The nucleus is the most prominent organelle in eukaryotic cells. Within it the DNA is organized level upon level, beginning with simple packaging into nucleosomes and continuing with subsequent organization into 30 nm filaments, and ultimately into chromatin loops. Organization occurs as the result of specific interactions between chromatin and internal or peripheral structures of the nucleus, interactions known to involve topoisomerase II, the nuclear lamina and the nuclear membrane. Although a number of individual nuclear components have been identified it has yet to be discovered how they are integrated at the molecular level into a functional nucleus. Recent evidence suggests that these structural elements, specifically the nuclear envelope and the nuclear matrix, play a critical role in regulating nuclear functions, including DNA replication, RNA transcription, and RNA processing. In this proposal we intend to investigate the way in which nuclear components interact to assemble a nucleus. For this, two cell- free extracts (derived from Xenopus eggs) have been developed to study both nuclear assembly and disassembly. The first extract can assemble nuclei around exogenously added DNA templates. The second can induce the ordered disassembly of nuclei into condensed chromosomes. These systems offer powerful tools for studying and experimentally manipulating the way in which the nucleus assembles, disassembles, and the structural components within the nucleus interact. Specifically, we intend to: 1) determine the role of topoisomerase II in organizing chromatin within the nucleus, 2) determine whether specific DNA sequences are important for attachment of chromatin to the nuclear periphery or matrix, 3) characterize the molecular mechanism regulating the assembly of a major nuclear structural element, the lamina, and 4) identify important proteins involved in regulating assembly and disassembly of the nuclear membrane. This work should provide valuable information not only on how the nucleus is organized, but also on the way in which the structural organization of the nucleus affects nuclear functions.
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