This revised proposal requests continuing support for a research effort to determine the three dimensional fine structure of mitotic chromosomes focussing primarily on the use of an in vitro chromosome condensation system developed recently by the PI s collaborators at UCSF. This study, now in its 13th year, represents a continuation and expansion of a well established research program to use both and improve tools for high resolution structural determination using intermediate voltage transmission electron microscopy, with a specific focus on three dimensional reconstructions from thick sections of embedded and stained chromosomes. Three major goals are set forth. First, with the further use of state of the art image reconstruction methods and electron microscopy tomography, the PI will focus on use of a Xenopus in vitro DNA condensation system in which chromosomes can be moved from interphase to mitosis and studied at various stages of condensation. The key focus here will be to use immunoelectron microscopic localizations and/or immunodepletion, to identify specific roles played by individual proteins. Beyond this, higher order chromatin structure will be further investigated focusing on the 130-nm fiber already seen both in telophase and interphase cells. The hope is that with an expansion and refinement of current imaging methods, the path of the three dimensional chromatin fiber within these higher order structures may be mapped. A second goal is to use the same set of tools to identify the structural organization of centrosomes, the structures that nucleate microtubule assembly in animal cells. The initial efforts will focus on gamma tubulin, as well as two centrosomal proteins previously discovered in Drosophila centrosomes. Here again, by focussing on in vitro methods, the consequences of the removal of specific components by immunodepletion will be assessed. Lastly, both the chromatin condensation and centrosomal efforts will be the focus of additional microscopic tool development with a goal of further improvements in immuno- tomography methodology.
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