Biochemical analysis of retinoblastoma related factor complexes in Drosophila melanogaster

视网膜母细胞瘤肿瘤抑制蛋白在细胞周期控制和肿瘤中起着重要作用。最近，人们发现pRb在多亚基复合体中起抑制转录和复制的作用。这些复合物中的一些通过组蛋白修饰或atp依赖性染色质重塑酶改变局部染色质结构。pRb复合物的组成和确切的作用机制目前尚不清楚。在许多（转化的）细胞系中，通常用于纯化哺乳动物蛋白复合物，因此，对pRb功能进行彻底的生化分析受到了阻碍。果蝇胚胎已被证明是纯化和分析野生型细胞蛋白质复合物的理想选择。果蝇含有两个pRb同源物。遗传分析表明，pRb途径的基本方面在苍蝇中是保守的。然而，果蝇视网膜母细胞瘤蛋白及其复合物尚未被研究。我建议研究果蝇视网膜母细胞瘤样因子（RBF）的生物化学。将从野生型果蝇胚胎提取物中纯化RBF蛋白复合物，并分析其亚基组成和酶活性。提出的项目是从非转化细胞分离的肿瘤抑制蛋白复合物的生化分析的第一个实例。

The Retinoblastoma tumour suppressor protein plays a fundamental role in cell cycle control and cancer. Recently, it has become apparent that pRb functions within multisubunit complexes to repress transcription and replication. Some of these complexes enzymatically alter local chromatin structure by histone modification or ATP-dependent chromatin remodelling. The composition and precise mechanism of action of pRb complexes is currently unknown. A thorough biochemical analysis of pRb function is hampered by the pRb inactivation encountered in many of the (transformed) cell lines typically used for purification of mammalian protein complexes. Drosophila embryos have proven to be ideally suited for the purification and analysis of protein complexes from wild-type cells. Drosophila contains two pRb homologues. Genetic analysis has demonstrated that fundamental aspects of the pRb pathway are conserved in the fly. However, the fly retinoblastoma proteins and complexes have not been studied. I propose to investigate the biochemistry of the Drosophila retinoblastoma like factor, RBF. RBF protein complexes will be purified from wild-type Drosophila embryo extracts and analysed for their subunit composition and enzymatic activities. The proposed project is the first instance of a biochemical analysis of tumour suppressor protein complexes isolated from non-transformed cells.