The proposed research builds on recent extensions to our hardware and software that allows, in a generalized resolution overlapping fashion, 3-dimensional reconstructions of subcellular structures at both the light and electron microscopic levels. We apply this methodology to the analysis of higher-order chromosome structure and function using the unique interphase polytene chromosomes of Drosophilia melanogaster. We focus on cytologically identified polytene chromosome bands, interbands and puffs (transcriptionally active, morphologically distinctive chromosome sites). We utilize Drosophila metamorphosis, triggered by the insect hormone ecdysone, as a way to target a characteristic transcriptional cascade. New technology, a 3-dimensional Bar Code, provides for a systematic approach to the study of 3-dimension diploid interphase chromosome topography in the nucleus on a cell-by-cell basis. Chromosome dynamics, in living nuclei, can now be studied using green fluorescent protein (GFP) to label specific chromosomal loci.
The specific aims of this proposal are: A.) Continue the determination of the 3-dimensional structure of specific polytene chromosome band, interbands and puffs using electron microscope tomography. B.) Study the in vivo structural dynamics (quantitative motion analysis) of pulse labeled DNA replication sites followed by ecdysone induced puff formation and regression. C.) Determine the 3-dimensional diploid chromosome topography in Drosophila early embryo nuclei using the 3- dimensional Bar Code methodology. D.) Analyze diploid chromosome nuclear topography within a PEV genetic perturbation on a cell- by-cell basis in eye imaginal disks. E.) Study the 4-dimensional dynamics of diploid chromosome behavior using the newly available GFP chromosome locus labeling methodology.
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