The object of this proposal is to determine the relationship between nuclear structure and nuclear function. In order to begin approaching this large problem we will carry out a rigorous biochemical analysis of the protein components involved in nuclear structure. Such a study is now possible due to our recent discovery that nuclei reconstitute de novo around naked DNA added to an extract made from Xenopus eggs. We will use this nuclear reconstitution system to analyze: 1) The relation between DNA sequence and its effect on the assembly of nuclear structure. 2) In determining the size and arrangement of chromatin loops within the nucleus. 3) To identify important structural components involved in nuclear reconstitution, and ask in what sequence they are incorporated into a reforming nucleus; and 4) Using cell cycle regulators, determine the relationship between nuclear organization in interphase nuclei and mitotic chromosomes. This work should provide a solid foundation upon which to further study the role of nuclear structure in DNA replication, transcription, and recombination. It should also be useful in furthering our understanding of the molecular regulators controling the transition of G2 to M phase of the cell cycle. As such it should be of general interest to both cell and molecular biologists working on basic problems of eukaryotic cell organization.
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