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72.
A simple and efficient method for the separation and detection of small DNA fragments by electrophoresis in formamide containing agarose gels and Southern blotting to DBM-paper. 总被引:5,自引:2,他引:3 下载免费PDF全文
We show in this report that DNA fragments smaller than 300 bp are separated with high resolution by electrophoresis in concentrated (up to 7%) agarose gels containing 50% formamide. The separated DNA fragments can subsequently be quantitatively transferred to DBM-paper [Alwine, J.C. et al., Proc. Natl. Acad. Sci. USA (1977) 74, 5350-54] using the Southern technique [Southern, E.M., J. Mol. Biol. (1975) 98, 503-517], while preserving the sharpness of the original gel pattern. Since thin (0.2-0.4 mm) and thick (up to 5 mm) agarose slab gels can be easily handled in vertical or horizontal apparatus, this method should prove to be a very useful extention of the Southern technique, applicable to a variety of analytical and preparative purposes. 相似文献
73.
74.
Qingwei Tian Jingting Wu Haifeng Xu Zhangli Hu Yangao Huo Liyan Wang 《The Journal of biological chemistry》2022,298(6)
The discovery of reduced flavin mononucleotide and fatty aldehydes as essential factors of light emission facilitated study of bacterial luminescence. Although the molecular mechanisms underlying bacterial luminescence have been studied for more than 60 years, the structure of the bacterial fatty acid reductase complex remains unclear. Here, we report the cryo-EM structure of the Photobacterium phosphoreum fatty acid reductase complex LuxC–LuxE to a resolution of 2.79 Å. We show that the active site Lys238/Arg355 pair of LuxE is >30 Å from the active site Cys296 of LuxC, implying that catalysis relies on a large conformational change. Furthermore, mutagenesis and biochemical experiments support that the L-shaped cleft inside LuxC plays an important role in substrate binding and reaction. We obtained a series of mutants with significantly improved activity as measured by in vitro bioluminescence assays and demonstrated that the double mutant W111A/F483K displayed the highest activity (370% of the WT). Our results indicated that the activity of LuxC significantly affects the bacterial bioluminescence reaction. Finally, we expressed this mutated lux operon in Escherichia coli but observed that the in vivo concentrations of ATP and NADPH limited the enzyme activity; thus, we conclude that the luminous intensity mainly depends on the level of metabolic energy. 相似文献
75.
Bingqing Xia Xurui Shen Yang He Xiaoyan Pan Feng-Liang Liu Yi Wang Feipu Yang Sui Fang Yan Wu Zilei Duan Xiaoli Zuo Zhuqing Xie Xiangrui Jiang Ling Xu Hao Chi Shuangqu Li Qian Meng Hu Zhou Yubo Zhou Xi Cheng Xiaoming Xin Lin Jin Hai-Lin Zhang Dan-Dan Yu Ming-Hua Li Xiao-Li Feng Jiekai Chen Hualiang Jiang Gengfu Xiao Yong-Tang Zheng Lei-Ke Zhang Jingshan Shen Jia Li Zhaobing Gao 《Cell research》2021,31(8):847-860
Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.Subject terms: Cell death, Molecular biology 相似文献
76.
Qiang Lv Shuang Han Lei Wang Jinchan Xia Peng Li Ruoyang Hu Jinzheng Wang Lei Gao Yuli Chen Yu Wang Jing Du Fang Bao Yong Hu Xingzhi Xu Wei Xiao Yikun He 《Nucleic acids research》2022,50(12):6820
Nitric oxide (NO) is a key player in numerous physiological processes. Excessive NO induces DNA damage, but how plants respond to this damage remains unclear. We screened and identified an Arabidopsis NO hypersensitive mutant and found it to be allelic to TEBICHI/POLQ, encoding DNA polymerase θ. The teb mutant plants were preferentially sensitive to NO- and its derivative peroxynitrite-induced DNA damage and subsequent double-strand breaks (DSBs). Inactivation of TEB caused the accumulation of spontaneous DSBs largely attributed to endogenous NO and was synergistic to DSB repair pathway mutations with respect to growth. These effects were manifested in the presence of NO-inducing agents and relieved by NO scavengers. NO induced G2/M cell cycle arrest in the teb mutant, indicative of stalled replication forks. Genetic analyses indicate that Polθ is required for translesion DNA synthesis across NO-induced lesions, but not oxidation-induced lesions. Whole-genome sequencing revealed that Polθ bypasses NO-induced base adducts in an error-free manner and generates mutations characteristic of Polθ-mediated end joining. Our experimental data collectively suggests that Polθ plays dual roles in protecting plants from NO-induced DNA damage. Since Polθ is conserved in higher eukaryotes, mammalian Polθ may also be required for balancing NO physiological signaling and genotoxicity. 相似文献
77.
云南主要有害实蝇种类及区划 总被引:1,自引:0,他引:1
采用诱剂诱捕辅以受害瓜果调查的方法,系统梳理了云南有害实蝇类群,确定主要有害种类25种。基于云南自然地理特征,采用聚类分析法深入探讨了云南实蝇区系,提出云南实蝇地理区划可分为德宏、版纳盆谷区,南部边缘中低山宽谷区,滇西南山原峡谷区,中东部高原、岩溶山原区以及北部高原山地区等5大区域。研究发现,受热量条件制约,云南主要有害实蝇类群呈南北向梯度递减;受纵向岭谷区"通道-阻隔"综合作用影响,云南西南部河谷地区是实蝇危害较为严重的区域。首次系统揭示了云南主要有害实蝇的地理分布规律。 相似文献
78.
Yihao Yang Ziyan Shen Youguang Li Chenda Xu Han Xia Hao Zhuang Shengyuan Sun Min Guo Changjie Yan 《植物学报(英文版)》2022,64(10):1860-1865
Rice eating and cooking quality(ECQ) is a major concern of breeders and consumers, determining market competitiveness worldwide. Rice grain protein content(GPC) is negatively related to ECQ,making it possible to improve ECQ by manipulating GPC. However, GPC is genetically complex and sensitive to environmental conditions; therefore, little progress has been made in traditional breeding for ECQ. Here, we report that CRISPR/Cas9-mediated knockout of genes encoding the grain storage protein gluteli... 相似文献
79.
Jun Ma Qiang Li Lei Zhang Sen Cai Yuanyuan Liu Juncheng Lin Rongfeng Huang Yongqiang Yu Mingzhang Wen Tongda Xu 《植物学报(英文版)》2022,64(12):2425-2437
Callus induction,which results in fate transition in plant cells,is considered as the first and key step for plant regeneration.This process can be stimulated in different tissues by a callus-inducing medium(CIM),which contains a high concentration of phytohormone auxin.Although a few key regulators for callus induction have been identified,the multiple aspects of the regulatory mechanism driven by high levels of auxin still need further investigation.Here,we find that high auxin induces callus ... 相似文献
80.