Proposed is a plan of research designed to determine the three-dimensional structure of polytene puffs, bands and inter-bands. The spatial arrangement of chromosomes within the the cell nucleus and its alteration as a function of cell cycle stage and development state will also be examined. A direct structural analysis is proposed using polytene interphase and diploid interphase, prophase, and metaphase chromosomes from Drosophila melanogaster. The structural analysis will rely extensively on recently developed computer methods for three-dimensional image reconstruction and enhancement. These methods are particularly powerful and require neither crystalline specimens nor internal symmetry. High-resolution three-dimensional data (100-500A) on polytene chromosomes will be collected by a HVEM tilt series on embedded, stained, thick sections. Low-resolution 3-D topology data will be collected by optical fluorescence microscopy using a computer-controlled Zeiss Axiomat microscope. Interpretation shall proceed by model building and comparison of structures from different biologically-defined chromosome states. Current research on problems of eukaryotic gene regulation has focused primarily on aspects related to DNA sequence organization and DNA modification. Nuclease sensitivity experiments have begun to hint at the importance of chromatin structural changes during gene activation. A more direct approach to understanding chromosome structure, its arrangement within the nucleus, and its degree of plasticity as a function of cell cycle stage, developmental state, and transcriptional activity is required. The proposed research is a first step in understanding these complex problems. As an outgrowth of this work development will continue on cellular tomographic techniques and computer modeling methods that should have a wide application to other areas of cell biology.
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