ESTABLISHMENT OF THE GERM CELL LINEAGE IN DROSOPHILA

本申请的长期目标是获得分子生物学活性。 了解果蝇的生殖细胞谱系是如何建立的 在胚胎发育过程中。 正确建立和维护细胞 命运对生物体的正常生长和发育至关重要。 这一基本过程的缺陷会导致癌症和出生缺陷。 这项建议的重点是遗传，分子和生物化学 检测一个已知建立 生殖细胞谱系，无生殖细胞基因。 无生殖细胞基因 编码一种mRNA，该mRNA定位于种质， 生殖细胞决定因素。 基于反义、过表达和 异位定位研究是必需的，并且能够启动 一些生殖细胞前体或极细胞形成的事件。 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中 在确定生殖细胞命运之前、期间和之后。 生殖细胞- 因此，较少的基因在细菌的建立中起着核心作用 细胞谱系 该项目的最初重点将是验证 无生殖细胞功能的特异性 细胞谱系分离强和无效突变体。 无生殖细胞 突变体将通过基于遗传的P元件逆转获得 筛选和基于标准EMS诱变剂的遗传筛选。 生殖细胞- 更少的突变体将在分子、遗传和表型上 其特征在于精确定义其在生殖细胞形成中的作用， 其他发展途径。 为了探索其生化功能， 无生殖细胞的蛋白质将被突变， 定位突变体，以测试是否需要珠心孔缔合， 功能 它也将异位表达在果蝇组织中 培养细胞以测定在调节核运输中的作用。 到 鉴定生殖细胞特化所需的其他基因， 将进行酵母双杂交筛选以鉴定 与无生殖细胞蛋白质发生物理相互作用。 基因鉴定 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，本发明还提供了一种方法， 还将进行遗传相互作用筛选， 这条路上的基因

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.