Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

This dissertation examines the interaction of the linker histone with DNA and with nucleosomes. The first goal of the project was to characterize the interaction of the linker histone with DNA. Three factors previously reported to influence the linker histone's interaction with DNA were examined: ratio of linker histone to DNA sites of binding, monovalent ions in the local environment, and conformation of the DNA molecules. Evidence obtained through gel mobility shift assays demonstrates the strong preference by the linker histone for DNA with superhelical torsion, i.e., supercoiling, and the negative cooperative mode of binding that the linker histone exhibits in association with supercoiled DNA. The second part of the dissertation examines the location of linker histone binding on the nucleosome, and documents the pronounced tendency of the linker histone to bind to two DNA duplex strands. A preparation of homogeneous nucleosome core particles, consisting of a defined 238 base pair DNA fragment and the core histone octamer positioned precisely on this DNA, was used as a substrate for the UV-induced crosslinking of the linker histone to the DNA of this nucleosome. By site-specific labeling of a single site on the DNA of the nucleosome, the linker histone was observed crosslinked at that labeled site, confirming that the linker histone binds at the pseudo-dyad axis of the nucleosome. This evidence was used to support a model of linker histone binding to the nucleosome that invokes the association of the linker histone with no fewer than two duplex strands of DNA of the nucleosome. / Graduation date: 2004

In order to better characterize linker histone interactions with DNA, avian erythrocyte-specific linker H5 and the trypsin-resistant globular domain of H5 (GH5) were used in DNA binding studies. To begin, H5 displayed a considerably higher binding affinity for DNA than the isolated globular domain (GH5), supporting the importance of the terminal tail domains in binding. This conclusion is based upon binding curves conducted in low-salt solution, and on the considerably-higher salt concentration required to prevent protein-DNA contact. Linker histones also induce DNA-protein aggregation in a process that was found to result in protein insolubility in 2% SDS, and included protein-protein interactions that did not require the terminal tail domains. In addition, DNA supercoiling appeared to impede the aggregation process; this that may be attributable to binding of linker histones in isolated clusters, as gauged by a limit in the number of observed dithiobis (succinimidyl) propionate (DSP)-crosslinkable contacts. In a related study, the property of GH5 to bind, then organize onto DNA was investigated. GH5 crosslinked onto DNA with dithiobis (succinimidyl propionate), then cleaved with chymotrypsin, displayed highly uniform contacts that appeared to involve the C-terminal four amino acids, and suggests protein-protein interactions are important for binding. This finding may be relevant since GH5 (and H5) were observed to self-associate free in solution in an arguably specific manner. Finally, the exposure of Phe 93 to chymotrypsin was used to identify the surface of the globular domain that contacts DNA for the binding of intact H5. Results suggests that the side of the protein opposite to the recognition helix preferentially binds to DNA, supporting a novel winged-helix protein DNA-binding mechanism. Furthermore, parallel studies with octamers reconstituted onto a DNA fragment with twelve copies of the 208 b.p. rDNA 5s gene from Lytechinus variegatus, shows that H5 had a high binding affinity with all detectable protein binding to the reconstituted complex. H5 binding conferred protection to a site located near the dyad axis from endonuclease digestion, supporting the contention that H5 binds near or at the nucleosome dyad axis. H5 binding also was observed to condense fibers as observed from agarose gel electrophoresis, although velocity analytical sedimentation studies indicate that H5 in itself was not sufficient to fully compact chromatin fibers; rather H5 and 30 mM NaCl, in combination, were required. Results indicate that the chromatin-reconstituted "208-12 DNA" makes an excellent model for analyzing the effect of linker proteins on chromatin morphology. / Graduation date: 1998

Thesis (Ph. D.)--California Institute of Technology, 1994. UM #94-06,216. / Advisor names found in the Acknowledgements pages of the thesis. Title from home page. Viewed 01/15/2010. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 110-115). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 135-149). Available also in a digital version from Dissertation Abstracts.

No description available.

The binding of a drug or other ligand to plasma proteins can effect their absorption, metabolism and excretion which can lead to a change in its toxicity and therapeutic action. Fluorescence is a technique that has been used to study such interactions and has the advantages of extreme sensitivity and specificity. Previously fluorescence has been monitored in the UV /vis range of the spectrum. However, a new development is long wavelength fluorescence (600-1000nm), which has the added benefits of a lower background, decreased scattering, decreased photodecomposition and the availability of inexpensive, solid state, optical components. Certain dyes including polymethines, xanthenes and phenoxazines that fluoresce in the long wavelength region (600-1000nm) of the spectrum were investigated for use as fluorescence probes. Nile Red, a strongly hydrophobic phenoxazine dye, was found to have an emission wavelength and intensity which was strongly dependent on the polarity of its environment. Consequently, it was bound to certain proteins including bovine and human serum albumin, aI-acid glycoprotein and B-lactoglobulin and provided both qualitative and quantitative information on the nature and type of binding site on the protein. It was also used in the study of ligand protein binding interactions in which competition for a binding site on the protein occurs between the probe and other ligands such as drugs or fatty acids. The project also involved a preliminary investigation into a novel double probe technique for the study of drug-protein interactions and the development of a flow injection analysis method involving gradient titration of a drug against a probe:protein system.

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: Abraham M. Lenhoff, Dept. of Chemical Engineering, and Eric W. Kaler, College of Engineering . Includes bibliographical references.

Thesis (M.Phil.) - University of St Andrews, March 2007.