This proposal concerns the three dimensional (3D) arrangement and detailed structural organization of components within the cell nucleus. The nucleus is a complex organelle that contains, in the form of specific DNA sequences, the information required for cell construction, maintenance and replication. Although recent advances in molecular biology have revealed many of the details of gene sequences and the molecular aspects of the decoding and transmission of genetic information, knowledge of the spatial framework within these mechanisms operate is still meager. This is impeding a full understanding of the most basic cell functions - gene activation, and gene transmission. Experiments are proposed to determine the 3D architecture and spatial interrelationships of chromatin structures within the nucleus. Electron microscopy of frozen samples will be used to determine the native hydrated conformation of chromatin subunits, and their fundamental folding properties. The same novel technique will be applied to a regulatory region of the albumen gene where precise positioning of subunits correlated with gene activation, and it will be possible to compare the 3D conformation of the active and inactive states. At a larger organizational scale, preparative techniques developed specifically for DNA-protein complexes will be used to examine the conformational transitions that occur when interphase chromatin is transformed into metaphase chromosomes. The detailed 3D information will be used to evaluate existing models of chromosome organization and formation.
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