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MBC in Press, published online ahead of print September 15, 2004
Mol. Biol. Cell 10.1091/mbc.E04-04-0289

A more recent version of this article appeared on December 1, 2004
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Submitted on April 7, 2004
Revised on September 2, 2004
Accepted on September 3, 2004

Long Range Interphase Chromosome Organization in Drosophila: A Study Using Color Barcoded FISH and Structural Clustering Analysis

Michael G. Lowenstein,* Thomas D. Goddard,{dagger} and John W. Sedat{ddagger}{sect}

*Graduate Group in Biophysics, {dagger}Computer Graphics Lab, and {ddagger}Department of Biochemistry & Biophysics, University of California San Francisco, San Francisco, CA 94143

Monitoring Editor: Lawrence Goldstein

We have developed a color barcode labeling strategy for use with fluorescence in situ hybridization (FISH), which 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 ~15-28 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 were also 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.

{sect}Corresponding author. E-mail: sedat{at}msg.ucsf.edu






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