PROTEIN REQUIRED FOR GERM CELL DETERMINATION

在许多生物体中，早期的决定性事件是由 信息分子的差异分布（细胞质 决定因素）。 在果蝇中，这些决定子的分布是 是由严格意义上的母性基因决定的 胚胎发育 在果蝇中，基因解剖，显微操作， 和分子克隆可以结合起来研究一个特定的发育 过程 因此，果蝇可作为研究基因的模型系统 产生这些决定因素和/或参与适当的 在胚胎中分配这些决定因素。 我们尤其 感兴趣的是研究母体效应基因， 原始生殖细胞的确定和 胚胎的前后轴。 一个这样的地点，都铎（tud），是 这一提议的基础。 tud基因座编码一个约8.0 kb的mRNA，该mRNA定位在 卵子发生中期卵母细胞的后半部。 使用抗体 针对tud蛋白，我们发现该蛋白质是在 最早的卵子发生阶段，并填补核周空间的阶段1- 卵母细胞 在胚胎发生早期（0-0.25 h），蛋白质定位于 后极细胞质（种质）。 在卵黄核内 分裂蛋白质被隔离在细胞核和线粒体沿着 胚胎的前后轴。 然而，蛋白质积累 优先在后极线粒体中。 实验概述 下面将增加我们对tud+函数的作用的理解。 首先，间接免疫荧光和免疫电子显微镜将 用于确定卵母细胞中tudor蛋白的亚细胞定位 以及从突变雌性收集的胚胎。 其次，胚胎将 分馏以分离纯化的细胞核和线粒体。 Tudor蛋白 将从这些级分中纯化并表征以确定是否 这种蛋白质还有其他的形式。 第三，P元素介导 各种tud基因组和cDNA构建体的插入将用于 鉴定序列：（1）互补tud表型的子集;和 (2)所需的靶向蛋白。 最后，一个反义构建体 将用于确定在生命周期内对tud的要求。

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.