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Study of the yeast Noc3p homolog in human cells

Authors Hu, Yun
Issue Date 2006
Summary Discovery of the double helix structure of DNA (deoxyribonucleic acid) unveils the molecular mechanism of inheritance and mutation. For a living cell to function properly, DNA, the genetic material for inheritance, must be duplicated precisely in vivo. So far two mechanisms have been discovered that ensure this: the existence of replication origins throughout the genome and the coordinated presence and absence of replication initiation proteins on the sites of replication origins. One of the key initiation proteins Noc3p (nucleolar complex associated proteins-3) has been reported by our lab to be required for the normal progression of DNA replication initiation in budding yeast. An earlier report found Noc3p to be one of the components of the machinery that participates in maturation of preribosomal particles and their subsequent transport from the nucleolus via the nucleoplasm into the cytoplasm. Recently, a human ortholog FAD24 (Factor for Adipocyte Differentiation 24) has been cloned and shown by another group to be involved in stimulating adipocyte differentiation. In this project, one set of seven siRNAs (small interfering RNA) have been designed and commercially synthesized before a titration experiment was performed to find out the optimal conditions for these siRNAs-based gene silencing in cultured HeLa cell lines. Two different siRNAs targeted to the coding and 3'-UTR region were transfected into FAD24-overexpressing stable cell lines to further test the gene silencing effect and the specificity of the siRNAs. After silencing the endogenous FAD24 by siRNAs, the proliferation of the HeLa cells was inhibited significantly as observed using the WST-1 assay. Furthermore, depletion of FAD24 gene expression in a cell population synchronized by double thymidine could lead to increased occurrence of the sub G1 peak, characteristic of apoptosis. Throughout the cell cycle, FAD24 binds to the chromatin region as indicated by chromatin binding assay. To conclude, this study shows that FAD24 is required for DNA replication initiation and cell proliferation in human cells. Inhibiting the expression of FAD24 could dramatically disrupt cell cycle progression and lead to apoptosis in cancer cells.
Note Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006
Language English
Format Thesis
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