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Dissertation Information for Brian David Athey

- Brian David Athey

- Ph.D.

- [No Discipline Recorded]

- University of Michigan, Ann Arbor (USA) (1990)

- None

- None

MPACT Status: Incomplete - Not_Inspected

Title: An EM study of chromatin fibers: Evidence in support of the double-helical crossed-linker model

Abstract: "Electron microscopy experiments were performed to test the validity of the proposed models for chromatin fiber structure. In one such experiment, chromatin fiber specimens from Thyone (sea cucumber) and Necturus (mudpuppy) were embedded into vitrified aqueous solutions of physiological ionic strength (124 mM) and imaged using cryo-electron microscopy procedures. Diameter measurements were performed using interactive digital image-processing techniques. Thyone and Necturus fibers had significantly different estimated mean outer diameters of 43.5 nm (CD = 4.2 nm; SEM = 0.61 nm) and 32.0 nm (SD = 3.0 nm; SEM = 0.36 nm), respectively. The same specimens were negatively-stained, and had estimated outer diameters of 38.7 nm (SD = 4.0 nm, SEM = 0.8 nm) and 29.0 nm (SD = 2.2 nm; SEM = 0.8 nm), respectively. These fibers suffered $\sim$10% shrinkage, and were found to be significantly flattened. The DNA linker length of Thyone briareus sperm ($N$ = 87 bp) is significantly longer ($\sim$13 nm) than that of Necturus maculosus erythrocytes ($N$ = 48 bp). The diameter difference of $\sim$12 nm suggested that a direct relationship between fiber diameter and DNA linker length exists. These diameter measurements and the published EM and x-ray data regarding chromatin fiber diameter support this hypothesis. The constant 30 nm diameter solenoid and twisted-ribbon models are inconsistent with these diameter data.

Scanning transmission electron microscopy (STEM) mass analysis of freeze-dried Thyone and Necturus fibers was also performed; linear mass densities were 12 $\pm$ 2 and 7.5 $\pm$ 1 nucleosomes/10 nm, respectively. This demonstrated that chromatin fiber mass per unit length also increases with linker length. Additionally, computer Fourier transforms of negatively-stained Thyone and Necturus fibers and of frozen-hydrated Thyone fibers indicated that chromatin helical pitch is independent of linker length, in agreement with x-ray diffraction results. The average spacings of the strongest layer line were 1/s = 13.9 nm (SD = 1.7 nm; $n$ = 11) and 1/s = 12.9 nm (SD = 0.4 nm; $n$ = 7) for negatively-stained Thyone and Necturus fibers, respectively. These data were most consistent with double-helical symmetry, with a pitch of 26-28 nm. Images of metal-shadowed air-dried Necturus fibers suggested that the $\sim$13 nm helical family in chromatin is left-handed, consistent with computed diffraction patterns of negatively-stained Thyone and Necturus fibers, and with model building studies. The left-handed double-helical crossed-linker model is in quantitative agreement with the fiber diameter, mass per unit length, and helical diffraction data."

MPACT Scores for Brian David Athey

A = 0
C = 1
A+C = 1
T = 0
G = 0
W = 0
TD = 0
TA = 0
calculated 2008-01-31 06:19:19

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Students under Brian David Athey

- None

- Ann S. Zimmerman - University of Michigan, Ann Arbor (2003)