103 leaves : ill. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1972

Thesis (Ph.D.) -- University of Adelaide, Department of Biochemistry, 1972.

Wuppertal, Universiẗat, Diss., 1999.

Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references (leaves 364-365). Also available online through Digital Dissertations.

No description available.

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Chromatin is a highly dynamic complex that facilitates the structural transitions required for specific gene expression. An emerging player in the regulation of such chromatin functions are histone H2A variants. These proteins alter the histone-histone and histone-DNA interactions within the nucleosome to generate specialized nucleosomes with dedicated function. In this regard, it is quite possible that the Cterminal tails of H2A proteins confer a direct structural effect by altering the stability or folding potential of nucleosome arrays. This thesis addresses this issue by presenting the biophysical characterization of chromatin particles reconstituted with three different histone H2A variants. H2A.2, an essential protein, destabilizes the nucleosome and reduces the salt-dependent folding propensity of chromatin. H2A-Bbd, a histone variant exclusive to transcriptionally active domains, destabilizes the nucleosome and is more mobile within the nucleus. MacroH2A, which is believed to be involved in transcriptional repression, stabilizes the nucleosome and displays a C-terminal domain that is enriched in a-helix and adopts a globular conformation. Using irnmunochemical analysis it was determined that macroH2A is only found in subphylum vertebrata, is evenly distributed throughout autosomal chromatin at various levels of structure, and has a mutually exclusive relationship with histone HI. Interestingly, the ADP-ribosylation of macroH2A results in a stoichiometric decrease from two copies to one copy of macroH2A in a specific nucleosome, suggesting that the post-translational modification of histone variants may directly regulate nucleosome integrity

Understanding the structure of the 30 nm fiber in chromatin is relevant to understanding eukaryotic replication and transcription. The major controversy among the models of the fiber concerns the disposition of the linker DNA, the DNA between adjacent nucleosomes, and the location of the linker histones. To determine if the location of the linker histones and the linker DNA was internal or external, chromatin was digested with immobilized proteases and nucleases. The chromatin was probed either in a low salt extended 10 nm fiber of nucleosomes or in progressive compactions (addition of increasing amounts of salt) to form a condensed (30 nm) fiber. Digestion experiments performed on linker histones either in chicken erythrocyte chromatin, or free in solution or bound in mononucleosomes revealed: (1) Histone H5 is more protected than histone H1 in the fiber; (2) The N-and C-terminal portions of H1 do not change their accessibility upon compaction of the fiber; the tails of H5, however, become significantly internalized in the 30 nm fiber; (3) phenylalanine in the globular domain of both H1 and H5 is inaccessible both in the fiber and in mononucleosomes. Sedimentation velocity measurements demonstrate that the conformation of the fiber at all its different condensation states is highly sensitive to cuts in even a few of the linker histone molecules. The structure of these chromatin fibers has also been probed using micrococcal nuclease, both membrane-immobilized and free in solution, under extremely mild digestion conditions. The linker DNA is almost completely protected against digestion in the 30 nm fibers, whereas it is readily accessible in the more extended structures, independent of whether immobilized or free enzyme is employed. To circumvent complications due to the sensitivity of the enzyme to the salt concentration, control experiments were performed in which chromatin fibers were glutaraldehyde-fixed under different ionic conditions and then digested in low salt. The results were very similar to the above. Experiments with fibers of intermediate degree of condensation revealed a direct relationship between the degree of compaction and the resistance of linker DNA to digestion. These results support models for chromatin structure in which access to the linkers is limited by local steric hindrance, rather than by internalization in the core of the fibers. / Graduation date: 1993

By using immobilized trypsin, I have been able to prepare well-defined, stable trypsinized nucleosomes. The difficulties of lacking of control in the extent of trypsinization, which were encountered in previous studies with the use of free trypsin, have been eliminated. The nucleosomes and oligonucleosomes prepared by immobilized trypsin are suitable for biochemical and biophysical studies to analyze the function of the histone N-terminal regions ("tails"), which are removed by trypsin treatment, on chromatin structure and stability. Studies were first conducted using the trypsinized nucleosome core particles to examine the role of the histone tails in the stabilization of the nucleosome core particle. While it was found that these tails have little effect on either the nucleosome dissociation or the conformational transition in salt, they play a very important role in determining thermal stability of the nucleosome. The differential effects of selective removal of these tails also provided more insight about histone-DNA interactions in the nucleosome core particle. Experiments have also been carried out to investigate the change in structure and hydration of nucleosome core particles which may be associated with the salt-dependent conformational transition. Changes in the tertiary structures are suggested to be responsible for the salt-dependent transition. Roles of the histone tails in determination of nucleosome positions along specific DNA sequences were examined by analysis of nucleosome positioning on a specific eukaryotic gene sequence (Lytechinus Variegatus 5S rRNA gene) after in vitro nucleosome reconstitution with native and trypsinized histone octamers. Data obtained indicate that the histone tails are not required for nucleosome positioning. Results also seem to restrict the portions of histones which are responsible for determining nucleosome positions to the globular regions of (H3/H4)₂ tetramer, and possibly H2B. Studies with different DNA templates strongly suggest that the most important determinants of nucleosome positioning are the mechanical properties (such as bending and flexibility) of the DNA molecule. Taking together, it seems that the N-terminal tails of the histones may play roles in stabilizing both nucleosome structure and the higher-order structure of chromatin. / Graduation date: 1991

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xv1, 151 p.; also includes graphics. Includes abstract and vita. Advisor: Mark Parthun, Dept. of Biochemistry. Includes bibliographical references (p. 138-151).