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Title: Reconstitution of the 30-nm chromatin fiber
Author: Huynh, V.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2002
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This thesis describes the production of folded and regular 30-nm chromatin fibers reconstituted from purified components of DNA tandem repeats, histone octamer, and H5. Obtaining evenly spaced nucleosome arrays saturated with H5 is feasible with the use an artificially derived sequence with a high affinity for histone octamer. This artificial "sequence 601" positions the nucleosome specifically and uniquely. Folding of the 30-nm chromatin fiber requires that each DNA repeat (200 bp) be found or saturated with exactly one histone octamer and one H5. The basis of the reconstitution system presented in this thesis is the addition of mixed sequence competitor (or carrier) DNA which allows the reconstitution to be fully controlled. A combination of techniques including bandshift assays, restriction enzyme digestion, and electron microscopy are used to analyze the regularity and fully saturated nature of the reconstituted chromatin. The reconstituted chromatin arrays are shown to fold to form compact 30-nm chromatin particles as visualized by electron microscopy. The results suggest that a DNA fragment containing at least 19 tandem 200 bp 601 repeats (200-19mer) is required to produce stably folded particles as 200-12mer DNA repeats did not show sufficient stability. Analysis by cryoelectron microscopy showed that the reconstitution of 200-19mer DNA repeats resulted in both single chromatin particles and oligomerized chromatin fibers. Furthermore, the reconstituted chromatin fibers have a morphology similar to that of native chromatin fibers with the exception of appearing to be more regular (as desired). The production of regular chromatin fibers is a critical step towards the structural determination of the 30-nm chromatin fiber. Furthermore, these folded and reconstituted chromatin fibers provide the ideal substrate to address future questions of the role of higher order chromatin structure in biological processes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available