The objective of this proposal is to continue to develop and apply in situ hybridization methodology for an understanding of the organization and expression of nucleic acid sequences within the cell. In situ hybridization, particularly in conjunction with non- isotopic detection, is a powerful tool for describing the molecular biolgoy of a single cell. As such advances in this technology have important ramifications spanning both basic and clinical sciences. Having built a firm methodological foundation during the last grant period, we are in a strong position for applying in situ hybridization in new ways to obtain biological information of a fundamental and far-reaching nature. The unusually gentle and sensitive hybridization methodology we have developed has allowed us to investigate the intracellular distribution of specific mRNAs, leading to the discovery that mRNAs for different cytoskeletal proteins exhibit specific and distinct patterns of localization. In the work proposed here we will extend this analysis of mRNA localization to include a variety of cell types as well as other mRNAs for cytoskeletal-associated and non- cytoskeletal proteins. Moreover, we will employ several approaches including light and electron microscopy to analyze the mechanism of mRNA association with the cytoskeleton. Furthermore, recent developments in our lab make it possible to detect with high resolution and efficiency, a single copy sequence within interphase nuclei or on chromosomes. This methodology in conjunction with image processing now makes it possible to study the organization of genes in their functional state within interphase chromatin, using the ordered array of nuclei within the skeletal myofibre as a model system. Hence, we can investigate the molecular cytology of gene expression as a continuum from the production of transcripts within the nucleus to localization and translation of mRNA in the cytoplasm. Finally, the particular hybridization and non-isotopic detection methodology we continue to advance has significant impact on several fields as diverse as clinical diagnostics, human gene mapping (prenatal diagnosis), and virology.
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