PROMOTER- BASED ESTIMATION ANALYSIS

描述（由申请人提供）：长期目标是发展一个

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.