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First published online 3 August 2005
doi: 10.1242/dev.01950
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1 Max Planck Institute for Biophysical Chemistry, Department of Molecular
Biology, 37070 Göttingen, Germany
2 King's College London, Randall Division of Cell and Molecular Biophysics, New
Hunt's House Guy's Campus, London SE1 1UL, UK
* Author for correspondence (e-mail: djovin{at}gwdg.de)
Accepted 21 June 2005
Fluorescence recovery after photobleaching (FRAP) microscopy was used to determine the kinetic properties of Polycomb group (PcG) proteins in whole living Drosophila organisms (embryos) and tissues (wing imaginal discs and salivary glands).
PcG genes are essential genes in higher eukaryotes responsible for the maintenance of the spatially distinct repression of developmentally important regulators such as the homeotic genes. Their absence, as well as overexpression, causes transformations in the axial organization of the body. Although protein complexes have been isolated in vitro, little is known about their stability or exact mechanism of repression in vivo.
We determined the translational diffusion constants of PcG proteins, dissociation constants and residence times for complexes in vivo at different developmental stages. In polytene nuclei, the rate constants suggest heterogeneity of the complexes. Computer simulations with new models for spatially distributed protein complexes were performed in systems showing both diffusion and binding equilibria, and the results compared with our experimental data. We were able to determine forward and reverse rate constants for complex formation. Complexes exchanged within a period of 1-10 minutes, more than an order of magnitude faster than the cell cycle time, ruling out models of repression in which access of transcription activators to the chromatin is limited and demonstrating that long-term repression primarily reflects mass-action chemical equilibria.
Key words: Polycomb group proteins, FRAP, Inverse FRAP, iFRAP, Transcription, Repression, Homeotic genes
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