RETINOBLASTOMA PROTEIN IN DROSOPHILA

这项拟议研究的长期目标是阐明控制细胞增殖和分化的机制。具体地说，这项拨款的研究调查了视网膜母细胞瘤(RB)蛋白家族在发育过程中的功能和调节。Rb家族被认为在细胞增殖和细胞分化中发挥重要作用。了解这一特定蛋白质家族的正常功能将增强我们对细胞增殖和分化如何控制的一般理解。建议的研究集中在果蝇遗传系统中的Rb家族同源基因。虽然RB-E2F途径已被广泛应用于生化研究，但RB、E2F和DP家族蛋白的体内功能及其功能相互作用的研究一直受到阻碍，因为每个家族中都存在大量的蛋白质，并且缺乏有效的方法来从功能上识别该途径中的基因。对果蝇Rb途径的研究绕过了这些问题，并补充了标准的生化方法。果蝇Rb途径的图像要简单得多，而且在此类研究中易于结合遗传、分子、细胞和发育方法，使果蝇成为开展拟议研究的理想模型系统。在初步研究中，我们克隆了一个与果蝇Rb家族同源的RBF，并证明了Rb-E2F途径的功能和调控在果蝇和哺乳动物之间是保守的。此外，我们还为拟议的研究制作了一些试剂，包括：RBF突变体、抗RBF的单抗和一些RBF过表达菌株。此外，还建立了基因筛查来识别Rb途径中的基因。在这项资助中，我们建议使用这个果蝇遗传系统来研究RBF的体内功能，并获得Rb途径的功能解剖。具体地说，我们建议(I)研究RBF在发育过程中的功能；(Ii)通过挽救RBF突变体的致死性来分析RBF的结构和功能；(Iii)研究E2F和RBF以及Dacapo和RBF之间的功能关系；(Iv)继续我们的遗传筛选，以确定Rb途径的基因。

The long-term objective of the proposed research is to elucidate the mechanisms by which cell proliferation and differentiation are controlled. Specifically, the research in this grant investigates the function and regulation of the Retinoblastoma (RB) family of proteins during development. The RB family is thought to play important roles in both cell proliferation and in cell differentiation. Understanding the normal function of this particular family of proteins will enhance our general understanding of how cell proliferation and differentiation are controlled. The proposed studies focus on the RB family homolog in a Drosophila genetic system. Although the RB-E2F pathway has been extensively studied using biochemical approaches, studies of the in vivo function of RB, E2F, and DP family of proteins and their functional interactions have been hampered by the presence of large number of proteins in each family and the lack of efficient approaches to functionally identify genes in the pathway. Studies of the RB pathway in Drosophila circumvents these problems and complements standard biochemical approaches. The much simpler picture of the RB pathway in Drosophila and the ease of combining genetic, molecular, cellular, and developmental approaches in such studies make Drosophila an ideal model system to carry out the proposed research. In preliminary studies we have cloned a Drosophila RB family homolog RBF and have demonstrated that the function and regulation of the RB-E2F pathway is conserved between Drosophila and mammals. Furthermore we have generated a number of reagents for the proposed research including: RBF mutants, monoclonal antibodies against RBF, and a number of RBF overexpression strains. In addition, genetic screens have been set up to identify genes in the RB pathway. In this grant we propose to use this Drosophila genetic system to study the in vivo function of RBF and to obtain a functional dissection of the RB pathway. Specifically, we propose (i) to study the function of RBF during development, (ii) to analyze the structure and function of RBF through rescuing the lethality of RBF mutant, (iii) to investigate the functional relationship between E2F and RBF, and between Dacapo and RBF, (iv) to continue our genetic screen to identify genes of the RB pathway.