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We have developed a color barcode labeling strategy for use with fluorescence in situ hybridization that enables the discrimination of multiple, identically labeled loci. Barcode labeling of chromosomes provides long-range path information and allows structural analysis at a scale and resolution beyond what was previously possible. Here, we demonstrate the use of a three-color, 13-probe barcode for the structural analysis of Drosophila chromosome 2L in blastoderm stage embryos. We observe the chromosome to be strongly polarized in the Rabl orientation and for some loci to assume defined positions relative to the nuclear envelope. Our analysis indicates packing approximately 15- to 28-fold above the 30-nm fiber, which varies along the chromosome in a pattern conserved across embryos. Using a clustering implementation based on rigid body alignment, our analysis suggests that structures within each embryo represent a single population and are effectively modeled as oriented random coils confined within nuclear boundaries. We also found an increased similarity between homologous chromosomes that have begun to pair. Chromosomes in embryos at equivalent developmental stages were found to share structural features and nuclear localization, although size-related differences that correlate with the cell cycle also were observed. The methodology and tools we describe provide a direct means for identifying developmental and cell type-specific features of higher order chromosome and nuclear organization.

molecular biology of the cell

Long-Range Interphase Chromosome Organization in<i>Drosophila</i>: A Study Using Color Barcoded Fluorescence In Situ Hybridization and Structural Clustering Analysis

Long-Range Interphase Chromosome Organization inDrosophila: A Study Using Color Barcoded Fluorescence In Situ Hybridization and Structural Clustering Analysis