Characterization of extrinsic variability in eukaryotic gene expression

描述（由申请人提供）：本项目的目标是表征基因调控网络中存在的两种外源变异性来源。转录和调控不断受到外部变异源的影响，基因网络如何在这些条件下可靠地发挥作用是一个悬而未决的问题。该提案的第一个具体目标是解决不同类型的基因调控网络中细胞周期动态引起的变异性。当细胞生长和分裂时，细胞体积的变化导致调节途径中活性反应物浓度的变化。由于潜在的生化反应的速率取决于反应物的浓度，基因网络的动力学将受到细胞周期的影响。该提议的第二个具体目标是当基因网络的转录由外部试剂诱导时，检查基因网络中的细胞间变异性。许多基因网络可以通过向生长培养基中引入化学诱导剂来开启。通常，这些网络上调负责诱导物一旦被引入就内化的转运蛋白。这种正反馈可以在引入诱导物和网络完全活跃之间的时间内产生高的细胞间变异性。这些目标将使用涉及实验和计算方法的多学科方法来解决。实验方面将有三个方面。首先，将使用标准分子生物学技术来创建所需的基因网络并添加荧光报告蛋白。其次，本实验室开发的现有微流体设备将用于控制细胞的生长条件，并使用荧光显微镜记录细胞数据。最后，将使用现有的细胞跟踪软件分析从显微镜获得的荧光图像，以获得单个细胞的轨迹。计算建模将需要对所涉及的网络的综合模型进行离散随机模拟，并将用于描述和预测实验的行为。然后，实验结果将被用于生成更完整的模型，并对所涉及的网络有更连贯的理解。系统生物学的主要目标之一是了解基因调控网络的动态。实现这一目标的一个障碍是外在变异的表征，以及对基因网络如何在噪声环境中可靠地发挥作用的理解。本提案的具体目标将涉及转录调控的这些基本方面。

DESCRIPTION (provided by applicant): The goal of this project is to characterize two sources of extrinsic variability that are present in gene regulatory networks. Transcription and regulation are constantly being affected by extrinsic sources of variability, and it is an open question how gene networks reliably function under these conditions. The first specific aim of this proposal addresses the variability caused by cell cycle dynamics in different types of gene regulatory networks. As a cell grows and divides, the changes in the volume of the cell cause variability in the concentrations of the reactants that are active in the regulatory pathways. Since the rates of the underlying biochemical reactions depend on the concentrations of the reactants, the dynamics of gene networks will be affected by the cell cycle. The second specific aim of this proposal is the examination of the cell-to-cell variability in gene networks when their transcription is induced by an outside agent. Many gene networks can be turned on by introducing a chemical inducer into the growth media. Often, these networks up-regulate the transport protein responsible for the internalization of the inducer once it has been introduced. This positive feedback can create high cell-to-cell variability in the time between introduction of the inducer and the time at which the network is completely active. These aims will be addressed using a multidisciplinary approach involving both experimental and computational methods. The experimental aspects will be threefold. First, standard molecular biology techniques will be used to create the needed gene networks and to add fluorescent reporter proteins. Second, existing microfludic devices, developed in this lab, will be used to both control the growth conditions of cells and record cell data with the use of fluorescent microscopy. Finally, the fluorescent images obtained from the microscopy will be analyzed with existing cell tracking software to obtain trajectories of individual cells. The computational modeling will entail discrete stochastic simulations of comprehensive models of the networks involved, and will be used to both describe and predict the behavior of the experiments. The results of experiments will then be used, in turn, to generate more complete models and a more coherent understanding of the networks involved. One of the main goals of systems biology is to understand the dynamics of gene regulatory networks. One obstacle to this goal is the characterization of extrinsic variability, and the understanding of how gene networks reliably function in noisy environments. The specific aims of this proposal will address these fundamental aspects of transcriptional regulation.