Wang S, Hazelrigg T
Department of Biological Sciences, Columbia University, New York, New York 10027.
Nature. 1994 Jun 2;369(6479):400-03. doi: 10.1038/369400a0.
Subcellular RNA localization in different cell types leads to asymmetric distribution of proteins in these cells. The localization of bicoid (bcd) messenger RNA to the anterior pole of the developing Drosophila oocyte gives rise in embryogenesis to a steep concentration gradient of the bcd protein, a transcription factor that activates expression of zygotic genes needed for anterior development. The exuperantia (exu) gene is necessary for this localization of bcd mRNA. Here we express a chimaeric gene encoding a fusion between the Acquorea victoria green fluorescent protein (GFP) and the exu protein (Exu) in female germ cells, and find that the fusion protein fluoresces strongly in both live and fixed cells during Drosophila oogenesis. The fusion protein rescues an exu null allele, restoring full fertility to females, and is expressed and localized in a temporal and spatial pattern similar to native Exu. The high sensitivity of the GFP tag provides important new details on the subcellular localization of Exu. The fusion protein is found in particles concentrated at ring canals, where transport occurs between nurse cells and the oocyte. Drugs such as colchicine and taxol that affect microtubule stability alter localization of the particles. We propose that the particles are ribonucleoprotein complexes or vesicles which transport bcd mRNA along microtubules and target it to the anterior oocyte cortex.
不同细胞类型中的亚细胞RNA定位导致这些细胞中蛋白质的不对称分布。果蝇胚胎发育过程中，bicoid（bcd）信使RNA定位于发育中的卵母细胞的前极，在胚胎发生过程中产生bcd蛋白的陡峭浓度梯度，bcd蛋白是一种转录因子，可激活前体发育所需的合子基因的表达。exuperantia（exu）基因对于bcd mRNA的这种定位是必需的。在这里，我们在雌性生殖细胞中表达了一个嵌合基因，该基因编码维多利亚水母绿色荧光蛋白（GFP）和exu蛋白（Exu）之间的融合蛋白，并发现该融合蛋白在果蝇卵子发生过程中的活细胞和固定细胞中均发出强烈荧光。该融合蛋白挽救了exu无效等位基因，恢复了雌性的完全生育能力，并且以与天然Exu相似的时间和空间模式表达和定位。GFP标签的高灵敏度提供了有关Exu亚细胞定位的重要新细节。融合蛋白存在于集中在环管处的颗粒中，环管是营养细胞和卵母细胞之间发生运输的地方。影响微管稳定性的秋水仙碱和紫杉醇等药物会改变颗粒的定位。我们认为这些颗粒是核糖核蛋白复合物或囊泡，它们沿着微管运输bcd mRNA并将其靶向卵母细胞前皮质。
