利用酵母双杂交技术筛选p12~(CDK2AP1)新的相互作用蛋白Nbp及生物信息学分析
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摘要
癌是严重威胁人类健康,甚至致人死亡的恶性疾病,其发病率逐年增高,并有年轻化的趋势,一直以来都是医学领域中研究的重要课题之一。口腔癌的发生率高,在世界范围内口腔癌的发生率排在所有癌的第六位,且由于发病部位特殊,严重影响病人的外形和功能,甚至威胁患者生命。近年来在分子水平上研究癌的发生机制及其与癌基因、抑癌基因的相互作用关系已成为研究癌的发病机制及基因治疗的主要方向之一。目前的研究成果已经指出,癌的发生与癌基因的表达和抑癌基因的失表达密切相关。CDK2AP1(cyclin dependent kinase 2 associating protein 1)基因就是一个与口腔癌发病有关的候选抑癌基因。CDK2AP1基因原称DOC-1基因(deleted in oral cancer-1,口腔癌缺失-1),于1995年被Todd等人在仓鼠口腔癌模型中发现、分离并证实的一个候选抑癌基因,p12~(CDK2AP1)蛋白是其编码蛋白,具有抑制癌细胞生长的作用。人类CDK2AP1基因位于染色体12q24,编码115个氨基酸,其表达蛋白p12~(CDK2AP1)分子量为12.4kDa。在利用仓鼠模型对CDK2AP1的研究中发现,CDK2AP1可以诱导仓鼠口腔中恶性角化细胞凋亡。其表达蛋白p12~(CDK2AP1)通过下调CDK2(细胞周期依赖性蛋白激酶2)的活性和表达水平进而减弱对Rb基因的抑制,起到抑制癌细胞生长的作用。在鳞状细胞癌细胞中异位表达P12CDK2AP1蛋白后,其与CDK2和DNA-polymeraseα(DNA聚合酶α)结合,抑制了这两种酶的活性,使细胞的恶性表型发生转变。目前,关于p12~(CDK2AP1)蛋白在人口腔鳞癌中的表达显著降低甚至缺失的机理尚不完全清楚,与其相互作用蛋白及其发挥作用的蛋白网络尚不明确。
本研究采用酵母双杂交技术在正常人肝cDNA文库中筛选与p12~(CDK2AP1)蛋白相互作用的蛋白质,从而发现与p12~(CDK2AP1)蛋白相互作用的相关蛋白,利用生物信息学手段分析预测互作蛋白的性质类型,为进一步阐明CDK2AP1基因作用的机理,研究CDK2AP1的功能,揭示p12~(CDK2AP1)蛋白的作用网络奠定基础。
实验目的:
本课题研究的目的是通过酵母双杂交技术在正常人肝cDNA文库中筛选与p12~(CDK2AP1)蛋白相互作用的蛋白质,从而进一步研究p12~(CDK2AP1)蛋白在口腔癌缺失中的相关蛋白及作用网络。利用生物信息学手段分析互作蛋白的性质类型。通过研究p12~(CDK2AP1)蛋白与互作蛋白的相互作用关系,为进一步阐明CDK2AP1基因作用的机理奠定基础。
实验方法:
1、优化已有的酵母双杂交筛选工作体系,使其进一步高效准确,为后续筛选提供有利条件。
2、利用分子克隆的方法重组针对p12~(CDK2AP1)蛋白的酵母双杂交诱饵质粒,并验证其实用性。同时扩增正常人肝cDNA文库,为下一步进行p12~(CDK2AP1)蛋白的相互作用蛋白筛选奠定基础。
3、利用酵母双杂交技术在正常人肝cDNA文库中筛选与p12~(CDK2AP1)蛋白相互作用的蛋白。
4、使用生物信息学技术相关数据库和软件预测和分析互作蛋白的信息。包括互作蛋白的物种分类、基因组定位、互作蛋白的理化性质、跨膜特性、亚细胞定位、信号肽表达、二级结构、可能的功能基序和功能位点以及可能的同源蛋白之间的同源性比对。
实验结果:
利用酵母双杂交的方法,在正常人肝组织cDNA文库中发现了一个与p12~(CDK2AP1)蛋白有相互作用的新的蛋白质(GENE ID LOC93622、hypothetical protein BC006130),长度为119个氨基酸,暂时命名为Nbp(New binding protein,新结合蛋白)。通过酵母双杂交回转实验及NCBI blast,排除实验结果假阳性、酵母蛋白干扰、去除非编码区,确定该段cDNA序列为人类DNA,DNA序列符合率100%,染色体定位于人类4号染色体。发现其染色体定位、核酸序列、氨基酸序列MORF4蛋白(mortality factor 4)相似,可能与MRG(Mortality Related Genes)蛋白家族有一定的关系。利用生物信息学的方法,分析预测了Nbp蛋白的理化性质、二级结构、亚细胞定位、信号肽分泌以及其氨基酸序列中存在的可能的功能位点。
实验结论:
利用酵母双杂交技术成功筛选出了一个与p12~(CDK2AP1)蛋白相互作用的新的蛋白质Nbp,通过生物信息学方法预测了Nbp蛋白的理化性质、二级结构、亚细胞定位、信号肽分泌以及其氨基酸序列中存在的可能的功能位点。利用分子克隆技术成功重组了Nbp蛋白的真核表达质粒,为继续深入研究CDKAP1基因、p12~(CDK2AP1)蛋白及其互作蛋白的功能和作用网络奠定了基础。
Cancer is a serious threat to human health and even the deadly malignant disease, its incidence increased year by year as well as a young trend, and cancer has always been one of the important topics in medicine. Oral cancer ranking sixth in the world cancer incidence, and because of it's special area, oral cancer seriously impact the of patients' life and survive. In recent years, tumors mechanism and research with cancer gene, tumor-suppressor genes interacting relationship at the molecular level has become one of the main direction in tumor pathogenesis and gene therapy research . Current research results have indicated that the occurrence of cancer and cancer gene expression and tumor-suppressor genes lost expression are closely related. CDK2AP1 (cyclin associating protein kinase 2 dependent gene is a 1) is an oral cancer incidence relevant candidate tumor-suppressor gene. CDK2AP1 gene originally called DOC - 1 (deleted in oral cancer-1), which was found, separated and the confirmed as a candidate for tumor-suppressor genes by Todd in 1995 in oral cancer model in hamsters. P12CDK2AP1 protein is its coding protein, has the role of suppress tumor growth. CDK2AP1 gene is located in 12q24 human chromosomes, coding 115 amino acids, its expression protein p12~(CDK2AP1) the molecular weight of 12.4 kDa. In the research of CDK2AP1 in hamster model, CDK2AP1 can induce cell apoptosis of the oral malignant keratinocyte .Through down-regulating CDK2 (cut protein cell cycle dependence protein kinase 2) activity and the expression level, its expression p12~(CDK2AP1) weaken the Rb gene suppression, plays the role of inhibiting tumor cell growth. In squamous cell cancer cells ectopic expression, after the P12CDK2AP1 protein with CDK2 and DNA - polymerase alpha (DNA polymerases alpha) union, it inhibit the these two kinds of enzyme activity and change the malignant cell phenotype . At present, mechanism study of significantly reduced even lack of p12~(CDK2AP1) protein expression in oral squamous cell carcinoma the was not reported, and its interaction proteins and its protein network still not very clear.
Our study adopts yeast two-hybrid technology to screen protein interact with p12~(CDK2AP1) in normal liver double cDNA library, thus to find p12~(CDK2AP1) protein interactions with the related factors, then we adopts bioinformatics analysis and prediction by means of interaction proteins, to further illustrate different types of CDK2AP1 genetic mechanism of the effects of CDK2AP1, research p12~(CDK2AP1) protein function, reveal the role of foundation network.
Objective:
The purpose of this research is through yeast hybrid technology in normal liver double cDNA library screening and p12~(CDK2AP1) protein in the interaction of protein, thus further research p12~(CDK2AP1) protein in the related factors, and lack of oral role network. Use bioinformatics method analysis of the different types of interaction proteins. To further illustrate CDK2AP1 genetic mechanism of the effects lay the foundation.
Method:
1, optimize existing yeast two-hybrid screening work system, make its further effective and accurate, provide favorable conditions for the subsequent screening.
2, use molecular cloning p12~(CDK2AP1) protein in the reorganization of yeast two-hybrid bait plasmid, and verify its practicability. Meanwhile lay the foundation by amplify normal liver cDNA library for further p12~(CDK2AP1) protein interaction proteins screening .
3, use yeast two-hybrid technology to screen protein interact with p12~(CDK2AP1) protein in normal liver cDNA library.
4, using the method of molecular clone to restructure the original nuclear expression plasmid of the interaction proteins and induce its expression. Restructure p12~(CDK2AP1) protein eukaryotic expression plasmid expressing, and induce its expression.
5, use bioinformatics technology related database and software to forecast and analyze the information of interaction proteins. Including interactions protein species classification, genome positioning, physical and chemical properties of interactions, transmembrane characteristics, sub-cellular localization, signal peptide expression, secondary structure, the possible functions of the base sequence and function of homologous protein and the possible sites between than the homology.
Results:
Using the yeast two-hybrid method, we found a new protein (GENE ID LOC93622、hypothetical protein BC006130), interact with p12~(CDK2AP1) protein in normal liver cDNA libraries, the protein with length of 119 amino acids, temporarily named Nbp. Through the yeast two-hybrid rotary experiment and NCBI blast, excluding experimental results of false positive, yeast protein interference, wipe off none-coding areas and confirmed its cDNA sequence a human DNA pac with 100% coincidence rate in DNA sequence, the chromosomal locates in human chromosome 4. We find the chromosomal location, nucleic acid sequence, MORF4 protein (amino acid sequence mortality factor 4) similar, may be related to MORF MRG (those with a certain't) protein family . Using the method of bioinformatics we analyzed and predicted the physical and chemical properties, secondary structure, sub-cellular localization, signal peptide secretion and its existing in the amino acid sequence of the possible functions of the site of Nbp protein. And we successfully reorganized the Nbp protein eukaryotic expression plasmid.
Conclusion:
Using the yeast two-hybrid method, we successfully found a new protein Nbp interact with p12~(CDK2AP1), through bioinformatic, we predicted the physical and chemical properties, secondary structure, sub-cellular localization, signal peptide secretion and its existing in the amino acid sequence of the possible functions of the site of Nbp protein. Using molecular cloning, we successfully reorganized Nbp protein eukaryotic expression plasmids, laid a foundation for further research on CDK2AP1 genes, p12~(CDK2AP1) protein and their interactions, protein function and role network .
本研究采用酵母双杂交技术在正常人肝cDNA文库中筛选与p12~(CDK2AP1)蛋白相互作用的蛋白质,从而发现与p12~(CDK2AP1)蛋白相互作用的相关蛋白,利用生物信息学手段分析预测互作蛋白的性质类型,为进一步阐明CDK2AP1基因作用的机理,研究CDK2AP1的功能,揭示p12~(CDK2AP1)蛋白的作用网络奠定基础。
实验目的:
本课题研究的目的是通过酵母双杂交技术在正常人肝cDNA文库中筛选与p12~(CDK2AP1)蛋白相互作用的蛋白质,从而进一步研究p12~(CDK2AP1)蛋白在口腔癌缺失中的相关蛋白及作用网络。利用生物信息学手段分析互作蛋白的性质类型。通过研究p12~(CDK2AP1)蛋白与互作蛋白的相互作用关系,为进一步阐明CDK2AP1基因作用的机理奠定基础。
实验方法:
1、优化已有的酵母双杂交筛选工作体系,使其进一步高效准确,为后续筛选提供有利条件。
2、利用分子克隆的方法重组针对p12~(CDK2AP1)蛋白的酵母双杂交诱饵质粒,并验证其实用性。同时扩增正常人肝cDNA文库,为下一步进行p12~(CDK2AP1)蛋白的相互作用蛋白筛选奠定基础。
3、利用酵母双杂交技术在正常人肝cDNA文库中筛选与p12~(CDK2AP1)蛋白相互作用的蛋白。
4、使用生物信息学技术相关数据库和软件预测和分析互作蛋白的信息。包括互作蛋白的物种分类、基因组定位、互作蛋白的理化性质、跨膜特性、亚细胞定位、信号肽表达、二级结构、可能的功能基序和功能位点以及可能的同源蛋白之间的同源性比对。
实验结果:
利用酵母双杂交的方法,在正常人肝组织cDNA文库中发现了一个与p12~(CDK2AP1)蛋白有相互作用的新的蛋白质(GENE ID LOC93622、hypothetical protein BC006130),长度为119个氨基酸,暂时命名为Nbp(New binding protein,新结合蛋白)。通过酵母双杂交回转实验及NCBI blast,排除实验结果假阳性、酵母蛋白干扰、去除非编码区,确定该段cDNA序列为人类DNA,DNA序列符合率100%,染色体定位于人类4号染色体。发现其染色体定位、核酸序列、氨基酸序列MORF4蛋白(mortality factor 4)相似,可能与MRG(Mortality Related Genes)蛋白家族有一定的关系。利用生物信息学的方法,分析预测了Nbp蛋白的理化性质、二级结构、亚细胞定位、信号肽分泌以及其氨基酸序列中存在的可能的功能位点。
实验结论:
利用酵母双杂交技术成功筛选出了一个与p12~(CDK2AP1)蛋白相互作用的新的蛋白质Nbp,通过生物信息学方法预测了Nbp蛋白的理化性质、二级结构、亚细胞定位、信号肽分泌以及其氨基酸序列中存在的可能的功能位点。利用分子克隆技术成功重组了Nbp蛋白的真核表达质粒,为继续深入研究CDKAP1基因、p12~(CDK2AP1)蛋白及其互作蛋白的功能和作用网络奠定了基础。
Cancer is a serious threat to human health and even the deadly malignant disease, its incidence increased year by year as well as a young trend, and cancer has always been one of the important topics in medicine. Oral cancer ranking sixth in the world cancer incidence, and because of it's special area, oral cancer seriously impact the of patients' life and survive. In recent years, tumors mechanism and research with cancer gene, tumor-suppressor genes interacting relationship at the molecular level has become one of the main direction in tumor pathogenesis and gene therapy research . Current research results have indicated that the occurrence of cancer and cancer gene expression and tumor-suppressor genes lost expression are closely related. CDK2AP1 (cyclin associating protein kinase 2 dependent gene is a 1) is an oral cancer incidence relevant candidate tumor-suppressor gene. CDK2AP1 gene originally called DOC - 1 (deleted in oral cancer-1), which was found, separated and the confirmed as a candidate for tumor-suppressor genes by Todd in 1995 in oral cancer model in hamsters. P12CDK2AP1 protein is its coding protein, has the role of suppress tumor growth. CDK2AP1 gene is located in 12q24 human chromosomes, coding 115 amino acids, its expression protein p12~(CDK2AP1) the molecular weight of 12.4 kDa. In the research of CDK2AP1 in hamster model, CDK2AP1 can induce cell apoptosis of the oral malignant keratinocyte .Through down-regulating CDK2 (cut protein cell cycle dependence protein kinase 2) activity and the expression level, its expression p12~(CDK2AP1) weaken the Rb gene suppression, plays the role of inhibiting tumor cell growth. In squamous cell cancer cells ectopic expression, after the P12CDK2AP1 protein with CDK2 and DNA - polymerase alpha (DNA polymerases alpha) union, it inhibit the these two kinds of enzyme activity and change the malignant cell phenotype . At present, mechanism study of significantly reduced even lack of p12~(CDK2AP1) protein expression in oral squamous cell carcinoma the was not reported, and its interaction proteins and its protein network still not very clear.
Our study adopts yeast two-hybrid technology to screen protein interact with p12~(CDK2AP1) in normal liver double cDNA library, thus to find p12~(CDK2AP1) protein interactions with the related factors, then we adopts bioinformatics analysis and prediction by means of interaction proteins, to further illustrate different types of CDK2AP1 genetic mechanism of the effects of CDK2AP1, research p12~(CDK2AP1) protein function, reveal the role of foundation network.
Objective:
The purpose of this research is through yeast hybrid technology in normal liver double cDNA library screening and p12~(CDK2AP1) protein in the interaction of protein, thus further research p12~(CDK2AP1) protein in the related factors, and lack of oral role network. Use bioinformatics method analysis of the different types of interaction proteins. To further illustrate CDK2AP1 genetic mechanism of the effects lay the foundation.
Method:
1, optimize existing yeast two-hybrid screening work system, make its further effective and accurate, provide favorable conditions for the subsequent screening.
2, use molecular cloning p12~(CDK2AP1) protein in the reorganization of yeast two-hybrid bait plasmid, and verify its practicability. Meanwhile lay the foundation by amplify normal liver cDNA library for further p12~(CDK2AP1) protein interaction proteins screening .
3, use yeast two-hybrid technology to screen protein interact with p12~(CDK2AP1) protein in normal liver cDNA library.
4, using the method of molecular clone to restructure the original nuclear expression plasmid of the interaction proteins and induce its expression. Restructure p12~(CDK2AP1) protein eukaryotic expression plasmid expressing, and induce its expression.
5, use bioinformatics technology related database and software to forecast and analyze the information of interaction proteins. Including interactions protein species classification, genome positioning, physical and chemical properties of interactions, transmembrane characteristics, sub-cellular localization, signal peptide expression, secondary structure, the possible functions of the base sequence and function of homologous protein and the possible sites between than the homology.
Results:
Using the yeast two-hybrid method, we found a new protein (GENE ID LOC93622、hypothetical protein BC006130), interact with p12~(CDK2AP1) protein in normal liver cDNA libraries, the protein with length of 119 amino acids, temporarily named Nbp. Through the yeast two-hybrid rotary experiment and NCBI blast, excluding experimental results of false positive, yeast protein interference, wipe off none-coding areas and confirmed its cDNA sequence a human DNA pac with 100% coincidence rate in DNA sequence, the chromosomal locates in human chromosome 4. We find the chromosomal location, nucleic acid sequence, MORF4 protein (amino acid sequence mortality factor 4) similar, may be related to MORF MRG (those with a certain't) protein family . Using the method of bioinformatics we analyzed and predicted the physical and chemical properties, secondary structure, sub-cellular localization, signal peptide secretion and its existing in the amino acid sequence of the possible functions of the site of Nbp protein. And we successfully reorganized the Nbp protein eukaryotic expression plasmid.
Conclusion:
Using the yeast two-hybrid method, we successfully found a new protein Nbp interact with p12~(CDK2AP1), through bioinformatic, we predicted the physical and chemical properties, secondary structure, sub-cellular localization, signal peptide secretion and its existing in the amino acid sequence of the possible functions of the site of Nbp protein. Using molecular cloning, we successfully reorganized Nbp protein eukaryotic expression plasmids, laid a foundation for further research on CDK2AP1 genes, p12~(CDK2AP1) protein and their interactions, protein function and role network .
引文
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30 M.L. Figueiredo, S. Dayan, et al Expression of Cell-Cycle Regulator CDK2-Associating Protein 1 (p12CDK2AP1) in Transgenic Mice Induces Testicular and Ovarian Atrophy In Vivo MOLECULAR REPRODUCTION AND DEVELOPMENT 73:987–997 (2006)
31 Fields S, Songo.A et al Novel genetic systemto detect protein-protein interactions[J ].Nature ,1989 ,340(6230) :245 - 246.
32 Haian Fu .蛋白质-蛋白质相互作用方法与应用张幼怡等译,第一版,北京:北京大学医学出版社,2008年9月,270-288
33王玉飞,张影,贾雷立,蛋白质相互作用实验指南,第一版,北京:化学工业出版社,2010年3月,63-64.
34李先昆,聂智毅,曾日中,酵母双杂交技术研究与应用进展,安徽农业科学, 2009 ,37 (7) :2867 - 2869 ,2926.
35张阳德,生物信息学,第一版,北京:科学出版社,2005年9月,1-4
36赵雨杰,医学生物信息学,第一版,北京:人民军医出版社,2002年11月.
37 Hua Dong, Hoicheong Siu, Li Luo, Investigation gene and microRNA expression in glioblastoma BMC Genomics 2010, 11(Suppl 3):S16.
38 Joongho Shin, Ziqian Yuan, Kenneth Fordyce,et al. A del T poly T (8) mutation in the 3’untranslated region (UTR) of the CDK2-AP1 gene is functionally significant causing decreased mRNA stability resulting in decreased CDK2-AP1 expression in human microsatellite unstable (MSI) colorectal cancer (CRC). Surgery 2007, 142:222-227.
39金伟,孙沫逸,王文勇的,等,CDK2AP1基因的激光捕获显微切割应用研究,口腔医学,2010,30(4),193-195.
40郑子瑞,侯宁波,何湘,等,酵母双杂交技术筛选与RIG-Ⅰ相互作用蛋白,军事医学科学院院刊,2009,33(1),10-13.
41 Bertram, M. J., N. G. Berube, Identification of a gene that reverses the immortal phenotype of a subset of cells and is a member of a novel family of transcription factor-like genes. Mol. Cell. Biol. 19:1479–1485.
42 PARDO P S , LEUNG J K, LUCCHESI J C , et al. MRG15 , a novel chromodomain protein , is present in two distinct multiprotein complexes involved in transcriptional activation [ J ] . J Biol Chem , 2002 , 277 ( 52) : 508602-508661.
43 Kaoru Tominaga, Emiko Tominaga, et al The cell senescence inducing gene product MORF4 is regulated by degradation via the ubiquitin / proteasome pathway. Exp Cell Res. 2010 January 1; 316(1): 92–102.
44 LEUNGJ K, BERUBEN G, VENABLE S , et al. MRG15 activates the B-myb promoter through formation of a nuclear complex with the retinoblastoma protein and the novel protein PAM14 [ J ] . J Biol Chem , 2001 , 276 :39171239178.
45 TOMINAGA K, OLGUN A , SMITH J R , et al. Genetics of cellular senescence [J ] . Mech Ageing Dev , 2002 , 123 (8) :92729361.
46 Xue Zhang,,Hensin Tsao, Takanori Tsuji,et al. Identification and mutationanalysis of Doc-1R, a Doc-1 grow th suppressor-related gene. Biochemical and Biophysical Research Communications 255, 59–63 (1999).
47 GIANNI POZZI, MARCO R. OGGIONI, ALEXANDER TOMASZ, DNA Probe for Identification of Streptococcus pneumoniae, JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1989, Vol. 27, No. 2, p370-372.
48 Martin Leverkus, Martin R. Sprick, Tina Wachter, et al Proteasome Inhibition Results in TRAIL Sensitization of Primary Keratinocytes by Removing the Resistance Mediating Blockof Effector Caspase Maturation MOLECULAR AND CELLULAR BIOLOGY, Feb. 2003, Vol. 23, No. 3, p. 777–790.
49 Etienne Moussay, Valérie Palissot, Laurent Vallar, et al Research Determination of genes and microRNAs involved in the resistance to fludarabine in vivo in chronic lymphocytic leukemia Moussay et al. Molecular Cancer 2010, 9:115,p.1-16.
50 Jasmine Zain, Owen A, O’Connor, Targeting histone deacetyalses in the treatment of B- and T-cell malignancies Invest New Drugs (2010) 28 (Suppl 1): S58–S78.
51 Zhang-Zhi Hu, Hongzhan Huang, Amrita Cheema, et al Integrated Bioinformatics for Radiation-Induced Pathway Analysis from Proteomics and Microarray Data, J, Proteomics Bioinform. 2008 May ; 1(2): 47–60.
52 Sharon K. Huang, Marlene M. Darfler, Michael B. Nicholl , et al, LC/MS-Based Quantitative Proteomic Analysis of Paraffin-Embedded Archival Melanomas Reveals Potential Proteomic Biomarkers Associated with Metastasis, PLoS ONE, February 2009, Volume 4 ,Issue 2, e4430.
53 Rob M Ewing, Peter Chu, Fred Elisma, et al, Large-scale mapping of human protein–protein interactions by mass spectrometry, Molecular SystemsBiology 3; Article number 89; doi:10.1038/msb4100134.
54 The MGC Project Team, The Status, Quality, and Expansion of the NIH Full-Length cDNA Project:The Mammalian Gene Collection (MGC), Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.2596504.
55 Steven D. Bryce, Vivienne Morrison, Nicola J. Craig, et al, A Mortality Gene(s) for the Human Adenocarcinoma Line HeLa Maps to a 130-kb Region of Human Chromosome 4q22-q23 , Neoplasia . Vol. 4, No. 6, 2002, pp. 544– 550.
56 Gregory S. Yochum, Donald E. Ayer, Role for the Mortality Factors MORF4, MRGX, and MRG15 in Transcriptional Repression via Associations with Pf1, mSin3A, and Transducin-Like Enhancer of Split, MOLECULAR AND CELLULAR BIOLOGY, Nov. 2002, Vol. 22, No. 22 p. 7868–7876.
57 Meizhen Chen , Kaoru Tominaga, Olivia M. Pereira-Smith, The Emerging Role of the MORF/MRG Gene Family in Various Biological Processes Including Aging , Ann N Y Acad Sci. 2010 June ; 1197: 134–141. doi:10.1111/j.1749-6632.2010.05197.x.
58 PENG ZHANG, JINGYUE ZHAO, BING WANG, et al, The MRG domain of human MRG15 uses a shallow hydrophobic pocket to interact with the N-terminal region of PAM14, Protein Science (2006), 15:2423–2434.
59孙沫逸孙文斌陈兴等. p12蛋白抗原决定簇短肽的预测.临床口腔医学杂志.2003;19(3):223-225.
60邵小钧陈伟孙沫逸等. GST-P12融合蛋白的抗体制备、鉴定及免疫组化分析.现代肿瘤医学,2007;15(4) :463-465.
61任军陆伟孙沫逸等.两种人源性doc1基因真核表达重组质粒的构建及表达.临床口腔医学杂志,2007;23(9):539-542.
62孙沫逸邵小钧李建虎等.口腔鳞癌中p12蛋白的表达.中国美容医学,2009;18(4):510-512.
63邵小钧孙沫逸李连科等. DOC-1基因的原核表达载体构建及其重组蛋白的表达纯化.中国美容医学,2007;16(4) :447-450.
64生秀杰姜莉周伟强等.应用基因组步移克隆小鼠Doc-1R基因组序列.中华遗传学杂志.2001;20(4):314-316.
65生秀杰周伟强姜莉等.小鼠Doc-1R基因的克隆及其表达.癌症.2002;21(2):122-126.
66周伟强姜莉生秀杰等.小鼠DOC-1R反义重组载体的构建及表达的研究.癌症.2002;21(3):240-244.
67曲丹蔡东联.细胞衰老相关基因的研究进展.国外医学卫生学分册,2008;35(2):75-78.
68 J.萨姆布鲁克,D.W.拉塞尔.分子克隆实验指南.黄培堂等译.第三版,北京:科学出版社,2002.8,45-50.
69刘先早,蛋白质相互作用研究的技术方法和应用,西南国防医药,2009年第19卷第2期,251-253.
70王秋颖,酵母双杂交系统及发展,河北旅游职业学院学报,2009年第1期,42-43.
71王改芳,酵母双杂交系统在生命科学中的应用,现代农业科技,2009年第4期,259,264.
72尚彤,国强华、景霞,常用医学生物信息学数据库,第一版,北京:北京大学医学出版社,2003年5月.
73胡松年,薛庆中,基因组数据分析手册,第一版,杭州:浙江大学出版社,2003年5月.
74郑红霞,窦同海,蔡燕燕,等,人类基因组miRNA转录调控区保守性DNA序列生物信息学分析,基因组学与应用生物学,2009年,第28卷,第6期,第1049-1055页.
75刘智珺,生物信息学中数据库技术的应用,计算机与数字工程,2009年第5期,总第235期,157-159.
76侯振江,血清蛋白质组学在肝细胞癌的应用进展,临床肝胆病杂志2009年第25卷第6期,471-473.
77刘妍,李官成,生物信息学方法大规模筛选肿瘤差异表达基因,第30卷第6期2009年12月,694-700.
78李铁求,冯春琼,邹亚光,等,基于文献挖掘的雄激素非依赖型前列腺癌特异表达基因的生物信息学分析,中华男科学杂志,2009, 15 (12) : 1102– 1107.
79陈谋通,刘建军,蛋白质相互作用的研究方法,生物技术通报,2009年第1期,50-54.
80郑军,miR-21在p12~(CDK2AP1)表达调控中的作用及其对细胞侵袭和增殖的影响,第四军医大学学位论文,2010年5月,95-97.注:有关生物信息学各网站部分内容来自各专业网站。
2 Tsuji T, Duh FM, Latif F, et al. Cloning, mapping, expression, function, and Mutation analyses of the human ortholg of the hamster putative tumor supressor gene doc-1. J Biol Chem. 1998,273(12):6704-6709.
3 Shintani S,Ohyama H,Zhang X, et al.p12(doc-1) is a novel cyclin-dependent kinase 2-associated protein. Mol Cell Biol .2000;20(17):6300-6307.
4 Hu MG, Hu GF, Kim Y, et al. Role of p12(CDK2-AP1) in transforming growth factor-beta1-mediated growth suppression. Cancer Res. 2004 ; 15;64(2): 490-499.
5 Kim Y, Ohyama H, Patel V, Figueiredo M, Wong DT. Mutation of Cys105 inhibits dimerization of p12CDK2-AP1 and its growth suppressor effect. J Biol Chem. 2005;280(24):23273-23279.
6 Kim Y, McBride J, Zhang R, Zhou X, Wong DT. p12(CDK2-AP1) mediates DNA damage responses induced by cisplatin. Oncogene. 2005;24(3):407-418.
7 Matsou K, Shintain S, Tsuji T, et al. p12(doc-1), a growth suppressor, associates with DNA polymerase alpha/primase. FASEB J .2000;14(10): 1318- 1324.
8 Buajeeb W, Zhang X, Ohyama H, et al. Interaction of the CDK2-associated proteinc, p12(DOC-1/CDK2AP1), with its homolog, p14(DOC-1R). Biochem Biophys Res Commun. 2004;19;315(4):998-1003.
9 Zhang X, Tsao H , Tsuji T, et al. Identification and mutation analysis of doc-1R, a doc-1 growth suppressor-related gene. Biochem Biophys Res Commun. 1999;255(1):59-63.
10 Kusk M, Ahmed R, Thomsen B et al.Interactions of protein kinase CK2βsubunit within the holoenzyme and with other proteins.Molecular and Cellular Biochemistry.1999;191:51-58.
11 Deshpande AM,Dai YS,Kim Y,et al. Cdk2ap1 is required for epigenetic silencing of Oct4 during murine embryonic stem cell differentiation. The Journal of Biological Chemistry,2009,284(10):6043-6047.
12 Xavier Le Guezennec, Michiel Vermeulen. et al MBD2/NuRD and MBD3/ NuRD, Two Distinct Complexes with Different Biochemical and Functional Properties. MOLECULAR AND CELLULAR BIOLOGY, Feb. 2006, p. 843–851.
13 Cwikla S, Tsuji T, McBride J, et al.Doc-1--mediated apoptosis in malignant hamster oral keratinocytes. J Oral Maxillofac Surg .2000;58(4):406-414.
14 Kohno Y, Patel V, Kim Y, et al.Apoptosis,proliferation and p12doc-1 profiles in normal,dysplastic and malignant squamous epithelium of the Syrian hamster cheek pouch model. Oral Oncology.2002;38(2):274–280.
15 Sotsky Kent T, Yuan Z, Miller A et al .Deleted in oral cancer-1 expression upregulates proapoptosis elements in microsatellite-unstable human colorectal cancer. Ann Surg Oncol. 2004 ;11(2):192-196.
16 Shintani S, Mihara M, Terakado N,et al. Reduction of p12 doc-1 expression is a negative prognostic indicator in patients with surgically rescected oral squamous cell carcinoma. Clin Cancer Res. 2001;7(9):2776-2782.
17 Shintani S , Mihara M, Nakahara Y, et al. Expression of cell cycle control proteins in normal epithelium,premalignant and malignant lesion of oral cavity. Oral Oncology. 2002 ;38(2):235–243.
18任军冯源孙沫逸等.口腔鳞癌组织中doc1基因mRNA表达的意义.口腔医学研究2008;24(1):46-48.
19 Daigo Y, Suzuki K, Maruyama O,et al.Isolation,mapping and mutation analysis of a human cDNA homologous to the doc-1 gene of the Chinese hamster,a candidate tumor suppressor for oral cancer.Genes Chromosomes Cancer.1997;20(2):204-207.
20 Yuan Z, Sotsky Kent T, Weber TK et al. Differential expression of DOC-1 in microsatellite-unstable human colorectal cancer. Oncogene. 2003, 18; 22(40): 6304 -6310.
21 Zolochevska O, Figueiredo ML. Cell cycle regulator cdk2ap1 inhibits prostate cancer cell growth and modifies androgen-responsive pathway function. Prostate. 2009 ;69(14):1586-97.
22 Zolochevska O, Figueiredo ML. Expression of cell cycle regulator cdk2ap1 suppresses tumor cell phenotype by non-cell-autonomous mechanisms. Oral Oncol. 2009;45(9):e106-12.
23 Hiyoshi Y, Watanabe M, Hirashima K, et al. p12CDK2-AP1 is associated with tumor progression and a poor prognosis in esophageal squamous cell carcinoma. Oncol Rep. 2009;22(1):35-9.
24 Choi MG, Sohn TS, Park SB, et al . Decreased expression of p12 is associated with more advanced tumor invasion in human gastric cancer tissues. Eur Surg Res. 2009;42(4):223-9. 201.
25 Peng H,Shintani S, Kim Y, et al. Loss of p12CDK2-AP1 expression in human oral squamous cell carcinoma with disrupted transforming growth factor-beta-Smad signaling pathway. Neoplasia.2006;8(12):1028-36.
26 Tsuji T, Ibaragi S, Shima K, et al . Epithelial-mesenchymal transition induced by growth suppressor p12CDK2-AP1 promotes tumor cell local invasion but suppresses distant colony growth. Cancer Res. 2008;68(24):10377-86.
27 Figueiredo ML, Kim Y, St John MA,et al .p12CDK2-AP1 gene therapy strategy inhibits tumor growth in an in vivo mouse model of head and neck cancer. Clin Cancer Res. 2005;11(10):3939-48.
28 Kim Y, McBride J, Kimlin L, et al .Targeted inactivation of p12, CDK2 associating protein 1, leads to early embryonic lethality. PLoS One.2009; 4(2):e4518.
29 Kim Y, Deshpande A, Dai Y. Cyclin-dependent kinase 2-associating protein 1 commits murine embryonic stem cell differentiation through retinoblastoma protein regulation. J Biol Chem. 2009;284(35):23405-14.
30 M.L. Figueiredo, S. Dayan, et al Expression of Cell-Cycle Regulator CDK2-Associating Protein 1 (p12CDK2AP1) in Transgenic Mice Induces Testicular and Ovarian Atrophy In Vivo MOLECULAR REPRODUCTION AND DEVELOPMENT 73:987–997 (2006)
31 Fields S, Songo.A et al Novel genetic systemto detect protein-protein interactions[J ].Nature ,1989 ,340(6230) :245 - 246.
32 Haian Fu .蛋白质-蛋白质相互作用方法与应用张幼怡等译,第一版,北京:北京大学医学出版社,2008年9月,270-288
33王玉飞,张影,贾雷立,蛋白质相互作用实验指南,第一版,北京:化学工业出版社,2010年3月,63-64.
34李先昆,聂智毅,曾日中,酵母双杂交技术研究与应用进展,安徽农业科学, 2009 ,37 (7) :2867 - 2869 ,2926.
35张阳德,生物信息学,第一版,北京:科学出版社,2005年9月,1-4
36赵雨杰,医学生物信息学,第一版,北京:人民军医出版社,2002年11月.
37 Hua Dong, Hoicheong Siu, Li Luo, Investigation gene and microRNA expression in glioblastoma BMC Genomics 2010, 11(Suppl 3):S16.
38 Joongho Shin, Ziqian Yuan, Kenneth Fordyce,et al. A del T poly T (8) mutation in the 3’untranslated region (UTR) of the CDK2-AP1 gene is functionally significant causing decreased mRNA stability resulting in decreased CDK2-AP1 expression in human microsatellite unstable (MSI) colorectal cancer (CRC). Surgery 2007, 142:222-227.
39金伟,孙沫逸,王文勇的,等,CDK2AP1基因的激光捕获显微切割应用研究,口腔医学,2010,30(4),193-195.
40郑子瑞,侯宁波,何湘,等,酵母双杂交技术筛选与RIG-Ⅰ相互作用蛋白,军事医学科学院院刊,2009,33(1),10-13.
41 Bertram, M. J., N. G. Berube, Identification of a gene that reverses the immortal phenotype of a subset of cells and is a member of a novel family of transcription factor-like genes. Mol. Cell. Biol. 19:1479–1485.
42 PARDO P S , LEUNG J K, LUCCHESI J C , et al. MRG15 , a novel chromodomain protein , is present in two distinct multiprotein complexes involved in transcriptional activation [ J ] . J Biol Chem , 2002 , 277 ( 52) : 508602-508661.
43 Kaoru Tominaga, Emiko Tominaga, et al The cell senescence inducing gene product MORF4 is regulated by degradation via the ubiquitin / proteasome pathway. Exp Cell Res. 2010 January 1; 316(1): 92–102.
44 LEUNGJ K, BERUBEN G, VENABLE S , et al. MRG15 activates the B-myb promoter through formation of a nuclear complex with the retinoblastoma protein and the novel protein PAM14 [ J ] . J Biol Chem , 2001 , 276 :39171239178.
45 TOMINAGA K, OLGUN A , SMITH J R , et al. Genetics of cellular senescence [J ] . Mech Ageing Dev , 2002 , 123 (8) :92729361.
46 Xue Zhang,,Hensin Tsao, Takanori Tsuji,et al. Identification and mutationanalysis of Doc-1R, a Doc-1 grow th suppressor-related gene. Biochemical and Biophysical Research Communications 255, 59–63 (1999).
47 GIANNI POZZI, MARCO R. OGGIONI, ALEXANDER TOMASZ, DNA Probe for Identification of Streptococcus pneumoniae, JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1989, Vol. 27, No. 2, p370-372.
48 Martin Leverkus, Martin R. Sprick, Tina Wachter, et al Proteasome Inhibition Results in TRAIL Sensitization of Primary Keratinocytes by Removing the Resistance Mediating Blockof Effector Caspase Maturation MOLECULAR AND CELLULAR BIOLOGY, Feb. 2003, Vol. 23, No. 3, p. 777–790.
49 Etienne Moussay, Valérie Palissot, Laurent Vallar, et al Research Determination of genes and microRNAs involved in the resistance to fludarabine in vivo in chronic lymphocytic leukemia Moussay et al. Molecular Cancer 2010, 9:115,p.1-16.
50 Jasmine Zain, Owen A, O’Connor, Targeting histone deacetyalses in the treatment of B- and T-cell malignancies Invest New Drugs (2010) 28 (Suppl 1): S58–S78.
51 Zhang-Zhi Hu, Hongzhan Huang, Amrita Cheema, et al Integrated Bioinformatics for Radiation-Induced Pathway Analysis from Proteomics and Microarray Data, J, Proteomics Bioinform. 2008 May ; 1(2): 47–60.
52 Sharon K. Huang, Marlene M. Darfler, Michael B. Nicholl , et al, LC/MS-Based Quantitative Proteomic Analysis of Paraffin-Embedded Archival Melanomas Reveals Potential Proteomic Biomarkers Associated with Metastasis, PLoS ONE, February 2009, Volume 4 ,Issue 2, e4430.
53 Rob M Ewing, Peter Chu, Fred Elisma, et al, Large-scale mapping of human protein–protein interactions by mass spectrometry, Molecular SystemsBiology 3; Article number 89; doi:10.1038/msb4100134.
54 The MGC Project Team, The Status, Quality, and Expansion of the NIH Full-Length cDNA Project:The Mammalian Gene Collection (MGC), Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.2596504.
55 Steven D. Bryce, Vivienne Morrison, Nicola J. Craig, et al, A Mortality Gene(s) for the Human Adenocarcinoma Line HeLa Maps to a 130-kb Region of Human Chromosome 4q22-q23 , Neoplasia . Vol. 4, No. 6, 2002, pp. 544– 550.
56 Gregory S. Yochum, Donald E. Ayer, Role for the Mortality Factors MORF4, MRGX, and MRG15 in Transcriptional Repression via Associations with Pf1, mSin3A, and Transducin-Like Enhancer of Split, MOLECULAR AND CELLULAR BIOLOGY, Nov. 2002, Vol. 22, No. 22 p. 7868–7876.
57 Meizhen Chen , Kaoru Tominaga, Olivia M. Pereira-Smith, The Emerging Role of the MORF/MRG Gene Family in Various Biological Processes Including Aging , Ann N Y Acad Sci. 2010 June ; 1197: 134–141. doi:10.1111/j.1749-6632.2010.05197.x.
58 PENG ZHANG, JINGYUE ZHAO, BING WANG, et al, The MRG domain of human MRG15 uses a shallow hydrophobic pocket to interact with the N-terminal region of PAM14, Protein Science (2006), 15:2423–2434.
59孙沫逸孙文斌陈兴等. p12蛋白抗原决定簇短肽的预测.临床口腔医学杂志.2003;19(3):223-225.
60邵小钧陈伟孙沫逸等. GST-P12融合蛋白的抗体制备、鉴定及免疫组化分析.现代肿瘤医学,2007;15(4) :463-465.
61任军陆伟孙沫逸等.两种人源性doc1基因真核表达重组质粒的构建及表达.临床口腔医学杂志,2007;23(9):539-542.
62孙沫逸邵小钧李建虎等.口腔鳞癌中p12蛋白的表达.中国美容医学,2009;18(4):510-512.
63邵小钧孙沫逸李连科等. DOC-1基因的原核表达载体构建及其重组蛋白的表达纯化.中国美容医学,2007;16(4) :447-450.
64生秀杰姜莉周伟强等.应用基因组步移克隆小鼠Doc-1R基因组序列.中华遗传学杂志.2001;20(4):314-316.
65生秀杰周伟强姜莉等.小鼠Doc-1R基因的克隆及其表达.癌症.2002;21(2):122-126.
66周伟强姜莉生秀杰等.小鼠DOC-1R反义重组载体的构建及表达的研究.癌症.2002;21(3):240-244.
67曲丹蔡东联.细胞衰老相关基因的研究进展.国外医学卫生学分册,2008;35(2):75-78.
68 J.萨姆布鲁克,D.W.拉塞尔.分子克隆实验指南.黄培堂等译.第三版,北京:科学出版社,2002.8,45-50.
69刘先早,蛋白质相互作用研究的技术方法和应用,西南国防医药,2009年第19卷第2期,251-253.
70王秋颖,酵母双杂交系统及发展,河北旅游职业学院学报,2009年第1期,42-43.
71王改芳,酵母双杂交系统在生命科学中的应用,现代农业科技,2009年第4期,259,264.
72尚彤,国强华、景霞,常用医学生物信息学数据库,第一版,北京:北京大学医学出版社,2003年5月.
73胡松年,薛庆中,基因组数据分析手册,第一版,杭州:浙江大学出版社,2003年5月.
74郑红霞,窦同海,蔡燕燕,等,人类基因组miRNA转录调控区保守性DNA序列生物信息学分析,基因组学与应用生物学,2009年,第28卷,第6期,第1049-1055页.
75刘智珺,生物信息学中数据库技术的应用,计算机与数字工程,2009年第5期,总第235期,157-159.
76侯振江,血清蛋白质组学在肝细胞癌的应用进展,临床肝胆病杂志2009年第25卷第6期,471-473.
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