Proposed is a plan of research designed to determine the three-dimensional structure of Drosophila melanogaster chromosomes in a variety of biologically well defined functional states. The long range goal is to understand the structural complexities that underly DNA condensation and its organization into structures that can allow as well as modulate transcriptional activity. Of central interest is determining the structure of diploid mitotic chromosomes and polytene puffs, bands and inter-bands. These structures will form the starting point for examining the structural transitions accompanying chromosomal condensation during the mitotic cell cycle as well as those changes accompanying transcriptional activation. A direct structural analysis is proposed using early embryonic diploid interphase, prophase, and metaphase chromosomes and larval Malpigian tubule polytene interphase chromosomes. In collaboration with Dr. J. W. Sedat, we plan to study the 3-D arrangement of diploid chromosomes within the nuclei of intact cells in embryos, both as a function of cell cycle and developmental state of the tissue. Chromosome pairing and topological arrangement will be studied. This work will complement Sedat's studies on polytenized tissues. High resolution (100-200 Angstrom) three-dimensional data will be collected by HVEM tilt series from either isolated critical point dried chromosomes (mitotic chromosomes) or embedded and stained thick plastic sections (both diploid and polytene chromosomes). Low resolution (1500-2000 Angstrom) 3-D in situ data within intact nuclei will be collected using our recently developed 3-D optical section microscopy. The structural analyses 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. As an outgrowth of this work, development will continue on both EM and optical cellular tomographic techniques and computer modeling methods that have a wide application to other areas of cell biology.
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