How chromatin is packaged within interphase and mitotic chromosomes remains a fundamental but largely unanswered question. Chromatin loop models have been based largely on extrapolations from radial loop models of mitotic chromosome structure or inferences from specialized chromosomes such as lampbrush or polytene chromosomes. A major technical impediment to progress in this field has been the low resolution of light microscopy and the lack of good DNA specific stains for transmission electron microscopy. We have proposed a chromonema model for interphase and mitotic chromosome structure in which 10 and 30 nm chromatin fibers are folded into large-scale chromatin fibers on the order of ~100 nm diameter. Our model initially was based on a strategy (more) in which we used light microscopy to document conditions for extracting soluble nucleoplasm from cells while preserving the appearance of interphase and mitotic chromosomes in living cells. The EM appearance of the large-scale chromatin fibers we were then able to visualize has since been confirmed more recently by independent approaches using DNA specific stains on nonextracted cells.
Further support for our chromonema model of interphase chromosome organization has come from live cell observations of engineered chromosome regions carrying lac operator repeats. These engineered chromosome regions can be visualized through expression of EGFP-lac repressor. Live cell microscopy reveals folding of large engineered chromosome regions into chromonema fibers extending over 5 microns in length. These fibers are relatively stable in their overall conformation over a time scale of hours, although regions of fibers can change orientation over a similar time scale. Over a time scale of seconds or less, rapid, short range movements are observed corresponding to conformational fluctuations of these large-scale structures. We have also confirmed the chromonema model by visualizing these engineered chromosome regions using a novel in vivo immunogold labeling procedure.
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