Objective.In order to demonstrate the binding of HBV X protein (HBX) with the general transcription factor TFIIB. Methods.In vitro glutathion S transferase (GST) resin Pull Down assay an...Objective.In order to demonstrate the binding of HBV X protein (HBX) with the general transcription factor TFIIB. Methods.In vitro glutathion S transferase (GST) resin Pull Down assay and Far Western Blotting assay, in vivo Co immunoprecipition assay were used. Results.The X199(51 99) domain of HBX is reponsible for HBX binding to TFIIB. While the d10 domain (125 295) of TFIIB is required for TFIIB binding to HBX. When the two basic amino acids(K) at position 178 and 189 of TFIIB were substituted by neutral amino acids(L), the binding of TFIIBK178L and K189L to HBX was siginificantly reduced. When the the basic amino acids were substituted by the acidic amino acids(E),the binding of TFIIB K178E and K189E to HBX were almost lost. In vitro results of HBX binding to TFIIB were further confirmed by in vivo co immunoprecipitation assay. Our results also indicated that the Woodchuck hepatitis virus X protein (WHX) interacts with TFIIB. Conclusion.These results suggested that the communication between HBX and general transcription factor TFIIB is one of the mechanisms which account for its transcriptional transactivation.展开更多
Pollen germination and embryogenesis are important to sexual plant reproduction. The processes require a large number of genes to be expressed. Transcription of eukaryotic nuclear genes is accomplished by three conser...Pollen germination and embryogenesis are important to sexual plant reproduction. The processes require a large number of genes to be expressed. Transcription of eukaryotic nuclear genes is accomplished by three conserved RNA polymerases acting in association with a set of auxiliary general transcription factors （GTFs）, including B-type GTFs. The roles of B-type GTFs in plant reproduction remain poorly understood. Here we report functional characterization of a novel plant-specific TFIIB-related gene PTF2 in Arabidopsis. Mutation in PTF2 caused failure of pollen germination. Pollen-rescue revealed that the mutation also disrupted embryogenesis and resulted in seed abortion. PTF2 is expressed prolifically in developing pollen and the other tissues with active cell division and differentiation, including embryo and shoot apical meristem. The PTF2 protein shares a lower amino acid sequence similarity with other known TFIIB and TFIIB-related proteins in Arabidopsis. It can interact with TATA-box binding protein 2 （TBP2） and bind to the double- stranded DNA （dsDNA） as the other known TFIIB and TFIIB-related proteins do. In addition, PTF2 can form a homodimer and interact with the subunits of RNA polymerases （RNAPs）, implying that it may be involved in the RNAPs transcription. These results suggest that PTF2 plays crucial roles in pollen germination and embryogenesis in Arabidopsis, possibly by regulating gene expression through interaction with TBP2 and the subunits of RNAPs.展开更多
[目的]探究BRF2与Wnt/β-catenin通路在非小细胞肺癌转移过程中的作用机制。[方法]从20例非小细胞肺癌患者中收集癌组织以及癌旁组织的实验样本,通过免疫组化和蛋白免疫印迹分析癌旁组织和癌组织中BRF2的表达水平。将A549细胞分为3组:si...[目的]探究BRF2与Wnt/β-catenin通路在非小细胞肺癌转移过程中的作用机制。[方法]从20例非小细胞肺癌患者中收集癌组织以及癌旁组织的实验样本,通过免疫组化和蛋白免疫印迹分析癌旁组织和癌组织中BRF2的表达水平。将A549细胞分为3组:si NC组、si BRF2组和KYA1797K组。通过CCK-8实验分析细胞的增殖能力,流式细胞仪分析细胞的凋亡率,蛋白免疫印迹分析Wnt/β-catenin通路相关蛋白的表达水平。[结果]BRF2在非小细胞肺癌组织中表达水平增加(0.47±0.01 vs 0.93±0.02,P<0.05);下调BRF2表达水平后能抑制A549细胞的增殖(2.68±0.05 vs 1.23±0.03 vs 1.28±0.01,P<0.05)和侵袭能力(128.23±5.01 vs 62.16±7.01 vs 58.16±7.01,P<0.05),并促进A549细胞凋亡(6.31±1.03 vs 14.68±2.01 vs 13.88±2.33%,P<0.05);BRF2能够抑制Wnt3α和β-catenin的表达(1.13±0.17 vs 0.51±0.02 vs 0.49±0.05;0.87±0.02 vs 0.39±0.01 vs 0.52±0.08,P<0.05)。[结论]BRF2在非小细胞肺癌患者体内的癌组织中表达上调,抑制非小细胞肺癌A549细胞中的BRF2表达后,A549细胞的增殖以及侵袭活性下降,同时细胞凋亡数量增多。BRF2抑制非小细胞肺癌A549细胞的作用与调控Wnt/β-catenin信号通路相关。展开更多
文摘Objective.In order to demonstrate the binding of HBV X protein (HBX) with the general transcription factor TFIIB. Methods.In vitro glutathion S transferase (GST) resin Pull Down assay and Far Western Blotting assay, in vivo Co immunoprecipition assay were used. Results.The X199(51 99) domain of HBX is reponsible for HBX binding to TFIIB. While the d10 domain (125 295) of TFIIB is required for TFIIB binding to HBX. When the two basic amino acids(K) at position 178 and 189 of TFIIB were substituted by neutral amino acids(L), the binding of TFIIBK178L and K189L to HBX was siginificantly reduced. When the the basic amino acids were substituted by the acidic amino acids(E),the binding of TFIIB K178E and K189E to HBX were almost lost. In vitro results of HBX binding to TFIIB were further confirmed by in vivo co immunoprecipitation assay. Our results also indicated that the Woodchuck hepatitis virus X protein (WHX) interacts with TFIIB. Conclusion.These results suggested that the communication between HBX and general transcription factor TFIIB is one of the mechanisms which account for its transcriptional transactivation.
基金the research grants from the Ministry of Sciences and Technology，the Natural Science Foundation of China，the Ministry of Education
文摘Pollen germination and embryogenesis are important to sexual plant reproduction. The processes require a large number of genes to be expressed. Transcription of eukaryotic nuclear genes is accomplished by three conserved RNA polymerases acting in association with a set of auxiliary general transcription factors （GTFs）, including B-type GTFs. The roles of B-type GTFs in plant reproduction remain poorly understood. Here we report functional characterization of a novel plant-specific TFIIB-related gene PTF2 in Arabidopsis. Mutation in PTF2 caused failure of pollen germination. Pollen-rescue revealed that the mutation also disrupted embryogenesis and resulted in seed abortion. PTF2 is expressed prolifically in developing pollen and the other tissues with active cell division and differentiation, including embryo and shoot apical meristem. The PTF2 protein shares a lower amino acid sequence similarity with other known TFIIB and TFIIB-related proteins in Arabidopsis. It can interact with TATA-box binding protein 2 （TBP2） and bind to the double- stranded DNA （dsDNA） as the other known TFIIB and TFIIB-related proteins do. In addition, PTF2 can form a homodimer and interact with the subunits of RNA polymerases （RNAPs）, implying that it may be involved in the RNAPs transcription. These results suggest that PTF2 plays crucial roles in pollen germination and embryogenesis in Arabidopsis, possibly by regulating gene expression through interaction with TBP2 and the subunits of RNAPs.
文摘[目的]探究BRF2与Wnt/β-catenin通路在非小细胞肺癌转移过程中的作用机制。[方法]从20例非小细胞肺癌患者中收集癌组织以及癌旁组织的实验样本,通过免疫组化和蛋白免疫印迹分析癌旁组织和癌组织中BRF2的表达水平。将A549细胞分为3组:si NC组、si BRF2组和KYA1797K组。通过CCK-8实验分析细胞的增殖能力,流式细胞仪分析细胞的凋亡率,蛋白免疫印迹分析Wnt/β-catenin通路相关蛋白的表达水平。[结果]BRF2在非小细胞肺癌组织中表达水平增加(0.47±0.01 vs 0.93±0.02,P<0.05);下调BRF2表达水平后能抑制A549细胞的增殖(2.68±0.05 vs 1.23±0.03 vs 1.28±0.01,P<0.05)和侵袭能力(128.23±5.01 vs 62.16±7.01 vs 58.16±7.01,P<0.05),并促进A549细胞凋亡(6.31±1.03 vs 14.68±2.01 vs 13.88±2.33%,P<0.05);BRF2能够抑制Wnt3α和β-catenin的表达(1.13±0.17 vs 0.51±0.02 vs 0.49±0.05;0.87±0.02 vs 0.39±0.01 vs 0.52±0.08,P<0.05)。[结论]BRF2在非小细胞肺癌患者体内的癌组织中表达上调,抑制非小细胞肺癌A549细胞中的BRF2表达后,A549细胞的增殖以及侵袭活性下降,同时细胞凋亡数量增多。BRF2抑制非小细胞肺癌A549细胞的作用与调控Wnt/β-catenin信号通路相关。
