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This dissertation, Expression Studies of NAP79: a New Member of Nucleosome Assembly Proteins by Sze-wan, Fong, 方詩韻, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Expression studies of NAP79, a new member of Nucleosome Assembly Proteins Submitted by Fong Sze Wan For the Degree of Master of Philosophy at The University of Hong Kong in August 2003 NAP79 was isolated in a differential screen to find genes expressed in human heart samples. It was found to be highly expressed in the adult but not fetal human heart and brain using Northern blot analysis. NAP79 is a novel protein of the nucleosome assembly protein (NAP) superfamily. All members in this superfamily consist of a highly conserved NAP domain and are suggested to be involved in regulation of DNA synthesis/ proliferation and transcription through regulation of chromatin structure. In the present studies, I have sequenced the open reading frame of NAP79 and subcloned it into various expression plasmids. From the sequence analysis, NAP79 contains four nuclear localization signals and is predicted to be in the nucleus. By tagging with green fluorescence protein at the C terminal, I found that the NAP79 fusion protein was present in the nucleus, with different nuclear localization patterns in transfected COS 7 and HEK293 cells. The subcellular localization of NAP79 was further confirmed by tagging with a hemagglutinin epitope at the N terminal and transfection into the above mammalian cell lines. NAP79 fusion protein appeared as very fine dots and homogenously spread in the nucleus during interphase. Later it localized as bright discrete domains and appeared to surround the intense foci of DAPI in the nucleus. These domains became larger and fused together as chromosome condensation proceeded. From these results, I propose that NAP79 might participate in chromosome condensation by organizing the chromatin fiber into a more compact structure. The mouse homologue of NAP79 was isolated by library screening. I performed RT-PCR and revealed that the expression level of Nap79 in embryonic heart increased with increasing age of gestation. The expression of Nap79 in the adult mouse heart and brain was examined by in situ hybridization. The staining of Nap79 in atrium was higher than that of the ventricle of the heart. For adult brain, positive staining was found in cerebral cortex, hippocampus and cerebellum. Polyclonal antibodies against mouse Nap79 were raised with synthetic peptides for studying the protein expression of Nap79. The staining pattern in adult and neonatal hearts was similar, except for the subcellular localization of Nap79. Nap79 was predominantly localized in the nucleus of cardiomyocytes of postnatal day 0 (P0) and P8 heart. However, the nuclear staining of Nap79 gradually declined in P14 and P22 heart, and became cytoplasmic in the adult stage. Staining in cardiac smooth muscle and valves was negative. In the adult brain, nuclear staining of Nap79 was found in cerebral cortex and hippocampus, whereas both cytoplasmic staining and nuclear staining were found in cerebellum. In addition, I also attempted to investigate the functional roles of NAP79 by identifying its potential interacting partners using yeast two-hybrid screening. Although this part is not fruitful, the arrest of cell growth in yeast and the above expression data supports the hypothesis that NAP79 is involved in regulating cell proliferation in postnatal cardiomyocytes. DOI: 10.5353/th_b2949377 Subjects: NucleoproteinsGene expression

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