着丝粒和端粒是真核细胞染色体的重要组成部分，他们都是由DNA和蛋白质组成的复合体。研究发现水稻的着丝粒DNA含有大量CentO卫星重复序列，而端粒DNA由富含鸟嘌呤的重复序列组成。他们的蛋白质成份在着丝粒和端粒发挥其功能的过程中起到非常重要的作用，然而对这些蛋白的了解却很少。该项研究通过使用affinity pull down技术和其他蛋白质组学方法捕捉到了一系列DNA特异性结合的蛋白；其中有86个与CentO序列结合和135个与水稻端粒重复序列结合的蛋白质被捕捉到。通过使用体外蛋白质与DNA结合验证方法，其中的一个蛋白Os02g0288200被证实了具有特异性结合着丝粒DNA的功能。同时，发现了4个水稻端粒结合蛋白。这些结果显示了affinity pull down技术能有效的应用于分离DNA特异结合蛋白，特别是着丝粒和端粒DNA结合蛋白的研究中。此外，在CenH3体外功能研究中，我们发现水稻内源CenH3蛋白对不同DNA序列的结合能力不同；与水稻rDNA序列相比，CenH3对着丝粒特异的DNA序列的结合能力更强。在研究DNA甲基化对CenH3与DNA结合能力的实验中，我们同时发现水稻CenH3蛋白对甲基化的CentO序列比未修饰的CentO序列的结合能力弱。这个结果同着丝粒功能区DNA次甲基化相吻合。 / Centromeres and telomeres are both DNA/protein complex and essential functional components of eukaryotic chromosomes. Previous researches have shown that rice centromeres and telomeres are occupied by CentO satellite repeat and G-rich telomere repeats, respectively. However, the protein components are not fully understood. DNA binding proteins associated with centromeric or telomeric DNA components will be most likely important for the understanding of centromere and telomere structure and functions. To capture DNA specific binding proteins, affinity pull down technique was applied in this research to isolate rice centromeric and telomeric DNA binding proteins. 86 proteins and 135 proteins were pulled down from CentO column and telomere repeat column respectively. One putative CentO binding protein, Os02g0288200, was demonstrated to bind to CentO specifically by in vitro assay. A conserved domain, DUF573 with unknown functions was identified in this CentO binding protein, and proven to be responsible for the specific binding to CentO sequence in vitro. Four proteins identified as telomere binding proteins in this research were studied by different groups and reported previously. These results demonstrate that the DNA affinity pull down technique is powerful in the isolation of sequence specific binding proteins and may be applicable in future studies of centromere and telomere proteins. In addition, the binding affinity of CenH3 to various forms of DNAs was analyzed by in vitro studies. The results show that rice endogenous CenH3 binds stronger to rice centromeric DNA sequences than rDNA sequence control, and prefers unmethylated CentO DNA sequence to methylated form. This phenomenon may provide explanation of the hypomethylation of centromeric DNAs in active centromeres. / Detailed summary in vernacular field only. / He, Qi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 50-55). / Abstracts also in Chinese. / List of Figures --- p.iii / List of Tables --- p.iv / List of Abbreviations --- p.v / Acknowledgements --- p.vii / Abstract --- p.viii / 摘要 --- p.ix / Chapter Chapter 1 --- LITERATURE REVIEW --- p.1 / Chapter Chapter 2 --- IDENTIFICATION OF CENTROMERE AND TELOMERE DNA BINDING PROTEINS IN RICE --- p.10 / Chapter Chapter 3 --- IN VITRO STUDIES OF CENH3 BINDING TO CENTRIMERIC DNA AND ITS METHYLATED FORM --- p.36 / Chapter Chapter 4 --- CONCLUSIONS AND PERSPECTIVES --- p.48 / References --- p.50
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328795 |
| Date | January 2012 |
| Contributors | He, Qi., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (ix, 67, 1 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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