REGULATED MRNA TRANSLATION IN DROSOPHILA BODY PATTERNING

果蝇胚轴的形成是由 决定因素在卵母细胞中以mRNAs的形式定位。这个 前后极性的形成是由 对这些mRNAs的翻译控制。麦克唐纳博士和他的团队致力于 本地化和翻译。果蝇胚胎的前形态原 是双曲面(BCD)，后方图案由Oskar(OSK)和 Nanos(否)。所有这三个mRNAs都在卵子发生过程中定位，并且只有 在本地化后翻译。平移控制在 至少有两个独立的组件。翻译在卵母细胞中被抑制，并且只有 当信使核糖核酸被本地化时，它是主动翻译的，而抑制 保持未定位的RNA。 这项建议的目的是研究OSK的平移控制。麦克唐纳博士 鉴定了OSK RNA 3‘端非编码区中对 几年前的翻译性压制。然后他鉴定了一种蛋白质，布鲁诺 (Bru)，与这些元素结合。在被逮捕的突变体(ARET)中，编码Bru， 在OSK的翻译控制发生之前，卵子发生停止。然而， 麦当劳已经开发出一种使用卵巢提取物的体外翻译系统， 并研究了布鲁诺在这一制度中的作用。在证明了 体外系统在体内重现结果，他已经证明了 Polya尾巴不影响Bru调节的翻译。他还 确定5‘端帽可能也不调节Bru功能。在一个 双杂交筛选寻找Bru的相互作用蛋白，鉴定了几种蛋白质。 其中之一是Apontic(APT)，它可能与另一个BRU冗余地工作 互动演员p50。另一个互动因素是翻译启动因子ELF-5。一个 第三个互动因素是鱿鱼，它在海狗的本地化过程中发挥着作用 核糖核酸。这些结果以及实验室的生化结果与 存在含有RNA定位的蛋白质复合体和 翻译控制蛋白。 茄子Aub已被确定为OSK的翻译激活剂。 麦克唐纳已经克隆了这个基因，并发现它编码精灵2C的翻译 启动因子在所有真核生物中都是保守的。麦克唐纳博士也 NOS调控所必需的3‘非编码区的识别序列(SRE) 不是翻译。使用生化方法，他已经鉴定出一种蛋白质， Smaug(SMG)，与SRE结合，并在体外研究了其功能 翻译系统。 该提案将继续采用这一一般方法。第一个目标是 体外结构功能分析。删除了Bru，然后点突变将 被用来定位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 抑制是它与翻译竞争结合ELF-5 机械设备。这种可能性将得到测试。目标二是跟进内部 通过在苍蝇体内的实验来证实它们的体外实验。 目的三是分离ELF-5基因的突变体。第四个目标是生化 含Bru的复合体的分离及其在翻译中的作用 压抑。这些实验已经在进行中。目标五是确定 蛋白质印迹显示该复合体中存在已知的蛋白质。来自未知蛋白质的多肽 将对条带进行测序，并克隆相应的基因。共同本地化 被确认为复合体一部分的蛋白质将以不同的方式进行分析 方法，在活体卵巢和固定材料中。 第六个也是最后一个目标是确定AUB活动的特征，特别是 确定其功能是否与elF2C相同。

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.