Quantitative electron microscopy of chromosome fibers: Determination of the radial distribution of density.

Chromatin is a large, flexible, irregular molecule, which does not lend itself to high resolution structural techniques such as x-ray crystallography and NMR. To circumvent the inability to directly obtain high resolution structural information about chromatin, low resolution electron microscopic techniques were employed and the results compared with predictions of proposed models. Three classes of models for chromatin exist: the solenoid; the twisted-ribbon; and the crossed-linker models. Crossed-linker models have two distinct structural characteristics that distinguish them from solenoid and twisted-ribbon models; (1) fiber diameter and mass per unit length dependence upon DNA linker length, and (2) density at the center of the structure. These differences are distinguishable using quantitative electron microscopy. Direct measurement of the fiber diameter of chromatin from two different tissues having different DNA linker lengths was performed using chromatin images obtained by dark-field scanning transmission electron microscopy (STEM) and bright-field energy-filtered cryo-electron microscopy (cryo-EM). The diameters were found to increase as the length of the linker DNA increased. Radial density distributions were determined from the STEM images, revealing relatively uniform density and no central hole. Analysis of frozen-hydrated tobacco mosaic virus (TMV) demonstrated for the first time that cryo-EM images could also be used to quantitatively determine the mass and internal densities of biological molecules, and achieve resolutions significantly higher than those obtained by STEM. Mass per unit lengths determined from chromatin images obtained by cryo-EM agreed quantitatively with STEM and x-ray measurements, showing a direct dependence upon linker length. Cryo-EM radial density distributions revealed positive scattering density at all radii, with the highest mass per unit volume at the fiber center. The double-stranded crossed-linker model was the only proposed chromatin model to correctly predict the mass per unit length, diameter, and internal structure of the chromatin fibers. Currently no reliable data contradicts the predictions of the double-stranded crossed-linker model.PhDBiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105791/1/9208659.pdfDescription of 9208659.pdf : Restricted to UM users only