Nuclear Cycle Regulation by Partner of Paired

我们正在研究一个新发现的泛素介导的蛋白质降解的F-box受体，配对的伙伴（Ppa），其性质表明在核周期调控以及在果蝇胚胎发育过程中的基因表达的节段模式的作用。初步研究表明ppa通过有丝分裂促进进展（见目标1），我们假设这是通过调节细胞周期蛋白降解而发生的（目标2）。我们以前已经建立了Ppa协调Pax转录因子Paired的降解。我们假设，在前13个快速的核周期，局部消除ppa表达周期14长间期期间创建一个发展窗口，其中Prd可以在没有有丝分裂的情况下发挥作用。我们假设这是重要的，因为Prd否则会对有丝分裂有害（目的3）。我们的具体目标是：（1）确定ppa是否促进有丝分裂。我们将测试局部ppa表达是否在时间和空间上与有丝分裂14的协调区域重叠。我们将检查ppa突变体中这些结构域中有丝分裂14的进程是否受到损害。我们也将建立在初步的结果表明，通过有丝分裂13的进展是延迟ppa突变体。(2)测试Ppa是否调节果蝇细胞周期蛋白。我们将确定是否PPA与特定的果蝇细胞周期蛋白的相互作用，通过测试蛋白质的相互作用，在双杂交和免疫共沉淀实验，并通过测试基因剂量实验中，PPA和细胞周期蛋白的水平被改变的遗传相互作用。在ppa突变体和ppa过表达转基因胚胎中通过western分析检测细胞周期蛋白水平，将测试ppa是否可以调节细胞周期蛋白水平。(3)目的：检测Prd功能是否对有丝分裂有害。通过比较的空间表达的Prd和PPA与本地化的有丝分裂领域通常观察到的核周期14，我们将测试是否Prd功能和有丝分裂是相互排斥的，如初步实验所建议的。然后，我们将测试，使用prd过表达转基因，是否异位Prd功能调节核循环。我们提出的分析将提供深入了解的多层监管网络，协调核循环和模式的形成在果蝇的发展，并建议可能的监管控制，为未来的脊椎动物的分析。

We are studying a newly-identified F-box receptor for ubiquitin- mediated protein degradation, Partner of paired (Ppa), whose properties suggest roles in nuclear cycle regulation as well as in segmental patterning of gene expression during Drosophila embryogenesis. Preliminary studies suggest that ppa promotes progression through mitosis (see Aim 1), and we hypothesize that this occurs through regulation of cyclin degradation (Aim 2). We have established previously that Ppa coordinates degradation of the Pax transcription factor, Paired. We hypothesize that after the first 13 rapid nuclear cycles, localized elimination of ppa expression during the long interphase of cycle 14 creates a developmental window in which Prd can function in the absence of mitosis. We hypothesize that this is important because Prd would otherwise be detrimental to mitosis (Aim 3). Our specific aims are: (1) To establish whether ppa promotes mitosis. We will test whether localized ppa expression overlaps in time and space the orchestrated domains of mitosis 14. We will examine whether in ppa mutants, progression of mitosis 14 in these domains is compromised. We will also build on preliminary results which suggest that progression through mitosis 13 is delayed in ppa mutants. (2) To test whether Ppa regulates Drosophila cyclins. We will determine whether Ppa interacts with particular Drosophila cyclins by testing for protein interactions in two-hybrid and co-immunoprecipitation experiments, and by testing for genetic interactions in gene dosage experiments in which ppa and cyclin levels are altered. Examination of cyclin levels by western analysis in ppa mutants and in embryos with ppa over-expression transgenes will test whether ppa can regulate cyclin protein levels. (3) To test whether Prd function is detrimental to mitosis. By comparing the spatial expression of Prd and Ppa with the localized mitotic domains normally observed in nuclear cycle 14, we will test whether Prd function and mitosis are mutually exclusive, as suggested by preliminary experiments. We will then test, using prd over-expression transgenes, whether ectopic Prd function modulates nuclear cycling. Our proposed analysis will provide insights into the multi-layered regulatory network that coordinates nuclear cycling and pattern formation in Drosophila development, and will suggest possible regulatory controls for future analysis of vertebrates.