导　　师： 王伟

学科专业： G1010

授予学位： 硕士

作　　者： ;

机构地区： 山西大学

摘　　要： 在酵母、动物、植物中存在不同的CHROMODOMAIN蛋白，CHROMODOMAIN结构域能够特异的识别组蛋白赖氨酸的甲基化。CHROMODOMAIN蛋白常作为蛋白质复合体组分、酶组分或者核酸的结合位点。 在四膜虫有性生殖过程中大核基因经过小核基因的选择性的删除、复制而来，在此过程中发生了大量的DNA重排。一些CHROMODOMAIN蛋白参与了和调控了这一过程。本研究对嗜热四膜虫CHROMODOMAIN蛋白TCD3和TCD4进行研究，主要获得以下结果: 1.嗜热四膜虫中TCD3和TCD4基因的生物信息学分析TCD3（TTHERM00585180）和TCD4(TTHERM_00585190)串联在同一条染色体上并且间隔...

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The chromodomain is one of the major histone mark readers, specialized for the lysine methyl mark. Some chromodomain proteins function as adapters to recruit protein complexes, some chromodomains are structural components of enzymes and some chromodomains appear to have significant nucleic acid binding activity. Various proteins containing chromodomain have been identified in yeast, animals and plants. Extensive DNA rearrangements occur during the sexual conjugation process and lead to the differentiation of the developing somatic macronuclear genome from the germ line micronuclear genome in Tetrahymena. Chromodomain proteins are necessary players in the process. In this study, we characterized the two new chromodomain containing genes TCD3/(Tetrahymena Chromo Domain3/) and TCD4/(Tetrahymena Chromo Domain4/). The main results obtained are as follows: 1. Bioinformatic analysis of TCD3and TCD4. TCD3/(TTHERM/_00585180/) and TCD4/(TTHERM/_00585190/) are in the same chromosome and237bp interval. TCD3and TCD4encoded predicted polypeptide of334and344amino acids. The predicted molecular weight is40.26kDa and41.11kDa, respectively. Tcd3p and Tcd4p contain an N-terminal chromodomain. Quantitative real-time PCR showed TCD3and TCD4specifically expressed during conjugation stage and reached a peak at8-10h. 2. Tcd3p and Tcd4p are essential for conjugation development. To analyze the role of Tcd3p and Tcd4p during conjugation, TCD3-TCD4germ line knockout strains were constructed. Different mating type△TCD3-4-Ⅰ and△TCD3-4-Ⅱ strains were mated, development of the mating cells was aborted after the zygotic formation. Then the paired cells separated and developing macronuclei and one micronucleus degenerated. These results indicate that TCD3and TCD4are essential for the conjugation process. 3. Dynamic localization of Tcd3p and Tcd4p. To explore the function of Tcd3p and Tcd4p, HA-tagged TCD3and HA-tagged TCD4construct were created. During conjugation, HA-Tcd3p and HA-Tcd4p appeared initially in the parental macronucleus. At8-10h postmixing, HA-Tcd3p and HA-Tcd4p were detected predominantly in the anlagen and disappeared from old macronuclear. In addition to macronuclear localization, HA-Tcd4p was also observed within cytoplasmic structures conjusome. 4. Analysis of Tcd3p and Tcd4p functional domain. To investigate the mechanism of Tcd3p and Tcd4p specific localization, we exchanged chromodomain between Tcd3p and Tcd4p. Two new chimeric proteins HA-3CD-Tcd4p and HA-4CD-Tcd3p was expressed in Tetrahymena cells. Chromodomain of Tcd4p could carry the C-terminal region of Tcd3p to conjusome. Surprisedly, N terminal chromodomain of Tcd3p and Tcd4p were deleted, truncted Tcd3pdel1-68and Tcd4pdel1-55still localized on the developing new macronuclei. But, truncted Tcd4pdel1-55disappear from conjusome. Only N terminal chromodomain of Tcd4p, Tcd4pdel56-344, disappeared from the developing new macronuclei and dispersed in the cytoplasm. These results suggested that chromodomain of Tcd4p determined its conjusome specific localization; C-terminal region of Tcd3p and Tcd4p determined their macronuclear localization. 5. Conjusome localization of Tcd4p is RNA dependent. HA-TCD3and HA-TCD4strains were treated with RNase A. localization of HA-Tcd4p disappeared from conjusome. The results imply that conjusome localization of Tcd4p may be involved in chromodomain-RNA interaction. Taken together, TCD3and TCD4showed similar sequence, conserved chromodomain location and the similar expression profile. Tcd3p and Tcd4p showed dynamic localization during conjugation. Disruption of TCD3and TCD4in micronuclei resulted in developmental abortion before the new macronucleus formation. C-terminal region of Tcd3p and Tcd4p could localize in macronuclear. TCD4chromodomain interacts with RNA in vivo, which contribute to Tcd4p localization into the conjusome. The results provide new data for further exploring the function of the chromodomain proteins.

关 键 词： 嗜热四膜虫 重排 基因敲除 亚细胞定位 基因 基因

分 类 号： [Q75]

领　　域： [生物学]