The genes encoding DNA-binding domain（BD） designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence, and cloned into ...The genes encoding DNA-binding domain（BD） designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence, and cloned into yeast expression vector pPIC9k. The resulted plasmid pTG was linearized and transfected into Pichia pastoris strains GS 115 by electroporation. High copies of transformants were obtained with Muts and HIS＋ phenotype identi- fication, PCR amplification and screening of G418. After flask culture and expression induced by methanol, the target protein named TG was well expressed and analyzed by SDS-PAGE and Western blot. Under optimized conditions, the yield of soluble recombinant protein was approximately 39.7 mg/L. DNA binding activity and cell transduction property of TG were analyzed by gel eleetrophoresis and fluorescent microscopy. The results show that the recombinant protein could bind strongly to the plasmid containing upstream activating sequence（UAS）. The cell experiments revealed that TG could deliver the binding plasmid into HEK-293 cells effectively. In summary, the work presented here suggests that TG is specific toward UAS containing plasmid and has the potential for use as nonviral DNA delivery agent.展开更多
Previous studies have shown that octamer-binding transcription factor 4（Oct4） plays a significant role in early embryonic development of mammalian animals, and different Oct4 expression levels induce multi-lineage d...Previous studies have shown that octamer-binding transcription factor 4（Oct4） plays a significant role in early embryonic development of mammalian animals, and different Oct4 expression levels induce multi-lineage differentiation which are regulated by DNA methylation. To explore the relationship between the methylation pattern of Oct4 gene exon 1 and embryonic development, in this work, five different tissues（heart, liver, lung, cerebrum and cerebellum） from three germ layers were chosen from low age（50–60 d） and advanced age（60–70 d） of fetal cattle and the differences between tissues or ages were analyzed, respectively. The result showed that the DNA methylation level of Oct4 gene exon 1 was significant different（P〈0.01） between any two of three germ layers in low age（〈60 d）, but kept steady of advanced age（P〉0.05）（〉60 d）, suggesting that 60-d post coital was an important boundary for embryonic development. In addition, in ectoderm（cerebrum and cerebellum）, there was no significant methylation difference of Oct4 gene exon 1 between low age and advanced age（P〉0.05）, but the result of endoderm（liver and lung） and mesoderm（heart） were on the contrary（P〈0.01）, which indicated the development of ectoderm was earlier than endoderm and mesoderm. The methylation differences from the 3rd, 5th and 9th Cp G-dinucleotide loci of Oct4 gene exon 1 were significantly different between each two of three germ layers（P〈0.05）, indicating that these three loci may have important influence on bovine embryonic development. This study showed that bovine germ layers differentiation was significantly related to the DNA methylation status of Oct4 gene exon 1. This work firstly identified the DNA methylation profile of bovine Oct4 gene exon 1 and its association with germ layers development in fetus and adult of cattle. Moreover, the work also provided epigenetic information for further studying bovine embryonic development and cellular reprogramming.展开更多
目的:探讨人染色质解旋酶DNA结合蛋白4(recombinant chromodomain helicase DNA binding protein 4,CHD4)基因表达对急性T细胞淋巴细胞白血病细胞增殖和凋亡的影响及其启动子鉴定。方法:使用siRNA-CHD4瞬时转染T淋巴细胞白血病细胞(Jurk...目的:探讨人染色质解旋酶DNA结合蛋白4(recombinant chromodomain helicase DNA binding protein 4,CHD4)基因表达对急性T细胞淋巴细胞白血病细胞增殖和凋亡的影响及其启动子鉴定。方法:使用siRNA-CHD4瞬时转染T淋巴细胞白血病细胞(Jurkat)敲低CHD4基因表达,实时荧光定量q RT-PCR和Western blot检测细胞转染后CHD4表达量,流式细胞术测定细胞凋亡率及细胞周期变化;CCK-8分析测定CHD4基因对Jurkat细胞增殖的影响。根据生物信息学分析,以Jurkat细胞提取的全基因组DNA为模板,PCR扩增CHD4基因候选启动子区2 091 bp片段,以pGL3-Basic为载体,克隆含有CHD4基因候选启动子区的序列,制备重组质粒,并且构建一系列含CHD4基因候选启动子5′侧翼区截短序列质粒。将含CHD4启动子序列的质粒及截短序列质粒转染至Jurkat细胞和人胚胎肾T细胞(HEK293T),双荧光素酶报告基因检测各片段的启动子活性,确定CHD4基因启动子最小活性区域,并通过生物信息学方法分析该区域转录因子结合位点,构建结合位点突变的质粒,通过双荧光素酶报告基因检测,分析结合位点对CHD4转录的影响。结果:流式细胞检测结果发现,与对照组比较,CHD4抑制Jurkat细胞凋亡,转染siRNA-CHD4的Jurkat细胞G0/G1期比例显著升高,而S期比例下降(P<0.01);CCK-8检测CHD4基因对Jurkat细胞的增殖有促进作用(P<0.05);成功构建含有CHD4基因候选启动子序列的质粒及其截短序列质粒,与pGL3-Basic空载体相比,含有CHD4基因候选启动子序列的质粒活性明显增加(P<0.05)。CHD4基因最小活性区域位于转录起始位点-233~-13 bp,其中包含NF-κB、MZF1等转录因子结合位点;NF-κB对CHD4启动子活性具有正向调控作用。结论:CHD4基因对Jurkat细胞凋亡有抑制作用,并且促进其增殖。CHD4最小活性区域位于转录起始位点-233~-13 bp,转录因子NF-κB对CHD4基因启动子活性具有正向调控作用。展开更多
文摘目的探索DNA损伤诱导转录因子4(DNA damage induced transcription factor 4,DDIT4)在毛乳头细胞氧化应激损伤中的保护作用及其机制。方法使用长波紫外线(ultraviolet radiation A,UVA)和过氧化氢(H 2 O 2)建立毛乳头细胞氧化应激模型;CCK-8检测不同处理条件下的细胞活力,利用2′,7′-二氯荧光素二乙酸酯(2′,7′-dichlorofluorescein diacetate,DCFH-DA)试剂检测细胞内活性氧(ROS)的变化;电镜观察自噬小体;Western blot观察DDIT4及自噬相关指标微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、泛素结合蛋白(ubiquitin-binding protein P62,P62)、雷帕霉素哺乳动物靶蛋白(mammalian target of rapamycin,mTOR)、p-mTOR的表达情况。结果UVA和H 2 O 2导致毛乳头细胞内ROS升高,活力下降(P<0.05);氧化应激环境下毛乳头细胞内的DDIT4表达增加(P<0.05),而抗氧化剂N-乙酰基-L-半胱氨酸(N-acetyl-L-cysteine,NAC)可有效抑制此效应(P<0.05);UVA或H 2 O 2处理后毛乳头细胞内自噬水平升高,表现为自噬体数量增多,LC3Ⅱ/Ⅰ比值增高(P<0.05),P62表达下降(P<0.05),3-甲基腺嘌呤(3-methyladenine,3-MA)阻断自噬可观察到细胞活力进一步降低(P<0.05),细胞内ROS增多(P<0.05),反之,雷帕霉素(rapamycin,RAPA)可增加自噬水平提升氧化条件下毛乳头细胞活力,减少细胞内ROS生成(P<0.05);下调DDIT4表达可使氧化应激环境下毛乳头细胞自噬减弱,LC3Ⅱ/Ⅰ减少(P<0.05),p-mTOR/mTOR、P62增加(P<0.05),细胞活力下降(P<0.05),细胞内ROS增多(P<0.05)。结论DDIT4可能通过自噬缓解毛乳头细胞氧化应激损伤。
基金Supported by National High-tech Research and Development Program of China(No.2007AA021004)
文摘The genes encoding DNA-binding domain（BD） designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence, and cloned into yeast expression vector pPIC9k. The resulted plasmid pTG was linearized and transfected into Pichia pastoris strains GS 115 by electroporation. High copies of transformants were obtained with Muts and HIS＋ phenotype identi- fication, PCR amplification and screening of G418. After flask culture and expression induced by methanol, the target protein named TG was well expressed and analyzed by SDS-PAGE and Western blot. Under optimized conditions, the yield of soluble recombinant protein was approximately 39.7 mg/L. DNA binding activity and cell transduction property of TG were analyzed by gel eleetrophoresis and fluorescent microscopy. The results show that the recombinant protein could bind strongly to the plasmid containing upstream activating sequence（UAS）. The cell experiments revealed that TG could deliver the binding plasmid into HEK-293 cells effectively. In summary, the work presented here suggests that TG is specific toward UAS containing plasmid and has the potential for use as nonviral DNA delivery agent.
基金supported by the Natural Science Foundation of Shaanxi Province, China (2014JQ3104)the National Natural Science Foundation of China (31000655)China Postdoctoral Science Foundation funded project (2014M560809)
文摘Previous studies have shown that octamer-binding transcription factor 4（Oct4） plays a significant role in early embryonic development of mammalian animals, and different Oct4 expression levels induce multi-lineage differentiation which are regulated by DNA methylation. To explore the relationship between the methylation pattern of Oct4 gene exon 1 and embryonic development, in this work, five different tissues（heart, liver, lung, cerebrum and cerebellum） from three germ layers were chosen from low age（50–60 d） and advanced age（60–70 d） of fetal cattle and the differences between tissues or ages were analyzed, respectively. The result showed that the DNA methylation level of Oct4 gene exon 1 was significant different（P〈0.01） between any two of three germ layers in low age（〈60 d）, but kept steady of advanced age（P〉0.05）（〉60 d）, suggesting that 60-d post coital was an important boundary for embryonic development. In addition, in ectoderm（cerebrum and cerebellum）, there was no significant methylation difference of Oct4 gene exon 1 between low age and advanced age（P〉0.05）, but the result of endoderm（liver and lung） and mesoderm（heart） were on the contrary（P〈0.01）, which indicated the development of ectoderm was earlier than endoderm and mesoderm. The methylation differences from the 3rd, 5th and 9th Cp G-dinucleotide loci of Oct4 gene exon 1 were significantly different between each two of three germ layers（P〈0.05）, indicating that these three loci may have important influence on bovine embryonic development. This study showed that bovine germ layers differentiation was significantly related to the DNA methylation status of Oct4 gene exon 1. This work firstly identified the DNA methylation profile of bovine Oct4 gene exon 1 and its association with germ layers development in fetus and adult of cattle. Moreover, the work also provided epigenetic information for further studying bovine embryonic development and cellular reprogramming.
