This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of heterochromatin and numerous proteinaceous subcompartments. The chromatin corral is formed of DNA condensed with nucleosomal histones H2A, H2B, H3, and H4. It has been shown that this ?chromatin higher-order structure? plays a critical role in many aspects of gene regulation. A full understanding of these manifestations of chromatin higher-order structure and their functional significance requires knowledge of the 3D arrangement of nuclear components. DNA viruses induce profound changes in the intranuclear structures of their host cells. We have used a combination of confocal microscopy imaging techniques including photobleaching and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon parvovirus infection, the expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. In this study, we will: 1) generate 3-D reconstructions of the mammalian cell nucleus, 2) visualize the 3D of morphology of nuclear envelope and chromatin, and 3) analyze virus-induced alterations in the morphology and distribution of nuclear envelope and chromatin. X-ray tomography will provide a novel and unique experimental approach that could yield significant new information on the structural aspects of nuclear components including chromatin and nuclear envelope in mammalian cells. In addition, these studies would illuminate virusinduced alterations of these structures.
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