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Yeast two hybrid

Peng Tan, Lian He, Jun Cui, Chen Qian, Xin Cao, Meng Lin, Qingyuan Zhu, Yinyin Li, Changsheng Xing, Xiao Yu, Helen Y Wang, Rong-Fu Wang
Mitochondrial antiviral signaling platform protein (MAVS) acts as a central hub for RIG-I receptor proximal signal propagation. However, key components in the assembly of the MAVS mitochondrial platform that promote RIG-I mitochondrial localization and optimal activation are still largely undefined. Employing pooled RNAi and yeast two-hybrid screenings, we report that the mitochondrial adaptor protein tripartite motif (TRIM)14 provides a docking platform for the assembly of the mitochondrial signaling complex required for maximal activation of RIG-I-mediated signaling, consisting of WHIP and protein phosphatase PPP6C...
October 19, 2017: Molecular Cell
Athanassios Tartas, Christoforos Zarkadas, Maria Palaiomylitou, Niki Gounalaki, Dimitris Tzamarias, Metaxia Vlassi
The Ssn6-Tup1 complex is a general transcriptional co-repressor formed by the interaction of Ssn6, a tetratricopeptide repeat (TPR) protein, with the Tup1 repressor. We have previously shown that the N-terminal domain of Ssn6 comprising TPRs 1 to 3 is necessary and sufficient for this interaction and that TPR1 plays critical role. In a subsequent study, we provided evidence that in the absence of Tup1, TPR1 is susceptible to proteolysis and that conformational change(s) accompany the Ssn6-Tup1 complex formation...
2017: PloS One
Lisa Yasmin Asseck, Niklas Wallmeroth, Christopher Grefen
Protein-protein interactions (PPIs) play fundamental roles in all cellular processes. Especially membrane proteins facilitate a range of important biological functions in stimuli perception, signaling, and transport. Here we describe a detailed protocol for the yeast mating-based Split-Ubiquitin System (mbSUS) to study PPIs of ER membrane proteins in vivo. In contrast to the prominent Yeast Two-Hybrid, mbSUS enables analysis of full-length membrane proteins in their native cellular context. The system is based on the ubiquitin proteasome pathway leading to the release of an artificial transcription factor followed by activation of reporter genes to visualize PPIs...
2018: Methods in Molecular Biology
María García-Rubio, Sonia I Barroso, Andrés Aguilera
DNA-RNA hybrids form naturally during essential cellular functions such as transcription and replication. However, they may be an important source of genome instability, a hallmark of cancer and genetic diseases. Detection of DNA-RNA hybrids in cells is becoming crucial to understand an increasing number of molecular biology processes in genome dynamics and function and to identify new factors and mechanisms responsible for disease in biomedical research. Here, we describe two different procedures for the reliable detection of DNA-RNA hybrids in the yeast Saccharomyces cerevisiae and in human cells: DNA-RNA Immunoprecipitation (DRIP) and Immunofluorescence...
2018: Methods in Molecular Biology
H Earl Petzold, Stephen B Rigoulot, Chengsong Zhao, Bidisha Chanda, Xiaoyan Sheng, Mingzhe Zhao, Xiaoyan Jia, Allan W Dickerman, Eric P Beers, Amy M Brunner
Cellular processes, such as signal transduction and cell wall deposition, are organized by macromolecule interactions. Experimentally determined protein-protein interactions (PPIs) and protein-DNA interactions (PDIs) relevant to woody plant development are sparse. To begin to develop a Populus trichocarpa Torr. & A. Gray wood interactome, we applied the yeast-two-hybrid (Y2H) assay in different ways to enable the discovery of novel PPIs and connected networks. We first cloned open reading frames (ORFs) for 361 genes markedly upregulated in secondary xylem compared with secondary phloem and performed a binary Y2H screen with these proteins...
October 10, 2017: Tree Physiology
Jonathan Woodsmith, Luise Apelt, Victoria Casado-Medrano, Ziya Özkan, Bernd Timmermann, Ulrich Stelzl
The identification of genomic variants in healthy and diseased individuals continues to rapidly outpace our ability to functionally annotate these variants. Techniques that both systematically assay the functional consequences of nucleotide-resolution variation and can scale to hundreds of genes are urgently required. We designed a sensitive yeast two-hybrid-based 'off switch' for positive selection of interaction-disruptive variants from complex genetic libraries. Combined with massively parallel programmed mutagenesis and a sequencing readout, this method enables systematic profiling of protein-interaction determinants at amino-acid resolution...
October 16, 2017: Nature Methods
Heng Sun, Lin Chen, Jianying Li, Meiling Hu, Abid Ullah, Xin He, Xiyan Yang, Xianlong Zhang
JASMONATE ZIM-domain (JAZ) family proteins are involved in regulating diverse biological processes in plants. However, their functions have not been well characterized in cotton (Gossypium spp.). In the present study, 13, 15, 25, and 30 JAZ genes were identified in Gossypium arboretum, Gossypium raimondii, Gossypium barbadense and Gossypium hirsutum respectively, based on gene homology. Selection and variation analyses showed that the SNP density of GhJAZ genes in wild species was much higher than that in domesticated species...
October 3, 2017: Plant & Cell Physiology
Kun Yin, Chao Xu, Gui-Hua Zhao, Ye Liu, Ting Xiao, Song Zhu, Ge Yan
C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C351-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation...
October 16, 2017: Bioscience Trends
Xiuli Yang, Meghna Thakur, Juraj Koci, Alexis A Smith, Preeti Singh, Xuran Zhuang, Kamoltip Promnares, Yan Wang, Ozlem Buyuktanir, Utpal Pal
The proteome of Borrelia burgdorferi undergoes dynamic alterations as the microbe cycles through and persists in diverse host or vector environments. Therefore, studies of B. burgdorferi proteome and protein-protein interactions, which play central roles in biological processes in diverse organisms, are critical in understanding biology and infectivity of spirochetes. Here, we describe the proteomic analysis of B. burgdorferi by two-dimensional (2-D) gel electrophoresis followed by protein identification via liquid chromatography-mass spectrometry and database searching...
2018: Methods in Molecular Biology
Yuancheng Qi, Chao Liu, Xiankai Sun, Liyou Qiu, Jinwen Shen
Pleurotus ostreatus laccase gene poxc is transcriptionally induced by copper, and several putative MREs have been found and confirmed in its promoter region. However, the related transcription factors mediating copper response via MREs have not been reported. To isolate MRE binding proteins, we carried out yeast one-hybrid screens. Reporter genes containing two trimers of the cMRE2 and cMRE3 element were prepared and introduced into a yeast strain. The yeast was transformed with library cDNA that represents RNA isolated from CuSO4-treated fungi of P...
November 2017: Fungal Biology
Casey Ward, Maciej Maselko, Christopher Lupfer, Meagan Prescott, Manoj K Pastey
Human Respiratory Syncytial Virus (HRSV) is a leading cause of bronchopneumonia in infants and the elderly. To date, knowledge of viral and host protein interactions within HRSV is limited and are critical areas of research. Here, we show that HRSV Matrix (M) protein interacts with the cellular adaptor protein complex 3 specifically via its medium subunit (AP-3Mu3A). This novel protein-protein interaction was first detected via yeast-two hybrid screen and was further confirmed in a mammalian system by immunofluorescence colocalization and co-immunoprecipitation...
2017: PloS One
Huiping Bi, Weijuan Fan, Peng Zhang
Sweepoviruses have been identified globally and cause substantial yield losses and cultivar decline in sweet potato. This study aimed to investigate the interaction between sweepovirus and plant host by analyzing the function of the viral protein C4 of Sweet potato leaf curl virus-Jiangsu (SPLCV-JS), a sweepovirus cloned from diseased sweet potato plants in East China. Ectopic expression of the C4 in Arabidopsis altered plant development drastically with phenotypic changes including leaf curling, seedling twisting, deformation of floral tissues and reduction of pollen fertility, and seed number...
2017: Frontiers in Plant Science
Peipei Zhang, Yingxin Zhang, Lianping Sun, Sittipun Sinumporn, Zhengfu Yang, Bin Sun, Dandan Xuan, Zihe Li, Ping Yu, Weixun Wu, Kejian Wang, Liyong Cao, Shihua Cheng
Meiosis is crucial in reproduction of plants and ensuring genetic diversity. Although several genes involved in homologous recombination and DNA repair have been reported, their functions in rice (Oryza sativa) male meiosis remain poorly understood. Here, we isolated and characterized the rice OsFIGNL1 (OsFidgetin-like 1) gene, encoding a conserved AAA-ATPase, and explored its function and importance in male meiosis and pollen formation. The rice Osfignl1 mutant exhibited normal vegetative growth, but failed to produce seeds and displayed pollen abortion phenotype...
2017: Frontiers in Plant Science
Ling Chen, Zhaoling Yan, Zihao Xia, Yuqin Cheng, Zhiyuan Jiao, Biao Sun, Tao Zhou, Zaifeng Fan
RNA silencing plays a critical role against viral infection. To counteract this antiviral silencing, viruses have evolved various RNA silencing suppressors. Meanwhile, plants have evolved counter-counter defense strategies against RNA silencing suppression (RSS). In this study, the violaxanthin de-epoxidase protein of maize (Zea mays L.) (ZmVDE) was shown to interact specifically with the helper component-proteinase (HC-Pro, a viral RNA silencing suppressor) of Sugarcane mosaic virus (SCMV) via its mature protein region by yeast two-hybrid assay, which was confirmed by co-immunoprecipitation in Nicotiana benthamiana cells...
October 11, 2017: Plant Physiology
Zhi-Wei Liu, Hui Li, Wen-Li Wang, Zhi-Jun Wu, Xin Cui, Jing Zhuang
We analyzed the changes of theanine content in post-harvest tea leaves under high temperature (38 °C), low temperature (4 °C), and shading spreadings by using the ultra-high-performance liquid chromatography. The differentially expressed proteins (DEPs), CsFd-GOGAT and CsNADH-GOGAT, which are involved in theanine biosynthesis pathway, were identified from the corresponding proteome data. The protein-protein interactions of CsFd-GOGAT and CsNADH-GOGAT, CsTS1, or CsNiR were verified by yeast two-hybrid technology...
October 11, 2017: Journal of Agricultural and Food Chemistry
Meixiang Han, Xuefeng Jin, Wei Yao, Lingjie Kong, Gan Huang, Yujin Tao, Lingfei Li, Xiaojing Wang, Yaqin Wang
Petal appearance is an important horticultural trail that is generally used to evaluate the ornamental value of plants. However, knowledge of the molecular regulation of petal growth is mostly derived from analyses of Arabidopsis thaliana, and relatively little is known about this process in ornamental plants. Previously, GEG (Gerbera hybrida homolog of the gibberellin [GA]-stimulated transcript 1 [GAST1] from tomato), a gene from the GA stimulated Arabidopsis (GASA) family, was reported to be an inhibitor of ray petal growth in the ornamental species, G...
2017: Frontiers in Plant Science
Xiao-Juan Liu, Xin Liu, Xiu-Hong An, Peng-Liang Han, Chun-Xiang You, Yu-Jin Hao
ABA is a crucial phytohormone for development and stress responses in plants. Snf1-related protein kinase 1.1 (SnRK1.1) is involved in the ABA response. However, the molecular mechanism underlying the SnRK1.1 response to ABA is largely unknown. Here, it was found that overexpression of the apple MdSnRK1.1 gene enhanced ABA sensitivity in both transgenic apple calli and Arabidopsis seedlings. Subsequently, a yeast two-hybrid screen demonstrated that MdCAIP1 (C2-domain ABA Insensitive Protein1) interacted with MdSnRK1...
July 25, 2017: Plant & Cell Physiology
Jian-Ping An, Xin Liu, Hao-Hao Li, Chun-Xiang You, Xiao-Fei Wang, Yu-Jin Hao
MdMYB1 is an important regulator for anthocyanin accumulation in apple (Malus × domestica). Here, an apple RING E3 ligase MdMIEL1 was screened out as a partner of MdMYB1 with a yeast two-hybrid approach. Pull-down, BiFC and coimmunoprecipitation assays further verified the interaction between MdMIEL1 and MdMYB1 proteins. Subsequently, in vitro and in vivo experiments indicated that MdMIEL1 functioned as an ubiquitin E3 ligase to ubiquitinate MdMYB1 protein, followed by degradation through a 26S proteasome pathway...
September 15, 2017: Plant & Cell Physiology
Hang Ge, Jing Zhang, Yi-Jin Zhang, Xian Li, Xue-Ren Yin, Donald Grierson, Kun-Song Chen
Lignin is an important component of many plant secondary cell walls. In the fruit of loquat (Eriobotrya japonica), lignification of cell walls in the fleshy tissue occurs when fruit are subjected to low-temperature storage, which is commonly used to avoid the rapid senescence that occurs at room temperature. In this study, two NAC domain genes, EjNAC3 and EjNAC4, were isolated and shown to be significantly induced at 0 °C, which was concomitant with an increase in the fruit lignification index. Lignification and expression of both EjNAC3 and EjNAC4 were inhibited by low-temperature conditioning and by heat treatment...
September 16, 2017: Journal of Experimental Botany
Ying-Ping Huang, Ying-Wen Huang, I-Hsuan Chen, Lin-Ling Shenkwen, Yau-Huei Hsu, Ching-Hsiu Tsai
To establish a successful infection, a virus needs to replicate and move cell-to-cell efficiently. We investigated whether one of the genes upregulated in Nicotiana benthamiana after Bamboo mosaic virus (BaMV) inoculation was involved in regulating virus movement. We revealed the gene to be a plasma membrane-associated cation-binding protein 1-like protein, designated NbPCaP1L. The expression of NbPCaP1L in N. benthamiana was knocked down using Tobacco rattle virus-based gene silencing and consequently the accumulation of BaMV increased significantly to that of control plants...
August 29, 2017: Journal of Experimental Botany
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