REGULATED MRNA TRANSLATION IN DROSOPHILA BODY PATTERNING

果蝇胚胎中轴的形成是由 决定簇在卵母细胞内以mRNA的形式定位。的 前-后极性的形成受到以下因素的次级调节： 这些mRNA的翻译控制。麦克唐纳博士和他的小组致力于 本地化和翻译。果蝇胚胎的前形态原 是双曲面（bcd），后部图案由oskar（osk）控制， 纳米（nos）。所有这三种mRNA都在卵子发生过程中定位， 翻译后，这个地方发生。翻译控制在 至少有两个独立的组件。翻译在卵母细胞中受到抑制， 当mRNA被定位时，它被主动翻译，而抑制mRNA的表达则是被动的。 未定位的RNA被保留。 本研究的目的是研究osk的翻译调控。麦唐纳医生 在osk RNA的3'UTR中鉴定了对于以下过程必需的元件： 几年前的翻译抑制然后他鉴定了一种蛋白质， (Bru)与这些元素绑定。在编码Bru的突变体中， 卵子发生在osk的翻译控制发生之前停止。然而，在这方面， Macdonald开发了一种使用卵巢提取物的体外翻译系统， 并研究了Bruno在该系统中的作用。在证明了 体外系统复制了体内结果，他已经表明， polyA尾部不影响Bru调节的翻译。他还 5 '帽可能也不调节Bru功能。中 寻找Bru相互作用物的双杂交筛选鉴定了几种蛋白质。 其中之一是Apontic（Apt），它可能与另一个Bru冗余运行 相互作用子p50。另一种相互作用物是翻译起始因子eIF-5。一 第三个互动者是squid，它在gurken的定位中发挥作用。 核糖核酸这些结果以及实验室的生化结果与 存在含有RNA定位的蛋白质复合物， 翻译控制蛋白。 Aubergine Aub已被鉴定为osk的翻译激活剂。 麦克唐纳克隆了该基因，发现它编码一个elf-2C翻译， 在所有真核生物中保守的起始因子。麦克唐纳博士也 nos的3 'UTR中的识别序列（SRE）， nos翻译。利用生物化学的方法，他已经确定了一种蛋白质， Smaug（Smg）与SRE结合，并在体外研究了其功能。 翻译系统 建议继续采用这一总的办法。第一个目标是 体外结构功能分析。布鲁和点突变体 用于绘制RNA结合、翻译抑制和特定蛋白质- 相互作用域。关于Bru如何在翻译中发挥作用的一个假设 抑制的另一个原因是它与翻译竞争结合elF-5， 机械.这种可能性将受到考验。目标二是跟进 体外实验通过在苍蝇体内实验证实它们。 目的三是分离elF-5基因中的突变体。目标四是生物化学 含有Bru和翻译功能的复合物的分离 镇压这些实验已经在进行中。目标五是确定 通过蛋白质印迹分析复合物中的已知蛋白质。来自未知蛋白质的肽 将对条带进行测序并克隆相应的基因。的共定位 作为复合物的一部分被鉴定的蛋白质将在各种不同的 方式，在活的卵巢以及固定的材料。 第六个也是最后一个目标是描述奥布活动的特征，特别是， 确定它是否像eIF2C一样起作用。

The formation of the embryonic axes is in Drosophila controlled by the localization of determinants in form of mRNAs within the oocyte. The formation of anterior-posterior polarity is secondarily regulated by translational control of these mRNAs. Dr. Macdonald and his group work on localization and translation. The anterior morphogen of the Drosophila embryos is bicoid (bcd) and the posterior patterning is controlled by oskar (osk) and nanos (nos). All three mRNAs are localized during oogenesis and are only translated after this localization occurs. The translational control has at least two separate components. Translation is repressed in the oocyte, and only when the mRNA is localized is it actively translated, while the repression of unlocalized RNA is maintained. This proposal's aim is to study the translational control of osk. Dr. Macdonald identified elements in the 3'UTR of osk RNA that are essential for translational repression several years ago. He then identified a protein, Bruno (Bru), binding to these elements. In mutants in arrest (aret), encoding Bru, oogenesis stops before the translational control of osk occurs. However, Macdonald has developed an in vitro translation system using ovary extracts, and has studied the function of Bruno in this system. After demonstrating that the in vitro system reproduces in vivo results, he has shown that the length of the polyA tail does not influence Bru regulated translation. He has also established that the 5'cap probably does not regulate Bru function either. In a two-hybrid screen looking for Bru interactors several proteins were identified. One of these is Apontic (Apt) that may function redundantly with another Bru interactor p50. Another interactor is a translation initiation factor elF-5. A third interactor is squid, which functions in the localization of the gurken RNA. These results as well as the lab's biochemical results are consistent with the existence of a protein complex containing RNA localization and translational control proteins. Aubergine Aub has been identified as a translational activator of osk. Macdonald has cloned the gene and found that it encodes an elf-2C translation initiation factor conserved in all eukaryotes. Dr. Macdonald has also identified sequences (SRE) in the 3' UTR of nos essential in the regulation of nos translation. Using a biochemical approach he has identified a protein, Smaug (Smg), binding to the SREs, and has studied its function in the in vitro translation system. The proposal is to continue with this general approach. The first aim is a structure function analysis in vitro. Deleted Bru and then point mutants will be used to map RNA binding, translational repression, and specific protein- interaction domains. One hypothesis of how Bru functions in translational repression is that it competes for binding elF-5 with the translation machinery. This possibility will be tested. Aim two is to follow up the in vitro experiments by confirming them through in vivo experiments in the fly. Aim three is to isolate mutants in the elF-5 gene. Aim four is the biochemical isolation of a complex containing Bru and functions in translational repression. These experiments are already underway. Aim five is to identify known proteins in the complex by western blots. Peptides from unknown protein bands will be sequenced and the corresponding genes cloned. The co-localization of the proteins identified as part of the complex will be analyzed in various ways, in live ovaries as well as fixed material. The sixth and last aim is to characterize Aub activity, in particular, to determine if it functions like elF2C.