OOCYTE DEVELOPMENT IN DROSOPHILA

果蝇卵母细胞的发育

基本信息

批准号：
2181944
负责人：
Lynn COOLEY
金额：
$ 24.18万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-07-01 至 1995-06-30
项目状态：
已结题

项目摘要

The long-term goal of this research is to understand how cells interact in the development of the Drosophila oocyte. The work will focus on the mechanism and regulation of cytoplasm transport from the nurse cells to the oocyte through the intercellular bridges that connect these cells. Aspects of the regulation of cytoplasm transport are likely to be common to many other cell types in invertebrate as well as vertebrate systems. In addition, the mechanism of communication between germ line cells connected by intercellular cytoplasmic bridges may be relevant to the cause of mammalian germ line tumors. The specific aims of the proposal are: (1) to determine the nucleotide sequences of two genes, chickadee and kelch, mutations in which disrupt the transport of cytoplasm to the oocyte; (2) to determine the expression pattern of chickadee and kelch at both the mRNA and protein levels; (3) to test models for the function of chickadee and kelch; (4) to search for additional mutants in cytoplasm transport using single P element insertional mutagenesis; and (5) to investigate the regulation of transport specificity. Using controlled single P element insertional mutagenesis, insertional alleles were recovered of two second chromosome genes, chickadee and kelch, that are clearly associated with nurse cell cytoplasm transport; the nurse cells of mutant egg chambers fail to empty their cytoplasm into the oocyte late in oogenesis. The genes were cloned by plasmid rescue of DNA flanking the inserted P elements and putative protein sequence will be examined for any similarities to known proteins or protein motifs. The expression pattern of chickadee and kelch will be examined by Northern hybridization and in situ hybridization. Additional mutants in nurse cell cytoplasm transport will be isolated by insertional mutagenesis of the autosomes with an "enhancer trap" P element construct. The mutants will be mapped cytogenetically and used in appropriate complementation tests to determine if other alleles of the genes exist. Phenotypic analysis will include staining nurse cell cytoplasmic actin filaments (required for cytoplasm transport) to determine whether the contractile apparatus is intact and determining whether the cytoplasmic bridges are "open" to cytoplasm flow by studying the distribution of maternal mRNA by in situ hybridization. Two classes of mRNA are transported into the oocyte: those that appear in the oocyte early in oogenesis and those that are sequestered in the nurse cells until the bulk of cytoplasm transport late in oogenesis. Mutant egg chambers will be examined by in situ hybridization for any effect on this transport specificity. The model that the sequestered mRNAs are actively translated will be tested by fractionating egg chamber extracts on sucrose gradients and examining polysome fractions for specific mRNAs.

这项研究的长期目标是了解细胞如何相互作用果蝇卵母细胞的发展。这项工作将集中于细胞质从护士细胞转移到的机理和调节卵母细胞穿过连接这些细胞的细胞间桥梁。方面细胞质转运的调节可能是许多人普遍的无脊椎动物和脊椎动物系统中的其他细胞类型。在补充，连接的生殖线细胞之间的通信机理通过细胞间细胞质桥，可能与哺乳动物种系肿瘤。该提案的具体目的是：（1）确定核苷酸两个基因的序列，山雀和kelch，突变中破坏了细胞质转运到卵母细胞；（2）确定表达式 mRNA和蛋白质水平上的山雀和kelch的模式；（3）到山雀和凯尔奇功能的测试模型；（4）搜索使用单P元素的细胞质转运中的其他突变体插入诱变；（5）调查运输调节特异性。使用受控的单个P元素插入诱变，插入等位基因被回收了两个第二秒染色体基因山雀和凯尔奇（Kelch）显然与护士细胞细胞质转运有关；护士突变蛋室的细胞无法将其细胞质清空为卵母细胞卵子发生后期。通过DNA侧面的质粒拯救将基因克隆将检查插入的P元素和推定的蛋白质序列的与已知蛋白质或蛋白质基序的任何相似之处。表达山雀和凯尔奇的模式将通过北部杂交检查和原位杂交。护士细胞质传输中的其他突变体将通过带有“增强子陷阱” P元素的常染色体的插入诱变构造。突变体将在细胞遗传学上映射，并用于适当的补充测试以确定是否其他等位基因存在基因。表型分析将包括染色护士细胞细胞质肌动蛋白丝（细胞质转运所需）以确定收缩设备是否完整并确定是否细胞质桥通过研究通过研究通过原位杂交分布母体mRNA。两类mRNA被运输到卵母细胞：出现在卵母细胞早期的卵母细胞和护士中隔离的卵母细胞细胞直至大部分细胞质转运在卵子发生后期。突变鸡蛋将会通过原位杂交对钱伯进行检查，以实现对此的任何影响运输特异性。隔离的mRNA积极的模型翻译将通过将卵室提取物分馏在蔗糖上测试梯度和检查特定mRNA的多级分数。