ESTABLISHMENT OF THE GERM CELL LINEAGE IN DROSOPHILA

这种应用的长期目标是获得一种分子 了解果蝇生殖细胞谱系是如何建立的 在胚胎发育过程中。适当建立和维护单元格 命运对于生物体的正常生长和发育是必不可少的。 这一基本过程中的缺陷会导致癌症和出生缺陷。 这一建议侧重于遗传、分子和生化方面的研究 对一种已知需要的基因的检测，以建立 生殖细胞谱系，即无生殖细胞基因。无生殖细胞基因 编码一种定位于种质的信使核糖核酸 生殖细胞的决定因素。 无生殖细胞的功能，基于反义、过度表达和 异位本地化研究是必需的，也是能够发起的 生殖细胞前体或极细胞形成的某些事件. 无生殖细胞蛋白的亚细胞分布分析 表明它定位于极细胞核的核孔。 在生殖细胞命运确定之前、期间和之后。生殖细胞- 因此，LESS基因在胚种的建立中起着核心作用。 细胞谱系。该项目最初的重点将是验证 无生殖细胞功能在胚种建立中的特异性 通过分离强突变体和零突变体进行细胞谱系划分。无细胞生殖细胞 突变体将通过基于N个选择性的P-元件反转来获得 筛选和基于标准EMS诱变剂的遗传筛选。生殖细胞- 较少的突变体将是分子、遗传和表型 其特征是准确地定义其在生殖细胞形成和 其他发育途径。为了探索它的生化功能， 这种无生殖细胞的蛋白质将发生突变，使其成为亚细胞 用于测试是否需要核孔关联的定位突变体 功能。它还将在果蝇组织中异位表达 培养细胞检测在管制核贩运方面的作用。至 确定生殖细胞规格所需的其他基因，a 将进行酵母双杂交筛选，以鉴定哪些蛋白质 与无生殖细胞的蛋白质发生物理上的相互作用。确定的基因 此屏幕将通过几个标准进行评估，以确定它们是否 在生殖细胞谱系的建立中发挥作用。此外, 还将进行遗传相互作用筛选，以识别其他 这条途径中的基因。

The long term objective of this application is to gain a molecular understanding of how the germ cell lineage of Drosophila is established during embryogenesis. Proper establishment and maintenance of cell fate is essential for normal growth and development of an organism. Defects in this fundamental process leads to cancers and birth defects. This proposal focuses on the genetic, molecular and biochemical examination of one gene known to be required for the establishment of the germ cell lineage, the germ cell-less gene. The germ cell-less gene encodes an mRNA that is localized to the germ plasm which contains the germ cell determinants. The function of germ cell-less, based on antisense, over-expression and ectopic localization studies is required for, and is capable of initiating some of the events of germ cell precursors, or ~pole cell~ formation. Analysis of the subcellular distribution of germ cell-less protein indicates that it is localized to the nuclear pores of the pole cell nuclei prior to, during and after germ cell fate is established. The germ cell- less gene therefore plays a central role in the establishment of the germ cell lineage. The initial focus of this project will be to verify the specificty of germ cell-less function for he establishment of the germ cell lineage by isolating strong and null mutants. Germ cell-less mutants will be obtained by a n lecular based, P-element reversion screen and a standard ems mutageneis based gentic screen. Germ cell- less mutants will be molecularly, genetically and phenotypically characerized to precisely define its role in germ cell formation and other developmental pathways. To explore its biochemical function, the germ cell-less protein will be mutated to make sub-cellular localization mutants to test if nucelar pore association is required for function. It will also be ectopically expressed in Drosophila tissue culture celis to assay for a role in regulating nuclear trafficking. To identify other genes which are required for germ cell specification, a yeast two-hybrid screen will be performed to identify proteins which physically interact with the germ cell-less protein. Genes identified by this screen will be evaluated by several criteria to determine if they have a role in the establishment of the germ cell lineage. In addition, a genetic interaction screen will be performed also to identify additional genes in this pathway.