Gene regulation in phage lambda: A real-time study with single-event resolution

描述（由申请人提供）：该系统由大肠杆菌及其病毒噬菌体lambda组成，作为基因调控许多方面的基本范例，范围从分子水平到生物体水平。在lambda系统中提出的问题通常会产生与“高级”真核系统相关的见解。利用遗传学和生物化学的传统工具对λ系统进行了广泛的表征，从而形成了一种优雅的、看似完整的描述观察到的细胞微观相互作用现象的叙述。然而，从定量科学家的角度来看，遗传和生化知识与观察到的群体表型之间存在巨大的理解差距。这种差距表现在系统数学模型的预测能力很差。为了尝试弥合知识差距，需要通过在单个活细胞中实时研究包含该生命周期的事件来“解构”噬菌体lambda的生命周期，以足够的分辨率量化细胞内动力学，以描述空间和时间上的单个事件。在这个提议中，我们建议表征lambda生命周期中的基因调控，集中在以下目标上：(1)表征在维持溶原（休眠）状态期间裂解/溶原开关的功能，以及在细胞损伤后诱导裂解（毒性）途径。(2)阐明影响生命周期的时空方面，如裂解途径基因组复制、基因表达、衣壳自组装和裂解的不同阶段如何在空间和时间上组织。(3)区分lambda生命周期中的精确性和随机性，通过将“真实”随机性（来自实际来源的不受控制的变异性）与“表观”随机性（来自我们自己无法检测和测量个体细胞之间生理参数的差异）区分开来。

DESCRIPTION (provided by applicant): The system comprised of the bacterium Escherichia coli and its virus, bacteriophage lambda, serves as the basic paradigm for many aspects of gene regulation, ranging in scale from the molecular to the organismic level. Questions asked within the lambda system often yield insights relevant to "higher" eukaryotic systems. The lambda system has been extensively characterized using the traditional tools of genetics and biochemistry, enabling the formation of an elegant and seemingly complete narrative of the observed phenomenology in terms of the microscopic interactions in the cell. However, from the point of view of a quantitative scientist there is an immense gap of understanding between the genetic and biochemical knowledge on the one hand, and the observed population phenotype on the other. This gap manifests itself in the poor predictive powers of mathematical models of the system. To try and bridge the knowledge gap it is required to "deconstruct" the life cycle of bacteriophage lambda by studying the events comprising this life cycle in real-time, in individual living cells, quantifying the intracellular dynamics with sufficient resolution to describe individual events in space and time. In this proposal we suggest to characterize gene regulation during the lambda life cycle, concentrating on the following aims: (1) Characterizing the function of the lysis/lysogeny switch during the maintenance of the lysogenic (dormant) state as well as the induction of the lytic (virulent) pathway following cell damage. (2) Elucidating spatiotemporal aspects affecting the life cycle, for example how the different stages of the lytic pathway genome replication, gene expression, capsid self-assembly and lysis are organized in space and time. (3) Distinguishing precision versus stochasticity in the lambda life cycle, by separating "real" stochasticity one resulting from actual sources of uncontrolled variability from "apparent" stochasticity, resulting from our own inability to detect and measure differences in physiological parameters between individual cells. PUBLIC HEALTH RELEVANCE: Filling the knowledge gap at the single-event level will in turn bring us closer to a quantitative understanding of whole-system (organism) characteristics in terms of the microscopic constituents, in the vain of "systems biology". Achieving this goal in a simple model system such as lambda can then be followed by similar endeavors in higher organisms.