The overall goal of this proposal is to address long-standing questions concerning genomic and nuclear organization using recently developed methods for sensitive, high-resolution localization of specific DNA and RNA sequences in conjunction with immunohistochemistry and biochemical fractionation techniques. Our plan is based on the philosophy (supported by preliminary work) that some of the greatest insights will be gained by an approach which integrates the investigation of genomic organization with that of RNA metabolism. Recently our lab has provided evidence that the metabolism of polymerase II transcripts is spatially localized within the nucleoplasm, and that the underlying genome is non-randomly organized with respect to poly(A) RNA/splicing factor rich """"""""transcript domains"""""""". Specific gene organization will be evaluated at levels of increasing depth, including their position within the 3-dimensional nucleus, the relationship to RNA pol II transcript domains, and the position and packaging with respect to loop/matrix structures. The spatial arrangement of an individual gene and its RNA track win be studied in detail, extending preliminary work indicating that tracks have a distinct polarity and represent sites of both transcription and splicing directly associated with the larger transcript domains. The integration of RNA metabolism and genomic organization with nuclear structure will be studied not only for its cell biological importance, but also in terms of its potential developmental significance. Using myogenesis as a model system, we will investigate whether there are functionally significant changes in the organization or packaging of specific genes that correlate with the developmental events of either determination or differentiation. Coupling of biochemical fractionation procedures with fluorescence hybridization will provide a novel approach to directly visualize specific DNA or RNA association with nuclear structure, such as differential packaging of different classes of DNA sequences within distended DNA loops. Other goals are directed at investigating the relationship of RNA metabolism to transport and nuclear structure, including an evaluation of whether genes and RNAs transcribed by pol Ill are spatially localized within the nucleoplasm. Longer-term goals involve extending these studies to tissues and early mouse embryos, as well as exploring the use of experimental manipulation of chromosome position effect to investigate the relationship between sequence context, gene packaging, and gene expression.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Cytology Study Section (CTY)
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University of Massachusetts Medical School Worcester
Anatomy/Cell Biology
Schools of Medicine
United States
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