Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Pathology and Laboratory Medicine

Supervisor

Knoll, Joan HM

Abstract

During mitosis, interphase chromatin structures change dramatically to allow formation of discrete chromosomes. The mechanisms that follow, allowing rapid and reproducible re-establishment of functional interphase organizations, remain elusive. Our laboratory identified locus-specific condensation differences (referred to as differential accessibility [DA]) in metaphase chromosome homologues by visualizing genome sequence-defined single-copy (sc) DNA probes using fluorescence in situ hybridization (FISH). Original identifications of DA loci were performed with individual scFISH probes (1.5–4kb) in lymphocyte chromosomes. In this study, we computationally designed multiple adjacent scFISH probes for 6 different DA loci and determined that DA occurs in domains. Domain lengths varied from ~16kb-130kb. DA was also investigated in other cell types (bone marrow and fibroblast samples) using 5 known DA probes and found to be maintained. DA is a conserved, structural feature in metaphase chromosomes that may play a role in the maintenance of chromosome memory from parent to daughter cells.

Summary for Lay Audience

Human DNA is organized into 23 different pairs of chromosomes. One member of each pair is inherited from each parent. Chromosome structure changes from being dispersed and string-like during periods of cell growth to highly compacted individual units during cell division. During the compaction phase, important functional structures disappear, however, chromosomes must return to their string-like form to function properly for cell growth. These changes in structure are necessary to form healthy daughter cells identical to the parent cell. This cycle continues for each new generation. The memory that allows this cycle of accurate chromosome reorganization from one cell generation to the next is not well understood.

Highly compacted chromosomes can be seen using a microscope and individual pairs of chromosomes can be identified. Our laboratory examines different regions of compacted chromosomes using a technique called FISH (fluorescence in situ hybridization) that identifies short regions of DNA by colouring the targeted region with fluorescence using DNA probes. Using FISH with short, unique human DNA probes, we previously identified novel non-random structural differences within individual compacted chromosomes pairs. Within a chromosome pair, using these DNA probes, particular regions of one chromosome of the pair shone brighter than the other. We refer to this finding as differential accessibility (DA). Identified in white blood cells in short individual regions on multiple chromosomes, DA results from different amounts of compaction at the same region between members of a chromosome pair.

In this study, new DA regions were identified. Six regions were expanded to include neighbouring, unique DNA sequences to determine if DA extended past the edges of regions identified by one probe. DA was found to extend outside of these individual regions. Five regions investigated in blood, bone and skin cells showed DA to be conserved at the same locations across the three tissues. This supports DA as a structural feature of compacted human chromosomes present during cell division that spans beyond the edges of individual unique regions at the same location in different cell types. DA may help to accurately transmit the memory of important structures between parent and daughter cells.

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