Developmental Regulation of the Cell Cycle in Drosophila

描述（申请人提供）：果蝇参与细胞周期的基因具有高度的功能保守性，加之其出色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。在果蝇早期胚胎发生过程中，有3个基因参与了这一发育阶段流线型“S-M”周期的调控：盘古（png）、钚（plu）和巨核（gnu）。携带这些基因突变的雌性是不育的，因为它们的胚胎不能进入有丝分裂，并过度复制DNA以形成“巨核”。PNG编码丝氨酸/苏氨酸蛋白激酶，plu和gnu编码小的新蛋白，PNG、plu和gnu与细胞周期蛋白B基因相互作用，在这些突变体中细胞周期蛋白B蛋白水平降低。我们最近证明PNG、PLU和GNU在体外相互作用形成一个活跃的激酶复合物，GNU通过诱导PNG-PLU复合物的二聚化来激活PNG。此外，我们发现GNU本身是PNG激酶的底物。在全基因组的生化筛选中，最近发现了几种具有脊椎动物同源物的PNG激酶底物，这表明PNG激酶复合物通过撞击进化上保守的成分来调节细胞周期。我们现在建议确定GNU是否通过体内二聚化机制激活PNG激酶，并测试它是否是具有潜在效应的底物。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，一个进化上保守的蛋白Mat89Bb被鉴定为PNG激酶的体外底物。果蝇对Mat89Bb的RNA干扰导致一种与巨核类基因相似的表型。我们将测试Mat89Bb是否是PNG的体外底物，并分离Mat89Bb突变体，以更精确地确定其在细胞周期调节中的作用。

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.