A systems analysis of novel essential cell cycle components in Caulobacter

描述(由申请人提供)：在新月弯杆菌中驱动和调整细胞周期进程的核心调控电路构成了一个高度集成的系统，旨在确保多个事件以时空调控的方式发生。尽管各种主要调控因子的鉴定和鉴定为细胞周期调控的分子基础提供了非凡的洞察力，但最近在弧菌基因组中发现的新的、必要的转录因子和非编码遗传元件强烈地表明了一种不完整的调控电路和新的、未被描述的调控模式。该项目的中心目标是确定新的基本调节元件在细胞周期控制中的作用。具体地说，我建议确定两项监管职能 利用微阵列分析鉴定细胞周期调控的新型Caulbacter转录因子。我将使用芯片和生物信息学分析来实验和计算确定每个转录因子的结合基序(S)。此外，我还将进行遗传和生化分析，以确定细胞周期进程中必需的非编码染色体元素的需求。最近在硫杆菌基因组中发现的基因间、非编码的“基本间隙”序列中，超过60%位于细胞周期调控基因附近，这表明这些序列在细胞周期控制中发挥作用。因此，我将根据细胞周期中相邻ORF被诱导的点将这个亚集分为五类，并通过缺失、互补、倒置和转座分析来表征每个集合中具有代表性的序列。这将使我能够通过操作各种参数(如长度、位置和方向)来探索这些序列的本质。由于初步证据表明，蛋白质可能会结合其中的一些序列，我将使用DNA采样，以交联和纯化特定结合每个序列的蛋白质。这一广泛的方法将使我能够确定如何将Caulbacter基因组的新基本元素整合到控制细胞生长和发育的核心遗传电路中。

DESCRIPTION (provided by applicant): The core regulatory circuitry that drives and paces cell cycle progression in the bacterium Caulobacter crescentus constitutes a highly integrated system designed to ensure that multiple events take place in a spatiotemporally regulated manner. Although identification and characterization of various master regulators has offered extraordinary insight into the molecular basis of cell cycle control, the recent identification of novel, essential transcription factors and non-coding genetic elements in the Caulobacter genome strongly suggest an incomplete regulatory circuit and novel, undescribed regulatory modalities. The central goal of this project is to determine the roles of novel essential regulator elements in cell cycle control. Specifically, I propose to identify the regulatory functions of two novel cell-cycle-regulated Caulobacter transcription factors by using microarray analysis to identify genes regulated by each. I will employ ChIP-chip and bioinformatic analysis to experimentally and computationally identify the binding motif(s) for each transcription factor. In addition, I will conduct genetic and biochemical analysis to determine the requirement for essential non-coding chromosomal elements in cell cycle progression. Among the intergenic, non-coding "essential gap" sequences recently identified in the Caulobacter genome, over 60% lie immediately adjacent to cell-cycle-regulated genes, suggesting a role for these sequences in cell cycle control. Therefore, I will divide this subset into five categories based on the points during the cell cycle at which adjacent ORFs are induced and characterize representative sequences from each set through deletion, complementation, inversion, and transposition analysis. This will allow me to probe the essential nature of these sequences by manipulating various parameters such as length, position, and orientation. As preliminary evidence suggests that proteins may bind some of these sequences, I will use DNA sampling in order to crosslink and purify proteins that bind each sequence specifically. This broad approach will enable me to determine how the new essential elements of the Caulobacter genome are integrated into the core genetic circuit that controls cell growth and development.