A comparison of the structure of isolated and in situ chromatin fibers was performed using starfish (Patiria miniata) sperm nuclei. The simple protein content consisting of five major histones was found. A DNA repeat length of 222bp was calculated. Compact chromatin fibers solubilized from detergent isolated nuclei showed diameters of 38 to 44nm by electron microscopy. Chromatin fibers observed in whole cells and in mildly treated nuclear preparations revealed fiber diameters of approximately 24nm when embedded in epoxy resin. Investigation of the chromatin isolation process uncovered a mechanism where fiber integrity was lost during nuclear isolation. Following nuclease digestion and chromatin release, fiber-like structures reformed in solution. The relationship of the isolated "fibers" to the native structures is not known. Nuclear permeability has been found to be a factor in both release of cleaved chromatin from nuclei and inward diffusion of nuclease. Permeability to various sized dextrans was consistent with a mechanism where cut chromatin exits nuclei as nucleosomal chains and aggregates in solution to form the isolated chromatin state. Attempts to isolate detergent free nuclei revealed that completely isolated nuclei were susceptible to the loss of chromatin fiber integrity suggesting that the medium was incapable of maintaining fiber structure. Other buffer compositions did not improve stability of in situ fibers underscoring a general lack of understanding of the nuclear environment.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-8369 |
| Date | 01 January 1992 |
| Creators | Giannasca, Paul J |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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