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λ组成，作为基因调控许多方面的基本范例，范围从分子水平到器官水平。在lambda系统中提出的问题往往会产生与“更高”的真核系统相关的见解。lambda系统已经广泛地使用遗传学和生物化学的传统工具进行了表征，使得能够在细胞中的微观相互作用方面形成对所观察到的现象学的优雅且看似完整的叙述。然而，从定量科学家的角度来看，一方面遗传和生化知识与另一方面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的数学模型的预测能力差。为了尝试和弥合知识差距，需要通过在单个活细胞中实时研究包括该生命周期的事件来“解构”噬菌体λ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中的单个事件。在这个建议中，我们建议在λ生命周期中表征基因调控，集中在以下目标：（1）表征溶原性（休眠）状态的维持过程中的溶解/溶原性开关的功能，以及诱导细胞损伤后的溶解（毒性）途径。(2)阐明影响生命周期的时空方面，例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λ生命周期中的精确性与随机性，通过将“真实的”随机性从“表观的”随机性中分离出来，“表观的”随机性是由于我们自己无法检测和测量个体细胞之间生理参数的差异。 公共卫生相关性：在单事件水平上填补知识空白将反过来使我们更接近于从微观成分的角度对整个系统（有机体）特征的定量理解，而不是“系统生物学”。在一个简单的模型系统（如λ）中实现这一目标之后，可以在高等生物中进行类似的努力。

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.