采用基于液相色谱-高分辨质谱的转录因子结合序列串联多拷贝双联DNA结合元件(Concatenated tandem array of transcription factor response elements,catTFRE)Pull down蛋白质组学技术,研究了曲妥珠单抗耐药胃癌细胞的转录因子及其调...采用基于液相色谱-高分辨质谱的转录因子结合序列串联多拷贝双联DNA结合元件(Concatenated tandem array of transcription factor response elements,catTFRE)Pull down蛋白质组学技术,研究了曲妥珠单抗耐药胃癌细胞的转录因子及其调控作用。以合成的串联转录因子结合序列DNA片段为亲和试剂,用生物素标记,作为"DNA诱饵",通过Pull down富集后,采用液相色谱-高分辨质谱检测捕获的转录因子,基于强度定量法(Intensity based absolute quantification,iBAQ)定量,筛选出与DNA结合活性显著变化的转录因子。利用WebGestalt(2017)数据库对激活转录因子调控的信号通路、家族分类、调控网络及转录因子-靶基因(TFs-targets)进行分析。结果表明,359个转录因子被定量检测,与亲本细胞相比,61个转录因子在曲妥珠单抗耐药胃癌细胞中与DNA结合活性显著变化,其中结合活性增强48个,活性降低13个。激活转录因子属于bZIP、bHLH、homebox、HMG box、Zine finger等家族。KEGG通路富集显示,癌症、MAPK、Wnt、TGF-β、凋亡等多条通路在耐药细胞中显著改变。TFs-targets分析表明,TCF7L2、TCF7、FOXC1、JUN、CYC、FOS、ELK4、NFκB2、DDIT3和ATF2在上皮-间质转化(Epithelial-mesenchymal transformation,EMT)、Wnt、MAPK信号通路促使胃癌曲妥珠单抗耐药过程中发挥重要调控作用,提示靶向这些转录因子所调控的信号通路可能是逆转胃癌曲妥珠单抗耐药的重要途径。展开更多
In this study, the entire mitochondrial DNA(mtDNA) control region(CR) of Pholis fangi was amplified via polymerase chain reaction followed by direct sequencing. The length of the mtDNA CR consensus sequence of P. fang...In this study, the entire mitochondrial DNA(mtDNA) control region(CR) of Pholis fangi was amplified via polymerase chain reaction followed by direct sequencing. The length of the mtDNA CR consensus sequence of P. fangi was 853 bp in length. In accordance with the recognition sites as were previously reported in fish species, the mtDNA CR sequence of P. fangi can be divided into 3 domains, i.e., the extended terminal associated sequence(ETAS), the central conserved sequence block(CSB), and the CSB domain. In addition, the following structures were identified in the mtDNA CR sequence of P. fangi: 2 ETASs in the ETAS domain(TAS and cTAS), 6 CSBs in the central CSB domain(CSB-F to CSB-A), and 3 CSBs in the CSB domain(CSB-1 to CSB-3). These demonstrated that the structure of the mtDNA CR of P. fangi was substantially different from those of most other fish species. The mtDNA CR sequence of P. fangi contained one conserved region from 656 bp to 815 bp. Similar to most other fish species, P. fangi has no tandem repeat sequences in its mtDNA CR sequence. Phylogenetic analysis based on the complete mtDNA CR sequences showed that there were no genetic differences within P. fangi populations of the same geographical origin and between P. fangi populations of different geographical origins.展开更多
Transcription factor and sequence specific DNA interactions play important roles in drug genome and transcription diagnosis. Gold nanoparticles show high sensitivity, stability and compatibility for biological molecul...Transcription factor and sequence specific DNA interactions play important roles in drug genome and transcription diagnosis. Gold nanoparticles show high sensitivity, stability and compatibility for biological molecules as electrochemical intercalators. Here unimolecular hairpin oligonucleotides were self-assembled onto Au electrode surface and elongation on solid phase was carried out to double strand oligonucleotides with transcription factor NF-r,13 binding site. Gold nanoparticle-catalyzed Ag deposition was detected by anodic stripping voltammetry （ASV） for NF-kB binding. It was indicated that this method for sequence specific DNA binding protein detection shows pronounced specificity, sensitivity and we can find application in transcription regulation research, open reading frame characterization and functional gene inspection by this method.展开更多
基金supported by the Public Science and Technology Research Funds Projects of Ocean (Nos. 201305043 and 201405010)
文摘In this study, the entire mitochondrial DNA(mtDNA) control region(CR) of Pholis fangi was amplified via polymerase chain reaction followed by direct sequencing. The length of the mtDNA CR consensus sequence of P. fangi was 853 bp in length. In accordance with the recognition sites as were previously reported in fish species, the mtDNA CR sequence of P. fangi can be divided into 3 domains, i.e., the extended terminal associated sequence(ETAS), the central conserved sequence block(CSB), and the CSB domain. In addition, the following structures were identified in the mtDNA CR sequence of P. fangi: 2 ETASs in the ETAS domain(TAS and cTAS), 6 CSBs in the central CSB domain(CSB-F to CSB-A), and 3 CSBs in the CSB domain(CSB-1 to CSB-3). These demonstrated that the structure of the mtDNA CR of P. fangi was substantially different from those of most other fish species. The mtDNA CR sequence of P. fangi contained one conserved region from 656 bp to 815 bp. Similar to most other fish species, P. fangi has no tandem repeat sequences in its mtDNA CR sequence. Phylogenetic analysis based on the complete mtDNA CR sequences showed that there were no genetic differences within P. fangi populations of the same geographical origin and between P. fangi populations of different geographical origins.
基金This research is financially supported by the National Natural Science Foundation （No. 90606027 60501010）.
文摘Transcription factor and sequence specific DNA interactions play important roles in drug genome and transcription diagnosis. Gold nanoparticles show high sensitivity, stability and compatibility for biological molecules as electrochemical intercalators. Here unimolecular hairpin oligonucleotides were self-assembled onto Au electrode surface and elongation on solid phase was carried out to double strand oligonucleotides with transcription factor NF-r,13 binding site. Gold nanoparticle-catalyzed Ag deposition was detected by anodic stripping voltammetry （ASV） for NF-kB binding. It was indicated that this method for sequence specific DNA binding protein detection shows pronounced specificity, sensitivity and we can find application in transcription regulation research, open reading frame characterization and functional gene inspection by this method.
