Paediatrics (RCH) - Theses
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ItemHuman centromeric and neocentromeric chromatin( 2000-09)Human centromeres contain large arrays of α-satellite DNA that are thought to provide centromere function. These arrays show size and sequence variations. However, the lower limit of the sizes of these DNA arrays in normal centromeres is unknown. Using a set of chromosome-specific α-satellite probes for each of the human chromosomes, interphase Fluorescence In Situ Hybridisation (FISH) was performed in a population screening study. This study demonstrated that extreme reduction of chromosome-specific α-satellite is unusually common in chromosome 21 (screened with the αRI probe), with a prevalence of 3.70%, compared to <=.12 % for each of chromosomes 13 and 17, and 0 % for the other chromosomes. No analphoid centromere was identified in over 17,000 morphologically normal chromosomes studied. All the low-alphoid centromeres are fully functional as indicated by their mitotic stability and binding to centromere proteins including CENtromere Protein-A (CENP-A), CENtromere Protein-B (CENP-B), CENtromere Protein-C (CENP-C), and CENtromere Protein-E (CENP-E). Sensitive metaphase FISH analysis of the low-alphoid chromosome 21 centromeres established the presence of residual αRI as well as other non-αRI α-satellite DNA suggesting that centromere function may be provided by (i) the residual αRI DNA, (ii) other non-αRI a-satellite sequences, (iii) a combination of i and ii, or (iv) an activated neocentromere DNA. (For complete abstract open document)
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ItemCharacterisation of the centromere protein FAM44A in human and mouse cells( 2012)The centromere is responsible for ensuring correct segregation of newly replicated sister chromatids into daughter cells. This structure is found in all eukaryotes ranging from single cell to complex multicellular organisms. Any errors in chromosome segregation, including mutations in proteins that have a role in the assembly of the spindle microtubule attachment site, known as the kinetochore, can result in daughter cells with an abnormal chromosomal number, or aneuploidy. In humans, changes in chromosome number significantly contribute to medical conditions such as spontaneous abortions, infertility and birth disorders (Hassold and Hunt, 2001), and is commonly linked with cancer via changes in copy numbers of oncogenes and tumour-suppressor genes. A novel centromere protein, FAM44A, was identified by the screening of a panel of patient sera with autoimmune antibodies that localise to the centromere. These sera were chosen for their presence of uncommon fragment sizes as visualised by Western blot. One such serum sample was subsequently used to probe a HeLa cDNA phage expression library. The 330 kDa FAM44A protein was identified, and contains the following chromatin domains; AT-hook motif, Cps15 domain, histone deacetylase interaction domain, and proline rich domain. The main aim of this study was to localise and functionally characterise the cell cycle roles of the FAM44A protein in mammalian cells. The cellular localisation of FAM44A using a FAM44A-specific antibody demonstrated that this protein is a centromeric and is present during all the mitotic stages. The functional study used RNAi-mediated down-regulation of FAM44A transcripts, which demonstrated a clear mitotic progression defect where accumulation of the cells at different mitotic stages was observed at 48 and 72 hours post siRNA knockdown. Further, several mitotic defects were observed to include, poor chromosome alignment during metaphase, lagging anaphases and chromatin bridges and an increase in the number of cells with micronuclei. These mitotic defects indicate that this protein plays an important role in the correct segregation of chromosomes during mitosis. Interestingly, further analysis showed most of these aberrant chromosomes and micronuclei were acentric, which suggest that FAM44A could be involved DNA repair, chromatin modification or remodelling processes. The identification and characterisation of FAM44A in this study contributes to the growing list of novel centromere proteins discovered in recent years. From the interesting mitotic phenotype observed, we can anticipate that further in-depth characterisation will fully define FAM44A as an important component of the centromere and chromatin that has multiple functions in the regulation of the cell cycle and chromosome segregation.