兰州大学机构库 >生命科学学院
Mre11蛋白相互作用分子的筛选及验证
Alternative TitleScreening and Validation of Proteins interacting with Mre11
吕明华
Thesis Advisor王勤 ; 秘晓林
2010-05-29
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
KeywordMre11N335蛋白 DNA损伤修复 酵母双杂交
Abstract生物体基因组DNA时常会受到一系列内源或外源DNA损伤试剂的作用而出现DNA断裂。如果DNA双链断裂不能得到及时有效地修复,会导致基因组不稳定性,如染色体缺失,多拷贝染色体等,引起多种人类疾病。在长期的进化过程中,生物有机体形成了一系列DNA损伤修复机制,以应对损伤的DNA。MRN复合物是参与DNA损伤修复的一个关键分子,由Mre11、NBS1和RAD50蛋白组成。研究发现MRN复合物在DNA双链损伤修复、同源重组、非同源末端连接、端粒结构维持、细胞周期检验点激活、DNA复制顺利进行,以及维持基因组的稳定性等方面都发挥着重要的作用,因此对于MRN复合物功能以及其发挥功能的机制的研究就显得至关重要。 本文以Mre11蛋白N端335个氨基酸残基作为诱饵蛋白(Mre11N335),通过酵母双杂交系统,从黑腹果蝇21小时胚胎cDNA文库中筛选与Mre11蛋白相互作用的分子,从而对其发挥功能的机制进行深入研究。以CG1945酵母菌作为宿主菌,通过SD-T-L-H-3A T营养缺陷培养基进行初筛,初筛得到的阳性克隆通过检测β半乳糖苷酶活性进一步筛选,将筛选到的阳性克隆连续涂三次SD-T-L -H-3AT营养缺陷培养板,丢失酵母菌中没有与Mre11N335蛋白发生相互作用的文库质粒。提取酵母菌中的质粒,用通用引物PCR扩增文库插入片段,HaeⅢ酶切PCR产物,根据PCR产物和酶切产物片段大小对克隆进行分组归类,去除重复。将分类后得到的质粒分别转化大肠杆菌,提取质粒,再次进行PCR扩增,HaeⅢ酶切,进一步进行分组归类。将分类后得到的质粒进行测序,测序结果与果蝇基因的cDNA序列进行比对,将与果蝇基因cDNA序列读码框一致的克隆挑选出来进行体内和体外再验证。体内通过共转AH109酵母菌、AH109酵母菌和Y187酵母菌杂交以及移码突变三个实验来验证。通过体内再验证筛选到五个与Mre11N335蛋白相互作用的基因,分别为CG4035 (eIF-4E), CG1216 (mri), CG4916 (me31B), CG5468 (TweedleM) 和CG3140 (Adk2)。体外通过免疫共沉淀实验验证这五个基因与Mre11N335蛋白之间的相互作用。CG1216 (mri), CG4916 (me31B)和CG3140 (Adk2)与Mre11N335之间相互作用已经得到证实。 关键字:Mre11N335蛋白;DNA损伤修复;酵母双杂交;
Other AbstractThe cellular genomes are subject to various types of exogenous or endogenous DNA-damaging agents that lead to changes in chromosome structure and DNA damage. If the DNA double strand break can not be repaired effectively and timely, that will lead to many human disorders. In the long-term evolution, the organism has developed a series of DNA damage repair mechanisms in response to DNA damage. MRN complex, including Mre11, NBS1 and RAD50, is one of the key factors involved in DNA damage repair. MRN complex participates in DNA damage repair, homologous recombination, non-homologous end joining, telomere structure maintenance, cell cycle checkpoint control, DNA replication and to maintain genomic stability. Therefore, it is important to study the functions of the MRN complex and ascertain the mechanism. In this paper, we used the N terminal 335 amino acid residues of Mre11 as a bait (Mre11N335), cDNA library of the Drosophila melanogaster 21h embryo as a prey pool, to do the Yeast Two-Hybrid screening. We used CG1945 yeast strain as a host, SD-T-L-H-3-AT dropout medium as selection medium, to do the first screening. β-galactosidase activity is another selection marker to be detected. We restreaked positive colonies on SD-L-T-H-3-AT plates 3 times to allow loss of some of the AD/library plasmids while maintaining selective pressure on both the DNA-BD and AD vectors. The positive colonies still contain duplicated library plasmids. We extracted plasmids from yeast strain, PCR amplified inserted fragments, digested with HaeⅢ to sort the colonies and eliminate duplicates. The screened plasmids were transformed into E.coli, and done PCR amplification, HaeⅢ digestion again to sort the colonies and eliminate duplicates. The screened positive colonies were sent to sequence, blasted with Drosophila cDNA sequence and selected the colonies that have the correct reading frame with the Drosophila gene. The positive colonies need to be retested. We did cotransfection in AH109 yeast strain, or yeast mating by AH109 and Y187 yeast strains, and frameshift mutation to retest the interaction in vivo. We got five interacting proteins, CG4035 (eIF-4E), CG1216 (mri), CG4916 (me31B), CG5468 (TweedleM) and CG3140 (Adk2) through above experiments. The interaction was also proved by co-immunoprecipitation in vitro. We have demonstrated the interaction between CG1216 (mri), CG4916 (me31B) or CG3140 (Adk2) and Mre11N335. Key words: Mre11N335 protein; DNA damage repair; Yeast Two...
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221941
Collection生命科学学院
Recommended Citation
GB/T 7714
吕明华. Mre11蛋白相互作用分子的筛选及验证[D]. 兰州. 兰州大学,2010.
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