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Screening of proteins that interact with human thrombopoietin receptor c-Mpl using yeast two-hybrid system

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Science China(Life Sciences) 2000年第3期43卷 239-244页

作者：赵新燕冯丽冰周伟国戴卫列李昌本赵寿元 State Key Laboratory of Genetic Engineering Institute of Genetics Fudan University Shanghai 200433 China

Thrombopoietin (TPO) is the major cytokine involved in platelet production and exerts its effects via the receptor c-Mpl. The yeast two-hybrid system has been used to screen the proteins interacting with c-Mpl. First, the cDNA fragment of c-Mpl intracellular domain was cloned into two-hybrid vector pAS2, and the resulting plasmid is designated as pASMM. Then a human placenta cDNA library was screened using the pASMM as a target plasmid. Seven positive clones were isolated from 150 000 independent transformants. Sequence analysis of one of the positive clones demonstrates that a part of coding sequence of vimentin from 611 bp to 3’ end and flanking non-translation region was obtained. Therefore, there is an interaction between vimentin and TPO receptor. The results suggest that cytoskeletal protein may play an important role in TPO signal transduction pathway.

关键词： thrombopoietin receptor c-Mpl yeast two-hybrid system vimentin interaction.

Isolation and characterization of a human apoptosis-inducing gene with yeast two-hybrid system

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Science China(Life Sciences) 2000年第3期43卷 310-320页

作者：齐兵齐义鹏 Masuo Yutsudo 刘青珍 Institute of Virology Wuhan University Wuhan 430072 China Research Institute for Microbial Diseases Osaka University 565 Osaka Japan

asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism of asy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system. The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels. Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5’ end and different 3’ end, and share a common open reading frame encoding a polypeptide of 236 amino acids. two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence. two highly hydrophobic regions encoding potentially two transmembrane domains are present. The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu). Immunoprecipitation assay confirmed the interaction

关键词： asy gene asyip gene apoptosis yeast two-hybrid system human lung cell cDNA library

Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

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Science China(Life Sciences) 2000年第2期43卷 169-175页

作者：石晓冰魏家绵沈允钢 Shanghai Institute of Plant Physiology Chinese Academy of Sciences Shanghai 200032 China

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding a, p, y, 8 and e subunits of Spinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding p-galactosidase was detected. Of all the combinations, that of y and e subunit genes showed the highest level of reporter gene expression, while those of a and p, a and e, p and e and p and 8 induced stable and significant reporter gene expression. The combination of 8 and e as well as that of 8 and y induced weak and unstable reporter gene expression. However, combinations of a and y, p and y and a and 8 did not induce reporter gene expression. These results suggested that specific and strong interactions between y and e, a and p, a and e, p and e and p and 8 subunits, and weak and transient interactions between 8 and e and 8 and y subunits occurred in the

关键词： ATP synthase subunit interaction yeast two-hybrid system.

Screening of BRD7 interacting proteins by yeast two-hybrid system

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Science China(Life Sciences) 2002年第5期45卷 546-552页

作者：余鹰周鸣朱诗国李小玲张必成李桂源 Cancer Research Institute Xiangya School of Medicine Central South University Changsha 410078 China

BRD7 gene is low or undetectably expressed in nasopharyngeal carcinoma tissue (NPC) and can obviously suppress the growth of NPC cell line HNE1. In this study, the proteins that interacted with BRD7 coding protein were screened by yeast two-hybrid system. The complete reading frame of BRD7 gene was subcloned into pAS2 vector (BRD7-BD). Then the human embryo brain cDNA library was screened by BRD7-BD bait. Eleven positive clones were obtained from 4.8×106 transformed clones. By sequencing directly, 6 interacting proteins of BRD7 coding protein were isolated (Bromodomain-containing 3 protein (BRD3), Bromodomain-containing 2 protein (BRD2), IκB kinase-beta, KIAA1375 protein, interleukin 7 and adaptor-related protein complex 3δ-1 subunit). These results suggest BRD7 protein might form heterogenous dimer or triplex polymer with BRD2 and/or BRD3 to participate in transcriptional regulation.

关键词： nasopharyngeal carcinoma, BRD7, two-hybrid system, interaction.

Use of bacterial two-hybrid system to investigate the molecular interaction between the regulators NifA and NifL of Enterobacter cloacae

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Science China(Life Sciences) 2002年第6期45卷 569-576页

作者：廖贡献沈善炯俞冠翘朱家璧 Laboratory of Molecular Genetics Shanghai Institute of Plant Physiology Shanghai Institutes of Biological Sciences Chinese Academy of Sciences Shanghai 200032 China

Expression of the nitrogen fixation (nif ) genes is tightly regulated by two proteins NifA and NifL in the (-subdivision of the proteobacteria. NifA is a transcriptional activator, which can be inactivated by NifL in the presence of oxygen or excess fixed nitrogen. A direct interaction between E. cloacae NifL and NifA was detected using the bacterial two-hybrid system. This interaction was accelerated in the presence of fixed nitrogen, while oxygen had no effect. NifL proteins, with their C-terminus being deleted, completely lost the ability to interact with NifA. The data suggest that the C-terminal domain of NifL acts as a sensor of the nitrogen status of the cell and mediates interaction with NifA.

关键词： bacterial two-hybrid system, NifL-NifA complex, environmental conditions, Enterobacter cloacae, Western blotting.

Shared and discrete interacting partners of ELL1 and ELL2 by yeast two-hybrid assay

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Advances in Bioscience and Biotechnology 2013年第7期4卷 774-780页

作者： Fortuna Arumemi Ian Bayles Joshua Paul Christine Milcarek Department of Immunology School of Medicine University of Pittsburgh Pittsburgh USA

ELL2 (eleven-nineteen lysine-rich leukemia transcription elongation factor), a component of a larger complex with pTEFb (cyclin T and CDK9) and AF4, is up-regulated in plasma cells where it influences mRNA processing by increasing exon skipping and enhancing proximal poly (A) site use. ELL2 is needed to produce the secretory-specific Ig heavy chain mRNA while ELL1 mRNA does not change in abundance with B cell stages. To investigate the potential interactions of other proteins with the ELL1 and ELL2 proteins, we preformed yeast two-hybrid studies. HSP40 and Testin were found to bind to ELL2 in its amino-terminal half. PCNA binds to ELL2 in a region encompassing amino acids 186 - 344. The potent transcription factors HIF1 α and ZNF622 interact with both ELL1 and 2 in the central, proline rich region. Meanwhile, BBS2 and ING3 interact with ELL1 but not ELL2 in this central proline-rich region. Many of the ELL-interacting-proteins uncovered in the two-hybrid screen are tumour suppressors that may work through the ELL: pTEFb complex to suppress or activate sets of genes in plasma cells.

关键词： Transcription Elongation Immunoglobulin Synthesis Yeast two-hybrid System

Construction of a Three-frame Yeast two-hybrid cDNA Library of Fusarium oxysporum

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Journal of Northeast Agricultural University(English Edition) 2019年第2期26卷 25-32页

作者： Luan Fei-shi Li Xiao-mei Zhu Zi-cheng Wang Xue-zheng Key Laboratory of Biology and Genetic Improvement Horticultural Crops(Northeast Region) Ministry of AgricultureHarbin 150030China College of Horticulture and Landscape Architecture Northeast Agricultural UniversityHarbin 150030China

A specialized test of two-hybrid library type three-frame cDNA yeast for Muskmelon Fusarium oxysporum using the switching mechanism at the 5'end of RNA template(SMART)technology was constructed to screen for interaction protein genes for wilt disease and to further research the molecular mechanisms of Fusarium oxysporum pathogenesis to explain the interactions between plant and pathogen.A 500-bp cDNA was purified and extracted using SMART and LD-PCR technology to synthesize ds cDNA and was then homogenized and purified to remove the *** processing,the ds cDNA was connected to three types of reading frame pGADT7-SfiI carriers,and the three connection products in *** Electrocell were used to build the primary cDNA *** titer of three ORF cDNA primary library storage capacities was 2.6×10^6,1.8×10^6 and 3×10^6 cfu;the PCR identification of the ORF 1 and 2 gene recombination rate was 94%,the ORF 3 gene recombination rate was 100%,and the insert length distribution was 0.5-4.0 kb as a single *** reach the quality requirements for library construction,three kinds of reading frame cDNA primary libraries were mixed and amplified,and the plasmid was transformed into the Y187 yeast *** titer of the Y187 yeast library was determined to be 3.5×107 cfu?mL-1,and the base of the yeast library was approximately 1 600 000 *** results showed that the construction of muskmelon Fusarium-specific two-hybrid library type three-frame cDNA yeast had a higher reservoir capacity and recombination rate and met the yeast two-hybrid screening requirements.

关键词： melon Fusarium oxysporum yeast two-hybrid cDNA library normalization

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Cell Research 2006年第6期16卷 539-547页

作者： Yankun Gao~1 Mei Jiang~1 Tao Yang~1 Jian Ni~1 Jiangye Chen~1 ~1State Key Laboratory of Molecular Biology,institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,320 Yue-yang Road Shanghai 200031,China State Key Laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China

hPFTAIRE1(PFTK1),a Cdc2-related protein kinase,is highly expressed in human *** exhibits cytoplasmicdistribution in Hela cells,although it contains two nuclear localization signals(NLSs)in its *** search for itssubstrates and regulatory components,we screened a two-hybrid library by using the full-length hPFTAIRE1 as a *** 14-3-3 isoforms(β,ε,η,τ)were identified interacting with the *** found a putative 14-3-3 bindingconsensus motif(RHSSPSS)in the hPFTAIRE1,which overlapped with its second *** of the RHSSPSS motifor substitution of Serwith Ala in the conserved binding motif abolished the specific interaction between the hPFTAIRE1and the 14-3-3 *** mutant S120A hPFTAIRE1 also showed a weak interaction to the 14-3-3 *** resultssuggested that the Seris crucial tor the interaction between hPFTAIRE1 and the 14-3-3 *** the hPFTAIRE1mutants distributed in cytoplasm of Hela cells and human neuroblastoma cells(SH-SYSY)when fused to the C-terminusof a green fluorescent protein(GFP),indicating that binding with the 14-3-3 proteins does not contribute to the subcel-lular localization of the hPFTAIRE1,although the binding may be involved in its signaling regulation.

关键词： hPFTAIRE1 14-3-3 proteins 14-3-3 binding motif two-hybrid

Identification of anti-Gram-negative bacteria agents targeting the interaction between ribosomal proteins L12 and L10

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Acta Pharmaceutica Sinica B 2018年第5期8卷 772-783页

作者： Weiwei Wang Chao Liu Ningyu Zhu Yuan Lin JiANDong Jiang Yanchang Wang Yan Li Shuyi Si Beijing Key Laboratory of Antimicrobial Agents Institute of Medicinal Biotechnology Peking Union Medical College and Chinese Academy of Medical Sciences State Key Laboratory of Bioactive Substances and Function of Natural Medicine Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College Department of Biomedical Sciences College of Medicine Florida State University

Gram-negative bacteria have become the main pathogens and cause serious clinical problems with increased morbidity and mortality. However, the slow discovery of new antimicrobial agents is unable to meet the need for the treatment of bacterial infections caused by drug-resistant strains. The interaction of L12 and L10 is essential for ribosomal function and protein synthesis. In this study, a yeast two-hybrid system was established to successfully detect the interaction between L12 and L10 proteins from gram-negative bacteria Escherichia coli, which allows us to screen compounds that specifically disrupt this interaction. With this system, we identified two compounds IMB-84 and IMB-87 that block L12-L10 interaction and show bactericidal activity against E. coli. We used glutathione-S-transferase(GST) pull-down and surface plasmon resonance(SPR) assays to demonstrate that these compounds disrupt L12-L10 interaction in vitro and the target of compounds was further confirmed by the overexpression of target proteins. Moreover, protein synthesis and elongation factor G-dependent GTPase activities are inhibited by two compounds. Therefore, we have identified two antibacterial agents that disrupt L12-L10 interaction by using yeast two-hybrid system.

关键词： Yeast two-hybrid Escherichia coli Ribosome L12/L10 Antimicrobial agents