PROMOTER- BASED ESTIMATION ANALYSIS

描述（由申请人提供）：长期目标是开发 基因调控中的综合计算，生物化学方法， 计算部分将大量基因表达数据与 基因组信息和生物化学部分将通过实验测试 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 启动子竞争测定。 基于启动子的估计分析是 由主要研究者（PI）开发，以模拟mRNA 从启动子DNA序列表达。 启动子竞争测定是 在PI的实验室开发，以测试特定顺式作用的作用， 基因表达调控元件。 在提案中， 一组顺式作用元件的意义将被预测为一个模型 系统中基于启动子的估计分析和预测将是 在启动子竞争测定中测试。 作为一个模型系统的基因表达的一个家庭的人类基质金属 将使用低蛋白酶，并将施加机械剪切， 环境压力 该项目的具体目标是：（1）模拟 基于启动子的MMPs mRNA表达的最小二乘建模 估计分析，以及（2）测试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 比色法 在MMPmRNA表达的模型中，mRNA水平将是 通过使用实时逆转录/聚合酶链反应测定。 在基于启动子的估计分析中，观察到的mRNA表达将 通过启动子DNA上顺式作用调控元件的频率来建模 序列的 一个标准的线性代数将被使用， 将进行分析，以预测具体的功能作用 剪切反应中的顺式作用元件。 顺式作用的预测作用 元件将在启动子竞争测定中进行测试，其中外源DNA 由特定顺式作用元件组成的片段将被瞬时地 转移到细胞。 在此开发的集成计算生化分析系统 该提案将用于解释许多基因的表达模式， 遗传或环境压力，并有助于阐明分子 复杂的细胞应激反应机制。

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.