PROTEIN REQUIRED FOR GERM CELL DETERMINATION

在许多生物体中，早期决定性事件是由 信息分子的不同分布(细胞质 决定因素)。在果蝇中，这些决定因素的分布是 是由严格意义上的母性基因建立起来的 发育中的胚胎。在果蝇中，基因解剖，微操作， 与分子克隆相结合可以研究特定的发育 进程。因此，果蝇是研究基因的一个模型系统 产生这些决定因素和/或适当地参与 在胚胎中分配这些决定因素。特别是，我们正在 有兴趣研究母体效应基因，这两者都是 原始生殖细胞的鉴定及培养体系的建立 胚胎的前后轴。都铎王朝(Tudor)就是这样的一个基因座 这项提议的基础。 TUD基因座编码一个~8.0kb的mRNA，定位于 卵子发生中期卵母细胞的后半部分。使用抗体 针对TUD蛋白，我们发现该蛋白是在 最早的卵子发生阶段，并填满了阶段1的核周空间- 卵母细胞。在胚胎发育早期(0-0.25h)，该蛋白定位于 后极质(生殖质)。在卵母细胞内核 分裂蛋白质被隔离在细胞核和线粒体中 胚胎的前后轴。然而，蛋白质会积累起来 优先分布于后极线粒体。概述的实验 下面将加深我们对TUD+功能作用的理解。 首先，间接免疫荧光和免疫电子显微镜将 用于确定Tudor蛋白在卵母细胞中的亚细胞定位 以及从突变雌性体内采集的胚胎。其次，胚胎将是 分离纯化的细胞核和线粒体。都铎蛋白 将从这些组分中提纯出来，并进行表征以确定 这种蛋白质有多种不同的形式。第三，P元素介导的 各种tud基因组和cdna构建物的插入将用于 鉴定序列：(1)TUD表型的互补亚群； (2)靶向蛋白质所必需的。最后，一个反义结构 将用于确定在生命周期内对TUD的需求。

In many organisms early determinative events are established by the differential distribution of informational molecules (cytoplasmic determinants). In Drosophila, the distribution of these determinants is established by genes that make a strictly maternal contribution to the developing embryo. In Drosophila, genetic dissection, micromanipulation, and molecular cloning can be combined to study a specific developmental process. Thus, Drosophila serves as a model system for studying genes that produce these determinants and/or are involved in properly distributing these determinants within the embryo. In particular, we are interested in studying maternal effect genes required both for the determination of the primordial germ cells and establishment of the anteroposterior axis of the embryo. One such locus, tudor (tud), is the basis of this proposal. The tud locus encodes an ~8.0 kb mRNA that is localized within the posterior half of the oocyte during mid-oogenesis. Using antibodies against tud protein we find that the protein is synthesized during the earliest oogenetic stages and fills the perinuclear space of stage 1- oocytes. In early embryogenesis (0-0.25 h) the protein is localized in the posterior pole plasm (germ plasm). During the intravitelline nuclear divisions the protein is sequestered in nuclei and mitochondria along the anteroposterior axis of the embryo. However, the protein accumulates preferentially in posterior pole mitochondria. Experiments outlined below will increase our understanding of the role of tud+ function. First, indirect immunofluorescence and immunoelectron microscopy will be used to determine the subcellular location of tudor protein in oocytes and embryos collected from mutant females. Second, embryos will be fractionated to isolate purified nuclei and mitochondria. Tudor protein will be purified from these fractions and characterized to determine if there are alternate forms of the protein. Third, P element-mediated insertion of various tud genomic and cDNA constructs will be used to identify sequences: (1) complementing subsets of the tud phenotype; and (2) required for targeting the protein. Finally, an antisense construct will be used to determine the requirement for tud during the life cycle.