PROMOTER- BASED ESTIMATION ANALYSIS

描述(由申请者提供)：长期目标是发展一个 基因调控中的综合计算、生化方法，其中一个 计算部分结合了大量的基因表达数据 基因组信息和生化部分将在实验中测试 在计算部分做出的预测。具体目标是发展 这种方法通过将基于启动子的估计分析与 启动子竞争分析。基于启动子的估计分析是 由首席调查员(PI)开发以对mRNA模式进行建模 从启动子DNA序列中进行表达。启动子竞争分析 在PI的实验室中开发，以测试特定顺式作用的角色 基因表达中的调控元件。在提案中，功能 将为模型预测一组顺式作用元件的重要性 系统在推动子的基础上进行了评估分析和预测 在启动子竞争试验中进行了测试。 一个人类基质金属家族的基因表达作为模型系统 将使用脂蛋白水解酶，并使用机械剪切作为 环境压力。该项目的具体目标是：(1)模拟 基于启动子的最小二乘模型在MMPs基因表达中的应用 估计分析，以及(2)检验特定的预测功能作用 利用启动子竞争研究剪切反应中的顺式作用元件 化验。在对基质金属蛋白酶mRNA的表达进行建模时，其mRNA水平将为 通过实时逆转录/聚合酶链式反应检测。 在基于启动子的估计分析中，观察到的mRNA表达将 以启动子DNA上的顺式作用调控元件的频率为模型 序列。将使用标准线性代数，生成值 将进行分析，以预测特定的功能角色 剪切响应中的顺式作用单元。顺式作用的预测作用 这些元素将在启动子竞争分析中进行测试，其中外源DNA 由特定顺式作用元件组成的片段将是瞬时的 转移到细胞中。 在此基础上开发的一体化计算机生化检测系统 提案将被用来解释许多基因的表达模式 遗传或环境应激并有助于阐明分子 复杂细胞应激反应的机制。

DESCRIPTION (provided by applicant): The long-term objective is to develop an integrated computational, biochemical approach in gene regulation, where a computational part incorporates a large volume of gene expression data with genomic information and a biochemical part will test experimentally the predictions made in the computational part. The specific goal is to develop such an approach by integrating a promoter-based estimation analysis with a promoter competition assay. The promoter-based estimation analysis was developed by the Principal Investigator (PI) to model a pattern of mRNA expression from promoter DNA sequences. The promoter competition assay was developed in the PI's laboratory to test a role of particular cis-acting regulatory elements in gene expression. In the proposal, functional significance of a group of cis-acting elements will be predicted for a model system in the promoter-based estimation analysis and the prediction will be tested in the promoter competition assay. As a model system the gene expression of a family of human matrix metal loproteinases will be used, and mechanical shear will be applied as environmental stress. The specific aims of the project are: (1) to model the mRNA expression of MMPs by using a least-square modeler in the promoter-based estimation analysis, and (2) to test predicted functional role of particular cis-acting elements in shear responses by using the promoter competition assay. In modeling the expression of MMP mRNA, the mRNA level will be determined by using real-time reverse-transcription/polymerase chain reaction. In the promoter-based estimation analysis, the observed mRNA expression will be modeled by a frequency of cis-acting regulatory elements on promoter DNA sequences. A standard linear algebra will be used, and the engenvalue analysis will be conducted to predict the functional role of specific cis-acting elements in shear responses. The predicted role of cis-acting elements will be tested in the promoter competition assay, where exogenous DNA fragments consisting of specific cis-acting elements will be transiently transferred to the cells. The integrated computational, biochemical assay system developed in this proposal will be used to interpret the expression pattern of many genes under genetic or environmental stress and contribute to elucidating molecular mechanisms in complex cellular stress responses.