Graduate Thesis Or Dissertation

 

Physical structure and reconstitution of chromatin core particles Public Deposited

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  • The nucleosome appears to be the basic structural unit of the eukaryotic chromosome. The usual nucleosome preparation, isolated from a micrococcal nuclease digest of nuclei, is heterogenous in DNA length and protein composition with some particles containing spacer DNA and associated protein. This heterogeneity complicated physical and reconstitution studies. As a first step in studying the physical structure of the nucleosome, I have isolated and studied the core particle. It was separated from those nucleosomes containing spacer DNA and histones HI and H5 by differential precipitation in 0.1 M NaCl. Large quantities of a very homogenous core particle preparation were later prepared by depleting chromatin of HI and H5 and then digesting it with micrococcal nuclease. The DNA in the core particle, while being more resistant than free DNA to thermal denaturation, denatures in two transitions. Forty base pairs of DNA in the core particle reversibly denature at 60°C in 1 mM cacodylate. The rest of the DNA, approximately 100 base pairs, denature irreversibly in the second transition at 74°C. Using circular dichroism spectroscopy to monitor the independent changes of the protein and DNA components, I found that the protein denatures only in the second transition. Nucleosomes containing spacer DNA were isolated and thermally denatured. The presence of H1 and H5 increased both the temperature and the amount of DNA melting in the second transition back to a value of 100 base pairs. Core histones and DNA were isolated and reconstituted by salt gradient dialysis to reform the core particle. The reconstituted particles, which could be prepared in 80-90% yield, were found to be identical to the native particles by the various physical and biochemical tests available. DNA molecules with 50 to 125 base pairs were isolated by preparative gel electrophoresis and reconstituted with core histones. Particles with less than 120 base pairs of DNA undergo intermolecular association reactions that depend on the size of the DNA and ionic strength. Particles containing 100 base pairs of DNA completely precipitate from solution at 0.15 M ionic strength while 120 base pair particles are completely soluble at the same ionic strength. Particles containing 110 base pair DNA undergo an intermediate degree of association. Core particles were reconstituted with two pieces of DNA each 65 base pairs long. While nearly identical to the native particle under most conditions, the 65 base pair particle appears to dissociate at very low ionic strength. This conformational change may be a more drastic example of a low ionic strength change observed in nucleosomes using electron microscopy or hydrodynamic methods.
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