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dc.contributor.authorVitharana, Samadhi Nipunika
dc.descriptionDissertation (Ph.D.)--University of Kansas, Chemistry, 2004.en_US
dc.description.abstractChromatin, the DNA-protein complex is believed to play a crucial role in the regulation of genes. The main focus of this study is to investigate the post-translational modifications (PTMs) of histones, the major class of protein associated with DNA in chromatin. Study of chromatin structure has been based on the context of Prader-Willi Syndrome (PWS), an imprinting genetic disorder. All the genes associated with this disease are clustered on the 15q11-13 region of chromosome 15.

As a preliminary step, an immunocytogenetic approach was taken to study the PTMs, especially acetylation in a specific genomic locus. Immunofluorescence (IF) was used as a tool to analyze the histone modifications while fluorescence in situ hybridization (FISH) with a single copy FISH probe was used to identify the genomic locus of interest. The resolution obtained by these immunocytogenetic tools was insufficient to provide information about the chemistry of chromatin at a molecular level.

These preliminary observations and the more recent findings about the possibility of multiple types of modifications of histories contributing to the regulation of genes led us to think in the direction of mass spectrometric analysis of histories. However, in order to analyze histories in a specific genomic locus such as the PWS region, histories need to be extracted from that particular region of the genome. Thus, sample preparation is the most critical step in such an analysis. In order to extract histories from a particular region of a chromosome, that region will have to be clipped from the rest and separated. This step would be a much easier task if the chromosome of interest is enriched. Therefore, the bulk of this dissertation is focused on the enrichment of chromosome 15 by two different approaches.

In one approach, the flow cytometric method, the main problem of lack of specificity, associated with the state-of the-art sorting of chromosomes by flow cytometry, which is based on the identification of chromosomes by fluorescent DNA binding dyes is addressed. Due to the poor specificity of this approach, there are many unresolvable groups of chromosomes that end up being sorted as a clump. In attempting to improve the purity of an individual chromosome by this method, the efficiency of sorting decreases dramatically.
dc.publisherUniversity of Kansasen_US
dc.rightsThis item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.en_US
dc.subjectPure sciencesen_US
dc.subjectBiological sciencesen_US
dc.subjectPrader-Willi syndromeen_US
dc.titleMapping chromatin structure: Isolation of a specific chromosomeen_US

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