，卵巢提取物的免疫沉淀将用于纯化与Mago和Tsunagi形成复合物的内源性rna。