Developmental Regulation of the Cell Cycle in Drosophila

描述(申请人提供)：参与细胞周期的基因功能高度保守，结合果蝇卓越的细胞生物学和遗传学，使其成为研究细胞周期调控的理想模式生物。在果蝇早期胚胎发育过程中，需要3个基因来调节这一发育阶段特有的“S-M”周期：盘古(PNG)、钚(Plu)和巨核(GNU)。携带这些基因中任何一种突变的雌性都是不育的，因为她们的胚胎无法进入有丝分裂，无法过度复制DNA形成“巨核”。PNG编码丝氨酸/苏氨酸蛋白激酶，PLU和GNU编码新的小蛋白，PNG、PLU和GNU与Cyclin B基因相互作用，Cyclin B蛋白水平在这些突变体中降低。我们最近证实，PNG、PLU和GNU在体外相互作用形成一个活性激酶复合体，GNU通过诱导PNG-PLU复合体的二聚化来激活PNG。此外，我们还证明了GNU本身是PNG激酶的底物。在全基因组生化筛查中，最近发现了几种具有脊椎动物同源物的PNG激酶底物，表明PNG激酶复合体通过作用于进化上保守的成分来调节细胞周期。我们现在建议确定GNU是否在体内通过二聚化机制激活PNG激酶，并测试它是否是具有潜在效应功能的底物。我们计划确定PNG激酶复合体是否调节后期促进复合体的活性，从而控制Cyclin B水平和进入有丝分裂。最后，进化上保守的蛋白质Mat89Bb已被确定为体外PNG激酶底物。在果蝇中，Mat89Bb的RNA干扰导致了一种使人想起巨核类基因的表型。我们将测试Mat89Bb是否为PNG的体外底物，并分离Mat89Bb突变株，以更准确地确定其在细胞周期调控中的作用。

DESCRIPTION (provided by applicant): The high degree of functional conservation of genes involved in the cell cycle combined with the superb cell biology and genetics of Drosophila make it an ideal model organism for studying cell cycle regulation. During early Drosophila embryogenesis, 3 genes are required for regulation of the streamlined "S-M" cycles characteristic of this developmental stage: pan gu (png), plutonium (plu), and giant nuclei (gnu). Females carrying mutations in any of these genes are sterile because their embryos fail to enter mitosis and over-replicate DNA to form "giant nuclei." PNG encodes a serine/threonine protein kinase, and plu and gnu encode small novel proteins, png, plu, and gnu interact genetically with cyclin B, and Cyclin B protein levels are decreased in these mutants. We recently demonstrated that PNG, PLU, and GNU interact to form an active kinase complex in vitro and that GNU activates PNG by inducing dimerization of PNG-PLU complexes. Additionally, we showed that GNU itself is a substrate for PNG kinase. In a genome-wide biochemical screen, several PNG kinase substrates with vertebrate homologs were recently identified, suggesting that the PNG kinase complex regulates the cell cycle by impinging on evolutionarily conserved components. We now propose to determine whether GNU activates PNG kinase via a dimerization mechanism in vivo and to test whether it is a substrate with potential function as an effector. We plan to determine whether the PNG kinase complex regulates activity of the Anaphase Promoting Complex so as to control Cyclin B levels and entry into mitosis. Finally, an evolutionarily conserved protein, Mat89Bb, has been identified as an in vitro PNG kinase substrate. RNA interference of Mat89Bb in Drosophila results in a phenotype reminiscent of that of the giant nuclei class of genes. We will test whether Mat89Bb is an in vitro substrate of PNG and will isolate Mat89Bb mutants to more precisely determine its role in cell cycle regulation.