The genes of eukaryotes exist as DNA-RNA-protein complexes known as chromatin. The structure of chromatin fluctuates to allow controlled access to genetic information while maintaining its important packaging function. Recent improvements in optics, image acquisition electronics, and live imaging techniques, as well as the introduction of fluorescent fusion proteins, have made it possible to use fluorescence light microscopy to study the dynamic nature of chromatin compaction in cells. Here we report the application of advanced fluorescence microscopy to characterize the effects of transcription on chromatin compaction in living yeast cells. Repressor protein-GFP fusion proteins which recognize specific operator sequences were used to fluorescently tag specific gene loci, and an OMX fluorescence light microscope was then used to track their positions in three dimensions. It was determined that image acquisition with the OMX microscope is rapid enough to track fluorescently tagged genomic loci in live yeast cells in 3D, and that it does so with a root mean squared (RMS) measurement error of 162 nanometers (nm). It was also determined that the OMX microscope can distinguish between strains with fluorescent spots separated by 40 or 70 kb genomic distances. Additionally, it was found that chromatin compaction of a 15 kb gene driven by the Gal1 promoter is correlated with the carbon source on which the cells are fed, and that three different carbon sources produce three different transcription-dependent chromatin structures. Reversible changes in end-to-end distance of ~500 nm within two seconds were detected in the induced strain. These findings indicate that improvements in light microscopy enable chromatin to be studied in living cells on a scale not previously possible.
|Date of Award||2010|
|Supervisor||Tom Owen-Hughes (Supervisor)|