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 ...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.展开更多
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 inter...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 fragments.After processing,the ds cDNA was connected to three types of reading frame pGADT7-SfiI carriers,and the three connection products in E.coli Electrocell were used to build the primary cDNA library.The 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 band.To 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 strain.The 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 cfu.The 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.展开更多
The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 (SOD1) using a yeast two-hybrid system. Using BLAST software, 15 real proteins which ...The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 (SOD1) using a yeast two-hybrid system. Using BLAST software, 15 real proteins which interacted with mutant SOD1 were obtained, including 8 known proteins (protein tyrosine-phosphatase non-receptor type 2, TBCl D4, protein kinase family, splicing factor, arginine/serine-rich 2, SRC protein tyrosine kinase Fyn, β-sarcoglycan; glycine receptor a2, microtubule associated protein/microtubule affinity-regulating kinase 1, ferritin H chain), and 7 unknown proteins. Results demonstrated interaction of mutant SOD1 with microtubule associated protein/microtubule affinity-regulating kinase 1 and β-sarcoglycan.展开更多
By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of re...By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of reporter genes were investigated.Total mRNA extracted from Hela cells was reversely transcribed into cDNA.Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector.The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.Th...展开更多
[Objective] The paper was to obtain host proteins interacting with porcine epidemic diarrhea virus (PEDV) N protein. [Method] The re-combinant vector pGBKT7-N of PEDV N gene was constructed and used as the bait plas...[Objective] The paper was to obtain host proteins interacting with porcine epidemic diarrhea virus (PEDV) N protein. [Method] The re-combinant vector pGBKT7-N of PEDV N gene was constructed and used as the bait plasmid to screen the proteins interacting with N protein ofPEDV from the cDNA library of porcine alveolar macrophage (PAM) by yeast two-hybrid method. [Result] There was no toxicity and self activationof bait protein in yeast hybridization system, and six proteins (FTH1, LGALS3, CORO1C, SNRPG, KRTAP5-3, ZNF598) interacting with N proteinwere indentified. It was confirmed that LGALS3 and SNRPG had specific interaction with N protein by return experiment and co-immunoprecipitation(CoIP) test. [Conclusion] The study lays a foundation for further studying the function of PEDV N protein and the pathogenic mechanism of PEDV.展开更多
Mitogen-activated protein kinases(MAPKs,also known as MPKs)regulate diverse cellular and physiological functions,and dual-specificity MAPK phosphatases(MKPs)modulate MAPK signalling through MAPK dephosphorylation and ...Mitogen-activated protein kinases(MAPKs,also known as MPKs)regulate diverse cellular and physiological functions,and dual-specificity MAPK phosphatases(MKPs)modulate MAPK signalling through MAPK dephosphorylation and inactivation.Due to lacking of overall understanding for the regulatory networks between Chrysanthemum morifolium MKPs(CmMKPs)and C.morifolium MAPKs(CmMPKs),we systematically studied the interactions between four groups of CmMPKs and eight identified CmMKPs in chrysanthemum and found that the interaction between the specific CmMKP and the specific CmMPK differed from those in other plants.Furthermore,the expression of CmMKP1 and CmMKP1-LIKE1showed opposite trends during the development of chrysanthemum flower buds under salt treatment and Alternaria alternata inoculation,but these genes could interact with the same CmMPKs,providing insight into the subfunctionalization of paralogues.Amino acid variations(M87V,T277P and V6L)in dual-specificity protein phosphatases(DsPTP1)-LIKE1/2/3 changed the interactions of these proteins with the four CmMPK groups in chrysanthemum,providing evidence for the de/neofunctionalization of paralogues in polyploids,suggesting that we can identify the key functional sites of proteins by studying polyploid paralogues.展开更多
The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease, Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase (beta-site amyloid pr...The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease, Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase (beta-site amyloid precursor protein-cleaving enzyme 1) and r-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of AIzheimer's disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1- interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer's disease.展开更多
BACKGROUND: Midkine is a heparin-binding growth factor that promotes the proliferation, survival, migration and differentiation of various target cells. Midkine plays an important role in tumorigenesis and tumor progr...BACKGROUND: Midkine is a heparin-binding growth factor that promotes the proliferation, survival, migration and differentiation of various target cells. Midkine plays an important role in tumorigenesis and tumor progression, and is overexpressed in many human malignant tumors. Patients with high tumor midkine expression frequently have a worse prognosis than those with low expression. The present study was designed to investigate the interaction network of midkine in hepatic cancer cells, and to elucidate its role in hepatocellular carcinoma. METHODS: DNA encoding full-length midkine was cloned into pDBLeu vector to serve as bait in yeast two-hybrid screening to identify interacting proteins. Candidate proteins were examined on SC-Leu-Trp-His+3-AT (20 mmol/L) plates and assayed for X-gal activity, then sequenced and classified according to the GenBank. Finally, identified proteins were expressed by the in vitro expression system pCMVTnT, and protein interactions were confirmed by co-immunoprecipitation. RESULTS: Using the yeast two-hybrid system, we found 6 proteins that interacted with midkine: NK-kappa-B inhibitor alpha (I-κ-B-α), Dvl-binding protein naked cuticle 2, granulin, latent active TGF-β binding protein 3, latent active TGF-β binding protein 4, and phospholipid scramblase 1. In vitro co-immunoprecipitation demonstrated that all identified proteins directly interacted with midkine.CONCLUSION: The identification of midkine-interacting proteins in hepatic cancer cells indicates that midkine is a multifunctional factor that may participate in cell migration, differentiation, and proliferation, and is also associated with the multicellular response feedback during the development of hepatocellular carcinoma.展开更多
AIM: To investigate the biological function of 14-3-3σ protein and to look for proteins that interact with 14-3-3σ protein in colon cancer stem cells. METHODS: Reverse transcription polymerase chain reaction was per...AIM: To investigate the biological function of 14-3-3σ protein and to look for proteins that interact with 14-3-3σ protein in colon cancer stem cells. METHODS: Reverse transcription polymerase chain reaction was performed to amplify the 14-3-3σ gene from the mRNA of colon cancer stem cells. The gene was then cloned into the pGEM-T vector. After being sequenced, the target gene 14-3-3σ was cut from the pGEM-T vector and cloned into the pGBKT7 yeast expression plasmid. Then, the bait plasmid pGBKT7-14-3-3σ was transformed into the yeast strain AH109. After the expression of the pGBKT7-14-3-3σ fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening assay was performed by mating AH109 with Y187 that contained a HeLa cDNA library plasmid. The interaction between the 14-3-3σ protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybridscreen. After extracting and sequencing the plasmids from the positive colonies, we performed a bioinformatics analysis. A coimmunoprecipitation assay was performed to confirm the interaction between 14-3-3σ and the proteins obtained from the positive colonies. Finally, we constructed 14-3-3σ and potassium channel modulatory factor 1 (KCMF1) siRNA expression plasmids and transfected them into colon cancer stem cells. RESULTS: The bait plasmid pGBKT7-14-3-3σ was constructed successfully, and the 14-3-3σ protein had no toxic or autonomous activation effect on the yeast. Nineteen true-positive colonies were selected and sequenced, and their full-length sequences were obtained. We searched for homologous DNA sequences for these sequences from GenBank. Among the positive colonies, four coding genes with known functions were obtained, including KCMF1 , quinone oxidore-ductase (NQO2 ), hydroxyisobutyrate dehydrogenase (HIBADH ) and 14-3-3σ . For the subsequent coimmu-noprecipitation assay, the plasmids PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH were successfully constructed, and the sequences were further confirmed by DNA sequencing. The Fugene 6 reagent was used to transfect the plasmids, and fluorescence-activated cell sorting analysis showed the transfection efficiency was 97.8% after 48 h. The HEK 293FT cells showed the stable expression of the PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH plasmids. After anti-Myc antibody immunoprecipitation with Myc-KCMF1, Myc-NQO2 and Myc-HIBADH from cell lysates, the presence of Flag-14-3-3σ protein in the immuno-precipitated complex was determined by western blot analysis. The knock-down expression of the 14-3-3σ and KCMF1 proteins significantly inhibited cell proliferation and colony formation of SW1116csc. CONCLUSION: Genes of the proteins that interactedwith 14-3-3σ were successfully screened from a HeLa cDNA library. KCMF1 and 14-3-3σ protein may affect the proliferation and colony formation of human colon cancer stem cells.展开更多
Two DNA fragments encoding PDZ domain (21-110 residues) and BAR domain ( 150-360 residues) from PICK1 (1-416 residues) were amplified by PCR and then introduced into vectors, pET-32M and pMAL-e2X respectively to...Two DNA fragments encoding PDZ domain (21-110 residues) and BAR domain ( 150-360 residues) from PICK1 (1-416 residues) were amplified by PCR and then introduced into vectors, pET-32M and pMAL-e2X respectively to generate recombinant plasmids, pE-pdz and pM-bar. Having been separately transferred into the hosts E. coli BL21 and E. coli JM109, these two strains can express fusion proteins: His-tagged PDZ(PDZ domain) and maltose binding protein-BAR( MBP-BAR domain) respectively, as confirmed by both SDS-PAGE and Wostem blotting. The interaction between these two domains is dose-dependence, as identified by a pull-down test. Moreover, it has been shown from the ELISA analysis that the actual amount of PDZ bound to MBP-BAR-amylose beads reaches ( 16 ± 0. 5)%, as calculated by the molar ratio of PDZ to MBP-BAR. In addition, the interaction between BAR(bait) and PDZ(prey) in vivo was also examined with a yeast two-hybrid system.展开更多
To further research the regulatory network of pyruvate dehydrogenase kinase (designated as TaPDK) in physiological male-sterility (PHYMS) of wheat induced by chemical hybridizing agent (CHA) SQ-1, an anther cDNA...To further research the regulatory network of pyruvate dehydrogenase kinase (designated as TaPDK) in physiological male-sterility (PHYMS) of wheat induced by chemical hybridizing agent (CHA) SQ-1, an anther cDNA library was constructed, and the proteins interacting with TaPDK were screened via yeast two-hybrid technique. Subsequently, a few candidate proteins in nucleotide expression levels were detected by real-time quantitative PCR. Yeast-two hybrid screening was performed by mating yeast strain Y2HGold containing BD-TaPDK bait plasmid with yeast strain Y187 including anther cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Ade/-His/-Leu/-Trp) (QDO), and further were incubated on QDO medium containing AbA and X-α-Gal. The interactions between TaPDK and the proteins obtained from positive colonies were further confirmed by co-transformation validation. After plasmids DNA were extracted from blue colonies and sequenced, the sequences results were analyzed by bioinformatic methods. Finally, 24 colonies were obtained, including eight genes, namely non-specific lipid-transfer protein precursor (TanLTP), polyubiquitin (TaPUbi), glyceraldehyde-3-phosphate dehydrogenase, proliferating cell nuclear antigen (TaPCNA), CBS domain containing protein (TaCBS), actin, guanine nucleotide-binding protein beta subunit, chalcone synthase, and three new genes with unknown function. The results of quantitative RT-PCR showed that the expression levels of TanLTP, TaPUbi, and TaPCNA were obviously up-regulated in PHYMS anther, and TaCBS expression was only increased at the tricellular stage in PHYMS anther compared with in fertile lines. Whereas, the expression of TaPDK was obviously down-regulated in PHYMS lines. Collectively, these datas indicated that the majority of candidate proteins might be related to pollen abortion in PHYMS lines, which further suggested that TaPDK plays multiple roles in pollen development, besides participating in regulating pyruvate dehydrogenase complex activity.展开更多
Liver-expressed antimicrobial peptide 2(LEAP-2)is a cationic peptide that plays an important role in a host’s innate immune system.We previously demonstrated that mudskipper(Boleophthalmus pectinirostris)LEAP-2(BpLEA...Liver-expressed antimicrobial peptide 2(LEAP-2)is a cationic peptide that plays an important role in a host’s innate immune system.We previously demonstrated that mudskipper(Boleophthalmus pectinirostris)LEAP-2(BpLEAP-2)induces chemotaxis and activation of monocytes/macrophages(MO/MΦ).However,the molecular mechanism by which BpLEAP-2 regulates MO/MΦ remains unclear.In this study,we used yeast twohybrid cDNA library screening to identify mudskipper protein(s)that interacted with BpLEAP-2,and characterized a sequence encoding motile sperm domain-containing protein 2(BpMOSPD2).The interaction between BpLEAP-2 and BpMOSPD2 was subsequently confirmed by co-immunoprecipitation(Co-IP).Sequence analyses revealed that the predicted BpMOSPD2 contained an N-terminal extracellular portion composed of a CRAL-TRIO domain and a motile sperm domain,a C-terminal transmembrane domain,and a short cytoplasmic tail.Phylogenetic tree analysis indicated that BpMOSPD2 grouped tightly with fish MOSPD2 homologs and was most closely related to that of the Nile tilapia(Oreochromis niloticus).The recombinant BpMOSPD2 was produced by prokaryotic expression and the corresponding antibody was prepared for protein concentration determination.RNA interference was used to knockdown BpMOSPD2 expression in the mudskipper MO/MΦ,and the knockdown efficiency was confirmed by quantitative real-time polymerase chain reaction(qRT-PCR)and western blotting.Knockdown of BpMOSPD2 significantly inhibited BpLEAP-2-induced chemotaxis of mudskipper MO/MΦand BpLEAP-2-induced bacterial killing activity.Furthermore,knockdown of BpMOSPD2 inhibited the effect of BpLEAP-2 on mRNA expression levels of BpIL-10,BpTNFα,BpIL-1β,and BpTGFβ in MO/MΦ.In general,BpMOSPD2 directly interacted with BpLEAP-2,and mediated the effects of BpLEAP-2 on chemotaxis and activation of mudskipper MO/MΦ.This is the first identification of MOSPD2 as a receptor for LEAP-2.展开更多
Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary ce...Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary cell wall formation. It has also been suggested that such three CesAs interact with each other to form plasma-membrane bound rosette complexes that are functional during cellulose production. However, in vivo demonstration of such assemblies of three CesAs into rosettes has not been possible. We used yeast two-hybrid assays to demonstrate the possible interactions among several CesAs from Arabidopsis and aspen via their N-terminal zinc-binding domains (ZnBDs). While strong positive interactions were detected among ZnBDs from secondary wall associated CesAs of both Arabidopsis and aspen, the intergeneric interactions between Arabidopsis and aspen CesAs were weak. Moreover, in aspen, three primary wall associated CesA ZnBDs positively interacted with each other as well as with secondary CesAs. These results suggest that ZnBDs from either primary or secondary CesAs, and even from different plant species could interact but are perhaps insufficient for specificities of such interactions among CesAs. These observations suggest that some other more specific interacting regions might exist within CesAs. It is also possible that some hitherto unknown mechanism exists in plants for assembling the rosette complexes with different compositions of CesAs. Understanding how cellulose is synthesized will have a direct impact on utilization of lignocellulosic biomass for bioenergy production.展开更多
The POU domain transcription factor Oct4 is a master regulator in maintaining self-renewal and pluripotency of embryonic stem (ES) cells. To further explore the functional network of Oct4, the yeast two-hybrid syste...The POU domain transcription factor Oct4 is a master regulator in maintaining self-renewal and pluripotency of embryonic stem (ES) cells. To further explore the functional network of Oct4, the yeast two-hybrid system was used to search for Oct4 interacting proteins. PH domain (containing POU domain and homeodomain) of human OCT4 was used as a bait. From the human testis cDNA library, we identified a strong interaction between OCT4 and karyopberin-alpha 2 (KPNA-2). KPNA2 is involved in active nuclear import of proteins. This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. The interaction between OCT4 and KPNA-2 was further mapped to multiple regions of the two proteins. In addition, we studied nuclear localization signal (NLS) of mouse Oct4 and demonstrated that it is essential for Oct4 nuclear localization. Thus, our data suggest that Oct4 nuclear localization may be mediated by its interaction with KPNA-2.展开更多
Fragile X-related protein 1(FXR1P) is a member of the FXR gene family,which also includes fragile X mental retardation protein and fragile X-related protein 2(FXR2P).To understand the functions of FXR1P,we screene...Fragile X-related protein 1(FXR1P) is a member of the FXR gene family,which also includes fragile X mental retardation protein and fragile X-related protein 2(FXR2P).To understand the functions of FXR1P,we screened FXR1P-interacting proteins using a yeast two-hybrid system.FXR1P was fused to pGBKT7 and used as the bait to screen a human fetal brain cDNA library.This screening revealed 10 FXR1P-interacting proteins including FTH1.FTH1 encodes Homo sapiens ferritin,heavy polypeptide 1.The interaction between FXR1P and FTH1 was confirmed by retesting in yeast using both a β-galactosidase assay and growth studies on selective media.A co-immunoprecipitation assay in mammalian cells further confirmed the FXR1P/FTH1 interaction.Moreover,the results revealed that FTH1 colocalized with FXR1P in the cytoplasm around the nucleus in mammalian cells.The present findings suggest that FXR1P plays an important role in iron metabolism in the brain by interacting with FTH1.This provides clues for elucidating the relationship between FXR1P function and fragile X syndrome.展开更多
Murine cytomegalovirus(MCMV) IE3 protein is a multifunctional viral protein that inter-acts with several target proteins of both viral and host cellular origin.To investigate the biological func-tion of IE3 in the p...Murine cytomegalovirus(MCMV) IE3 protein is a multifunctional viral protein that inter-acts with several target proteins of both viral and host cellular origin.To investigate the biological func-tion of IE3 in the pathogenesis of the brain disorders caused by CMV,a screening for host cellular pro-teins that could interact with IE3 was performed.By yeast two-hybrid screening,ankyrin repeats do-main 17(Ankrd17,also known as Gtar) was identified as a host factor that could interact with IE3.This interaction was verified by yeast two-hybrid assay and chemiluminescent co-immunoprecipitaion.Map-ping analysis suggested that the 1-148 residues of IE3 were responsible for the interaction.These results suggested that the interaction between Ankrd17 and IE3 may play a key role in the pathogenesis of MCMV-associated disease.展开更多
Rice black-streaked dwarf virus (RBSDV) is a recognized member of the genus Fijivirus, family Reoviridae. Its genome has ten double-stranded RNA (dsRNA) segments ($1-$10), in which the fifth genome segment ($5...Rice black-streaked dwarf virus (RBSDV) is a recognized member of the genus Fijivirus, family Reoviridae. Its genome has ten double-stranded RNA (dsRNA) segments ($1-$10), in which the fifth genome segment ($5) contains two open reading frames (ORFs) with a partially overlapping region. The second ORF of RBSDV S5 encodes a viral nonstructural protein named p5b with unknown function. To reveal the function of p5b, its gene was ligated into the bait plasmid pGBKT7 and an expression library containing rice cDNAs was constructed using plasmid pGADT7 for yeast two-hybrid assay. The bait protein p5b was detected in yeast by western blot, and the result of an auto-activation test showed that p5b could not autonomously activate the expression of reporter genes in yeast. Then the bait protein p5b was used for screening the cDNA expression libraries of rice. Gene fragments of some pivotal enzymes involved in photosynthesis, respiration and other important metabolic processes, were identified to interact with p5b in yeast, suggesting that these interactions may play roles in symptom development in infected plants.展开更多
Abstract The broad-spectrum rice blast resistance gene Pik2-H4 was cloned from the highly resistant line H4. The full-length sequence of Pik2-H4 was 3 066 bp, which was different from the corresponding allele sequence...Abstract The broad-spectrum rice blast resistance gene Pik2-H4 was cloned from the highly resistant line H4. The full-length sequence of Pik2-H4 was 3 066 bp, which was different from the corresponding allele sequence of Nipponbare. Pik2-H4 belonged to NBS-LRR genes and coded a protein containing NB-ARC domain and leueine-rieh repeats. To find the interaction proteins with Pik2-H4 from the rice, the yeast two-hybrid system was used and three important proteins ( LOC- Os08939300, LOCOs03g25960 and LOC-Os09929130)were identified. The results could provide some new information for the mechanism of rice blast resistance mediated by Pik2-H4.展开更多
Integrin α11 (ITGA11) is one of the collagen-binding integrin α chains;however, its biological significance remains unknown. To determine the functions of ITGA11, we performed a yeast two-hybrid screen using the cyt...Integrin α11 (ITGA11) is one of the collagen-binding integrin α chains;however, its biological significance remains unknown. To determine the functions of ITGA11, we performed a yeast two-hybrid screen using the cytoplasmic domain of ITGA11 as bait and transformed an EGY48 yeast strain with the bait-containing plasmid using the plasmid from a human lung fibroblast cDNA library. This screen identified calcium- and integrin-binding protein 1 (CIB1) as prey. Recombinant ITGA11 and CIB1 were expressed in mammalian cells and used in coimmunoprecipitation experiments, which showed that full-length ITGA11 and CIB1 are also associated in vivo. Over-expression of CIB1 in the human lung myofibroblast MRC-5 cells decreased the expression of α-smooth muscle actin and fibronectin. Using a mouse model of pulmonary fibrosis (bleomycin-treatment), we detected elevated expression of CIB1 in lung tissues compared with controls. These data suggest that CIB1 may regulate pulmonary fibrosis in concert with IT-GA11.展开更多
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...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展开更多
文摘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.
基金Supported by the National Nature Science Foundation of China(31372088)the "Academic Backbone" Project of Northeast Agricultural University(15XG05)China Agriculture Research System(CARS-26-02)
文摘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 fragments.After processing,the ds cDNA was connected to three types of reading frame pGADT7-SfiI carriers,and the three connection products in E.coli Electrocell were used to build the primary cDNA library.The 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 band.To 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 strain.The 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 cfu.The 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.
基金the National Natural Science Foundation of China, No. 30300116
文摘The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 (SOD1) using a yeast two-hybrid system. Using BLAST software, 15 real proteins which interacted with mutant SOD1 were obtained, including 8 known proteins (protein tyrosine-phosphatase non-receptor type 2, TBCl D4, protein kinase family, splicing factor, arginine/serine-rich 2, SRC protein tyrosine kinase Fyn, β-sarcoglycan; glycine receptor a2, microtubule associated protein/microtubule affinity-regulating kinase 1, ferritin H chain), and 7 unknown proteins. Results demonstrated interaction of mutant SOD1 with microtubule associated protein/microtubule affinity-regulating kinase 1 and β-sarcoglycan.
基金supported by grants from National Natural Sciences Foundation of China(No.30672227,30600668)"973"Program of China(No.2009CB521800)Joint Research Fund for Overseas Chinese,Hong Kong and Macao Young Scholars(No.30628029)
文摘By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of reporter genes were investigated.Total mRNA extracted from Hela cells was reversely transcribed into cDNA.Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector.The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.Th...
基金Supported by National Natural Science Foundation of China(31201915,31502071)Key Project of Science and Technology Promoting Agriculture in Shanghai City[HNKGZ(2013)No.3-6,No.5-5]
文摘[Objective] The paper was to obtain host proteins interacting with porcine epidemic diarrhea virus (PEDV) N protein. [Method] The re-combinant vector pGBKT7-N of PEDV N gene was constructed and used as the bait plasmid to screen the proteins interacting with N protein ofPEDV from the cDNA library of porcine alveolar macrophage (PAM) by yeast two-hybrid method. [Result] There was no toxicity and self activationof bait protein in yeast hybridization system, and six proteins (FTH1, LGALS3, CORO1C, SNRPG, KRTAP5-3, ZNF598) interacting with N proteinwere indentified. It was confirmed that LGALS3 and SNRPG had specific interaction with N protein by return experiment and co-immunoprecipitation(CoIP) test. [Conclusion] The study lays a foundation for further studying the function of PEDV N protein and the pathogenic mechanism of PEDV.
基金funded by the National Natural Science Foundation of China(Grant Nos.31870694,31870279,32002083)the Natural Science Fund of Jiangsu Province(Grant No.BK20190076)+2 种基金the National Key R&D Program of China(Grant No.2018YFD1000403)the Foundation of Central Laboratory of Xinyang Agriculture and Forestry University(Grant No.FCL202002)a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institution。
文摘Mitogen-activated protein kinases(MAPKs,also known as MPKs)regulate diverse cellular and physiological functions,and dual-specificity MAPK phosphatases(MKPs)modulate MAPK signalling through MAPK dephosphorylation and inactivation.Due to lacking of overall understanding for the regulatory networks between Chrysanthemum morifolium MKPs(CmMKPs)and C.morifolium MAPKs(CmMPKs),we systematically studied the interactions between four groups of CmMPKs and eight identified CmMKPs in chrysanthemum and found that the interaction between the specific CmMKP and the specific CmMPK differed from those in other plants.Furthermore,the expression of CmMKP1 and CmMKP1-LIKE1showed opposite trends during the development of chrysanthemum flower buds under salt treatment and Alternaria alternata inoculation,but these genes could interact with the same CmMPKs,providing insight into the subfunctionalization of paralogues.Amino acid variations(M87V,T277P and V6L)in dual-specificity protein phosphatases(DsPTP1)-LIKE1/2/3 changed the interactions of these proteins with the four CmMPK groups in chrysanthemum,providing evidence for the de/neofunctionalization of paralogues in polyploids,suggesting that we can identify the key functional sites of proteins by studying polyploid paralogues.
基金supported by the National Natural Science Foundation of China, No. 81171192XMU Basic Training Program of Undergraduate, No. CXB2011019Visiting Scholar Fellowship of Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering of Xiamen University, No. 201101
文摘The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease, Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase (beta-site amyloid precursor protein-cleaving enzyme 1) and r-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of AIzheimer's disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1- interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer's disease.
基金supported by grants from the National Natural Science Foundation of China (30772534)the Natural Science Foundation of Huzhou (2010YS05)
文摘BACKGROUND: Midkine is a heparin-binding growth factor that promotes the proliferation, survival, migration and differentiation of various target cells. Midkine plays an important role in tumorigenesis and tumor progression, and is overexpressed in many human malignant tumors. Patients with high tumor midkine expression frequently have a worse prognosis than those with low expression. The present study was designed to investigate the interaction network of midkine in hepatic cancer cells, and to elucidate its role in hepatocellular carcinoma. METHODS: DNA encoding full-length midkine was cloned into pDBLeu vector to serve as bait in yeast two-hybrid screening to identify interacting proteins. Candidate proteins were examined on SC-Leu-Trp-His+3-AT (20 mmol/L) plates and assayed for X-gal activity, then sequenced and classified according to the GenBank. Finally, identified proteins were expressed by the in vitro expression system pCMVTnT, and protein interactions were confirmed by co-immunoprecipitation. RESULTS: Using the yeast two-hybrid system, we found 6 proteins that interacted with midkine: NK-kappa-B inhibitor alpha (I-κ-B-α), Dvl-binding protein naked cuticle 2, granulin, latent active TGF-β binding protein 3, latent active TGF-β binding protein 4, and phospholipid scramblase 1. In vitro co-immunoprecipitation demonstrated that all identified proteins directly interacted with midkine.CONCLUSION: The identification of midkine-interacting proteins in hepatic cancer cells indicates that midkine is a multifunctional factor that may participate in cell migration, differentiation, and proliferation, and is also associated with the multicellular response feedback during the development of hepatocellular carcinoma.
基金Supported by The Medical Guidance Projects of Shanghai Science Committee,No.10411961800National Natural Science Foundation of China,No.81101617
文摘AIM: To investigate the biological function of 14-3-3σ protein and to look for proteins that interact with 14-3-3σ protein in colon cancer stem cells. METHODS: Reverse transcription polymerase chain reaction was performed to amplify the 14-3-3σ gene from the mRNA of colon cancer stem cells. The gene was then cloned into the pGEM-T vector. After being sequenced, the target gene 14-3-3σ was cut from the pGEM-T vector and cloned into the pGBKT7 yeast expression plasmid. Then, the bait plasmid pGBKT7-14-3-3σ was transformed into the yeast strain AH109. After the expression of the pGBKT7-14-3-3σ fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening assay was performed by mating AH109 with Y187 that contained a HeLa cDNA library plasmid. The interaction between the 14-3-3σ protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybridscreen. After extracting and sequencing the plasmids from the positive colonies, we performed a bioinformatics analysis. A coimmunoprecipitation assay was performed to confirm the interaction between 14-3-3σ and the proteins obtained from the positive colonies. Finally, we constructed 14-3-3σ and potassium channel modulatory factor 1 (KCMF1) siRNA expression plasmids and transfected them into colon cancer stem cells. RESULTS: The bait plasmid pGBKT7-14-3-3σ was constructed successfully, and the 14-3-3σ protein had no toxic or autonomous activation effect on the yeast. Nineteen true-positive colonies were selected and sequenced, and their full-length sequences were obtained. We searched for homologous DNA sequences for these sequences from GenBank. Among the positive colonies, four coding genes with known functions were obtained, including KCMF1 , quinone oxidore-ductase (NQO2 ), hydroxyisobutyrate dehydrogenase (HIBADH ) and 14-3-3σ . For the subsequent coimmu-noprecipitation assay, the plasmids PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH were successfully constructed, and the sequences were further confirmed by DNA sequencing. The Fugene 6 reagent was used to transfect the plasmids, and fluorescence-activated cell sorting analysis showed the transfection efficiency was 97.8% after 48 h. The HEK 293FT cells showed the stable expression of the PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH plasmids. After anti-Myc antibody immunoprecipitation with Myc-KCMF1, Myc-NQO2 and Myc-HIBADH from cell lysates, the presence of Flag-14-3-3σ protein in the immuno-precipitated complex was determined by western blot analysis. The knock-down expression of the 14-3-3σ and KCMF1 proteins significantly inhibited cell proliferation and colony formation of SW1116csc. CONCLUSION: Genes of the proteins that interactedwith 14-3-3σ were successfully screened from a HeLa cDNA library. KCMF1 and 14-3-3σ protein may affect the proliferation and colony formation of human colon cancer stem cells.
基金the National Natural Science Foundation of China(No 30400065)
文摘Two DNA fragments encoding PDZ domain (21-110 residues) and BAR domain ( 150-360 residues) from PICK1 (1-416 residues) were amplified by PCR and then introduced into vectors, pET-32M and pMAL-e2X respectively to generate recombinant plasmids, pE-pdz and pM-bar. Having been separately transferred into the hosts E. coli BL21 and E. coli JM109, these two strains can express fusion proteins: His-tagged PDZ(PDZ domain) and maltose binding protein-BAR( MBP-BAR domain) respectively, as confirmed by both SDS-PAGE and Wostem blotting. The interaction between these two domains is dose-dependence, as identified by a pull-down test. Moreover, it has been shown from the ELISA analysis that the actual amount of PDZ bound to MBP-BAR-amylose beads reaches ( 16 ± 0. 5)%, as calculated by the molar ratio of PDZ to MBP-BAR. In addition, the interaction between BAR(bait) and PDZ(prey) in vivo was also examined with a yeast two-hybrid system.
基金supported by the National High-Tech R&D Program of China(2011AA10A106)the National Natural Science Foundation of China(31071477,31171611)the Key Scientific and Technological Innovation Special Projects of Shaanxi"13115",China(2010ZDKG-68,2011KTZB02-01-01)
文摘To further research the regulatory network of pyruvate dehydrogenase kinase (designated as TaPDK) in physiological male-sterility (PHYMS) of wheat induced by chemical hybridizing agent (CHA) SQ-1, an anther cDNA library was constructed, and the proteins interacting with TaPDK were screened via yeast two-hybrid technique. Subsequently, a few candidate proteins in nucleotide expression levels were detected by real-time quantitative PCR. Yeast-two hybrid screening was performed by mating yeast strain Y2HGold containing BD-TaPDK bait plasmid with yeast strain Y187 including anther cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Ade/-His/-Leu/-Trp) (QDO), and further were incubated on QDO medium containing AbA and X-α-Gal. The interactions between TaPDK and the proteins obtained from positive colonies were further confirmed by co-transformation validation. After plasmids DNA were extracted from blue colonies and sequenced, the sequences results were analyzed by bioinformatic methods. Finally, 24 colonies were obtained, including eight genes, namely non-specific lipid-transfer protein precursor (TanLTP), polyubiquitin (TaPUbi), glyceraldehyde-3-phosphate dehydrogenase, proliferating cell nuclear antigen (TaPCNA), CBS domain containing protein (TaCBS), actin, guanine nucleotide-binding protein beta subunit, chalcone synthase, and three new genes with unknown function. The results of quantitative RT-PCR showed that the expression levels of TanLTP, TaPUbi, and TaPCNA were obviously up-regulated in PHYMS anther, and TaCBS expression was only increased at the tricellular stage in PHYMS anther compared with in fertile lines. Whereas, the expression of TaPDK was obviously down-regulated in PHYMS lines. Collectively, these datas indicated that the majority of candidate proteins might be related to pollen abortion in PHYMS lines, which further suggested that TaPDK plays multiple roles in pollen development, besides participating in regulating pyruvate dehydrogenase complex activity.
基金supported by the National Natural Science Foundation of China(31972821,31772876)Zhejiang Provincial Natural Science Foundation of China(LZ18C190001)+1 种基金Scientific Innovation Team Project of Ningbo(2015C110018)K.C.Wong Magna Fund in Ningbo University。
文摘Liver-expressed antimicrobial peptide 2(LEAP-2)is a cationic peptide that plays an important role in a host’s innate immune system.We previously demonstrated that mudskipper(Boleophthalmus pectinirostris)LEAP-2(BpLEAP-2)induces chemotaxis and activation of monocytes/macrophages(MO/MΦ).However,the molecular mechanism by which BpLEAP-2 regulates MO/MΦ remains unclear.In this study,we used yeast twohybrid cDNA library screening to identify mudskipper protein(s)that interacted with BpLEAP-2,and characterized a sequence encoding motile sperm domain-containing protein 2(BpMOSPD2).The interaction between BpLEAP-2 and BpMOSPD2 was subsequently confirmed by co-immunoprecipitation(Co-IP).Sequence analyses revealed that the predicted BpMOSPD2 contained an N-terminal extracellular portion composed of a CRAL-TRIO domain and a motile sperm domain,a C-terminal transmembrane domain,and a short cytoplasmic tail.Phylogenetic tree analysis indicated that BpMOSPD2 grouped tightly with fish MOSPD2 homologs and was most closely related to that of the Nile tilapia(Oreochromis niloticus).The recombinant BpMOSPD2 was produced by prokaryotic expression and the corresponding antibody was prepared for protein concentration determination.RNA interference was used to knockdown BpMOSPD2 expression in the mudskipper MO/MΦ,and the knockdown efficiency was confirmed by quantitative real-time polymerase chain reaction(qRT-PCR)and western blotting.Knockdown of BpMOSPD2 significantly inhibited BpLEAP-2-induced chemotaxis of mudskipper MO/MΦand BpLEAP-2-induced bacterial killing activity.Furthermore,knockdown of BpMOSPD2 inhibited the effect of BpLEAP-2 on mRNA expression levels of BpIL-10,BpTNFα,BpIL-1β,and BpTGFβ in MO/MΦ.In general,BpMOSPD2 directly interacted with BpLEAP-2,and mediated the effects of BpLEAP-2 on chemotaxis and activation of mudskipper MO/MΦ.This is the first identification of MOSPD2 as a receptor for LEAP-2.
文摘Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary cell wall formation. It has also been suggested that such three CesAs interact with each other to form plasma-membrane bound rosette complexes that are functional during cellulose production. However, in vivo demonstration of such assemblies of three CesAs into rosettes has not been possible. We used yeast two-hybrid assays to demonstrate the possible interactions among several CesAs from Arabidopsis and aspen via their N-terminal zinc-binding domains (ZnBDs). While strong positive interactions were detected among ZnBDs from secondary wall associated CesAs of both Arabidopsis and aspen, the intergeneric interactions between Arabidopsis and aspen CesAs were weak. Moreover, in aspen, three primary wall associated CesA ZnBDs positively interacted with each other as well as with secondary CesAs. These results suggest that ZnBDs from either primary or secondary CesAs, and even from different plant species could interact but are perhaps insufficient for specificities of such interactions among CesAs. These observations suggest that some other more specific interacting regions might exist within CesAs. It is also possible that some hitherto unknown mechanism exists in plants for assembling the rosette complexes with different compositions of CesAs. Understanding how cellulose is synthesized will have a direct impact on utilization of lignocellulosic biomass for bioenergy production.
基金supported by the Shanghai Science & Technology Developmental Foundations (No.06dj14001)the National High Technology Research and Development Program of China (No. 2006AA02Z197, 2006CB943901, and 2007CB947904)
文摘The POU domain transcription factor Oct4 is a master regulator in maintaining self-renewal and pluripotency of embryonic stem (ES) cells. To further explore the functional network of Oct4, the yeast two-hybrid system was used to search for Oct4 interacting proteins. PH domain (containing POU domain and homeodomain) of human OCT4 was used as a bait. From the human testis cDNA library, we identified a strong interaction between OCT4 and karyopberin-alpha 2 (KPNA-2). KPNA2 is involved in active nuclear import of proteins. This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. The interaction between OCT4 and KPNA-2 was further mapped to multiple regions of the two proteins. In addition, we studied nuclear localization signal (NLS) of mouse Oct4 and demonstrated that it is essential for Oct4 nuclear localization. Thus, our data suggest that Oct4 nuclear localization may be mediated by its interaction with KPNA-2.
基金the National Natural Science Foundation of China, No. 30370795
文摘Fragile X-related protein 1(FXR1P) is a member of the FXR gene family,which also includes fragile X mental retardation protein and fragile X-related protein 2(FXR2P).To understand the functions of FXR1P,we screened FXR1P-interacting proteins using a yeast two-hybrid system.FXR1P was fused to pGBKT7 and used as the bait to screen a human fetal brain cDNA library.This screening revealed 10 FXR1P-interacting proteins including FTH1.FTH1 encodes Homo sapiens ferritin,heavy polypeptide 1.The interaction between FXR1P and FTH1 was confirmed by retesting in yeast using both a β-galactosidase assay and growth studies on selective media.A co-immunoprecipitation assay in mammalian cells further confirmed the FXR1P/FTH1 interaction.Moreover,the results revealed that FTH1 colocalized with FXR1P in the cytoplasm around the nucleus in mammalian cells.The present findings suggest that FXR1P plays an important role in iron metabolism in the brain by interacting with FTH1.This provides clues for elucidating the relationship between FXR1P function and fragile X syndrome.
基金supported by a grant from National Natural Sciences Foundation of China (No. 30671859)
文摘Murine cytomegalovirus(MCMV) IE3 protein is a multifunctional viral protein that inter-acts with several target proteins of both viral and host cellular origin.To investigate the biological func-tion of IE3 in the pathogenesis of the brain disorders caused by CMV,a screening for host cellular pro-teins that could interact with IE3 was performed.By yeast two-hybrid screening,ankyrin repeats do-main 17(Ankrd17,also known as Gtar) was identified as a host factor that could interact with IE3.This interaction was verified by yeast two-hybrid assay and chemiluminescent co-immunoprecipitaion.Map-ping analysis suggested that the 1-148 residues of IE3 were responsible for the interaction.These results suggested that the interaction between Ankrd17 and IE3 may play a key role in the pathogenesis of MCMV-associated disease.
基金funded by the National Science and Technology Support Program of China(Grant No.2012BAD19B03)the National High Technology Research and Development Program of China(Grant No.2007AA10Z414)+4 种基金the National Basic Research Program of China(Grant No.2010CB126203)the International Science and Technology Cooperation Project(Grant No.2007DFB30350)the Special Fund for Agro-scientific Research in the Public Interest of China(Grant No.201003031)the Zhejiang Provincial Science and Technology Project,China(Grant No.2010C12027)the Zhejiang Provincial Foundation for Natural Science,China(Grant Nos.Z305165and Y3090657)
文摘Rice black-streaked dwarf virus (RBSDV) is a recognized member of the genus Fijivirus, family Reoviridae. Its genome has ten double-stranded RNA (dsRNA) segments ($1-$10), in which the fifth genome segment ($5) contains two open reading frames (ORFs) with a partially overlapping region. The second ORF of RBSDV S5 encodes a viral nonstructural protein named p5b with unknown function. To reveal the function of p5b, its gene was ligated into the bait plasmid pGBKT7 and an expression library containing rice cDNAs was constructed using plasmid pGADT7 for yeast two-hybrid assay. The bait protein p5b was detected in yeast by western blot, and the result of an auto-activation test showed that p5b could not autonomously activate the expression of reporter genes in yeast. Then the bait protein p5b was used for screening the cDNA expression libraries of rice. Gene fragments of some pivotal enzymes involved in photosynthesis, respiration and other important metabolic processes, were identified to interact with p5b in yeast, suggesting that these interactions may play roles in symptom development in infected plants.
基金Supported by Specialized Research Fund for the Doctoral Program of Higher Education"Resistance Mechanism of Rice Blast Resistance Gene Pik-m"(20124404120007)
文摘Abstract The broad-spectrum rice blast resistance gene Pik2-H4 was cloned from the highly resistant line H4. The full-length sequence of Pik2-H4 was 3 066 bp, which was different from the corresponding allele sequence of Nipponbare. Pik2-H4 belonged to NBS-LRR genes and coded a protein containing NB-ARC domain and leueine-rieh repeats. To find the interaction proteins with Pik2-H4 from the rice, the yeast two-hybrid system was used and three important proteins ( LOC- Os08939300, LOCOs03g25960 and LOC-Os09929130)were identified. The results could provide some new information for the mechanism of rice blast resistance mediated by Pik2-H4.
文摘Integrin α11 (ITGA11) is one of the collagen-binding integrin α chains;however, its biological significance remains unknown. To determine the functions of ITGA11, we performed a yeast two-hybrid screen using the cytoplasmic domain of ITGA11 as bait and transformed an EGY48 yeast strain with the bait-containing plasmid using the plasmid from a human lung fibroblast cDNA library. This screen identified calcium- and integrin-binding protein 1 (CIB1) as prey. Recombinant ITGA11 and CIB1 were expressed in mammalian cells and used in coimmunoprecipitation experiments, which showed that full-length ITGA11 and CIB1 are also associated in vivo. Over-expression of CIB1 in the human lung myofibroblast MRC-5 cells decreased the expression of α-smooth muscle actin and fibronectin. Using a mouse model of pulmonary fibrosis (bleomycin-treatment), we detected elevated expression of CIB1 in lung tissues compared with controls. These data suggest that CIB1 may regulate pulmonary fibrosis in concert with IT-GA11.
文摘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