scholarly journals Interaction of Mouse Pem Protein and Cell Division Cycle 37 Homolog

2005 ◽  
Vol 37 (11) ◽  
pp. 784-787 ◽  
Author(s):  
Fen Guo ◽  
Yue-Qin Li ◽  
Shi-Qian Li ◽  
Zhi-Wen Luo ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Cell Division ◽  
Amino Acid ◽  
Homeobox Gene ◽  
Interaction Model ◽  
Cell Division Cycle ◽  
Amino Acid Residues ◽  
Yeast Two Hybrid ◽  
Glutathione S Transferase ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

Abstract Mouse Pem, a homeobox gene, encodes a protein consisting of 210 amino acid residues. To study the function of mouse Pem protein, we used the yeast two-hybrid system to screen the library of 7-day mouse embryo with full-length mouse Pem cDNA. Fifty-two colonies were obtained after 1.57×108 colonies were screened by nutrition limitation and β-galactosidase assay. Seven individual insert fragments were obtained from the library, and three of them were identified, one of which was confirmed to be the cell division cycle 37 (Cdc37) homolog gene by sequencing. The interaction between mouse Pem and Cdc37 homolog was then confirmed by glutathione S-transferase pull-down assay, and the possible interaction model was suggested.

scholarly journals Interaction of Vav with ENX-1, a putative transcriptional regulator of homeobox gene expression.

1996 ◽  
Vol 16 (6) ◽  
pp. 3066-3073 ◽  
Author(s):  
O Hobert ◽  
B Jallal ◽  
A Ullrich
Keyword(s):  
Gene Expression ◽  
Critical Role ◽  
Homeobox Gene ◽  
Transcriptional Regulators ◽  
Polycomb Group ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

The proto-oncogene product Vav plays a critical role in hematopoietic signal transduction. By using the yeast two-hybrid system, we identified a novel human protein, ENX-1, which interacts specifically with Vav both in vitro and in vivo. ENX-1 represents the human homolog of the Drosophila Enhancer of zeste gene, a member of the Polycomb group of genes, which are transcriptional regulators of homeobox gene expression. Interaction with ENX-1 suggests that Vav functions as an upstream element in the transcriptional regulation of homeobox genes, known to be important effectors in the hematopoietic system.

scholarly journals Role of the Carboxy Terminus of Escherichia coli FtsA in Self-Interaction and Cell Division

2000 ◽  
Vol 182 (22) ◽  
pp. 6366-6373 ◽  
Author(s):  
Lucía Yim ◽  
Guy Vandenbussche ◽  
Jesús Mingorance ◽  
Sonsoles Rueda ◽  
Mercedes Casanova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Biological Function ◽  
Wild Type ◽  
Carboxy Terminus ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Carboxy Terminal ◽  
Two Hybrid

ABSTRACT The role of the carboxy terminus of the Escherichia coli cell division protein FtsA in bacterial division has been studied by making a series of short sequential deletions spanning from residue 394 to 420. Deletions as short as 5 residues destroy the biological function of the protein. Residue W415 is essential for the localization of the protein into septal rings. Overexpression of theftsA alleles harboring these deletions caused a coiled cell phenotype previously described for another carboxy-terminal mutation (Gayda et al., J. Bacteriol. 174:5362–5370, 1992), suggesting that an interaction of FtsA with itself might play a role in its function. The existence of such an interaction was demonstrated using the yeast two-hybrid system and a protein overlay assay. Even these short deletions are sufficient for impairing the interaction of the truncated FtsA forms with the wild-type protein in the yeast two-hybrid system. The existence of additional interactions between FtsA molecules, involving other domains, can be postulated from the interaction properties shown by the FtsA deletion mutant forms, because although unable to interact with the wild-type and with FtsAΔ1, they can interact with themselves and cross-interact with each other. The secondary structures of an extensive deletion, FtsAΔ27, and the wild-type protein are indistinguishable when analyzed by Fourier transform infrared spectroscopy, and moreover, FtsAΔ27 retains the ability to bind ATP. These results indicate that deletion of the carboxy-terminal 27 residues does not alter substantially the structure of the protein and suggest that the loss of biological function of the carboxy-terminal deletion mutants might be related to the modification of their interacting properties.

A novel testis protein, RSB-66, interacting with INCA1 (inhibitor of Cdk interacting with cyclin A1)

2008 ◽  
Vol 86 (4) ◽  
pp. 345-351 ◽  
Author(s):  
Xu Chen ◽  
Tinghui Hu ◽  
Gang Liang ◽  
Maojun Yang ◽  
Shudong Zong ◽  
...  
Keyword(s):  
Round Spermatid ◽  
Human Testis ◽  
Amino Acid Residues ◽  
Yeast Two Hybrid ◽  
Cyclin A1 ◽  
Yeast Two Hybrid System ◽  
Testis Cdna Library ◽  
Testis Cdna ◽  
Α Helix ◽  
Two Hybrid

rsb-66 is a novel gene from a suppression subtracted hybridization (SSH) library of round spermatid-specific cDNAs against those of primary spermatocytes. It was found to be specifically expressed in round spermatids. To explore the function of RSB-66, a yeast two-hybrid system was used to screen for potential interacting partners in a human testis cDNA library. HSD45, also known as INCA1 (inhibitor of Cdk interacting with cyclin A1), was identified as one of the positive clones. The interaction between RSB-66 and INCA1 was demonstrated to occur by GST pull down and coimmunoprecipitation. Using immunofluorescence, RSB-66 was found to be specifically expressed in round spermatids, mainly in the cytoplasm. When being transfected into HeLa cells, RSB-66 and INCA1 were found to be co-localized principally in the cytoplasm. The α helix in the RSB-66 C terminal and two amino acid residues (tyr117 and his119) appear to be crucial for its function.

scholarly journals Functional Analyses of the Pto Resistance Gene Family in Tomato and the Identification of a Minor Resistance Determinant in a Susceptible Haplotype

2002 ◽  
Vol 15 (3) ◽  
pp. 281-291 ◽  
Author(s):  
Jeff H. Chang ◽  
Yin-Shan Tai ◽  
Adriana J. Bernal ◽  
Daniel T. Lavelle ◽  
Brian J. Staskawicz ◽  
...  
Keyword(s):  
Cell Death ◽  
Amino Acid ◽  
Hybrid System ◽  
Dependent Manner ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
A Minor ◽  
Two Hybrid

Pto is a member of a multigene family and encodes a serine/threonine kinase that mediates gene-for-gene resistance to strains of Pseudomonas syringae pv. tomato expressing avrPto. The inferred amino acid sequence of the Pto homologs from both resistant (LpimPth2 to LpimPth4,) and susceptible (LescFen, LescPth2 to LescPth5) haplotypes suggested that most could encode functional serine/threonine kinases. In addition, the activation segments of the homologs are similar in sequence to that of Pto, and some have residues previously identified as required for binding of AvrPto by Pto in the yeast two-hybrid system. The Pto homologs were therefore characterized for transcription, for the ability of their products to interact with AvrPto in the yeast two-hybrid system, for their autophos-phorylation activity, and for their potential to elicit cell death in the presence of and absence of a ligand, as well as their dependence on Prf. LpimPth5, LpimPth4, and LescPth4 were not transcribed at levels detectable by reverse transcription-polymerase chain reaction. The interaction with AvrPto was unique to Pto in the yeast two-hybrid system. LescPth2 autophosphorylated in vitro as a fusion protein. LpimPth2, LpimPth3, LpimPth4, LescPth3, and LescPth4 did not autophosphorylate in vitro. Transient expression of wild-type Fen and wild-type LpimPth3, as well as LescFen, LescPth3, and LescPth5 with perturbations in their P+1 loop caused cell death in Nicotiana benthamiana. LpimPth3 and LescPth3 with amino acid substitutions in the P+1 loop also elicited cell death in tomato; this was dependent on the presence of wild-type Prf. Consequently, some homologs could potentially encode functional resistance proteins. LescPth5 induced cell death specifically in response to expression of AvrPto in tobacco in a Prf-dependent manner; this is consistent with a homolog from a ‘susceptible’ haplotype encoding a minor recognition determinant.

scholarly journals Study of the Interaction between Bacteriophage T4 asiA and Escherichia coli ς70, Using the Yeast Two-Hybrid System: Neutralization of asiA Toxicity to E. coli Cells by Coexpression of a Truncated ς70 Fragment

1999 ◽  
Vol 181 (18) ◽  
pp. 5855-5859 ◽  
Author(s):  
Umender K. Sharma ◽  
Sudha Ravishankar ◽  
Radha Krishan Shandil ◽  
P. V. K. Praveen ◽  
T. S. Balganesh
Keyword(s):  
Escherichia Coli ◽  
Hybrid System ◽  
Bacteriophage T4 ◽  
Amino Acid Residues ◽  
Yeast Two Hybrid ◽  
E Coli ◽  
Yeast Two Hybrid System ◽  
C Terminus ◽  
T4 Phage ◽  
Two Hybrid

ABSTRACT The interaction of T4 phage-encoded anti-sigma factor, asiA, andEscherichia coli ς70 was studied by using the yeast two-hybrid system. Truncation of ς70 to identify the minimum region involved in the interaction showed that the fragment containing amino acid residues proximal to the C terminus (residues 547 to 603) was sufficient for complexing to asiA. Studies also indicated that some of the truncated C-terminal fragments (residues 493 to 613) had higher affinity for asiA as judged by the increased β-galactosidase activity. It is proposed that the observed higher affinity may be due to the unmasking of the binding region of asiA on the sigma protein. Advantage was taken of the increased affinity of truncated ς70 fragments to asiA in designing a coexpression system wherein the toxicity of asiA expression in E. coli could be neutralized and the complex of truncated ς70 and asiA could be expressed in large quantities and purified.

Identifications of DNA Sequences Encoding Key Region of SCR Interacting with SRK Extracellular Domain by Using Yeast Two-Hybrid System

2013 ◽  
Vol 38 (9) ◽  
pp. 1583-1591
Author(s):  
Li-Yan XUE ◽  
Bing LUO ◽  
Li-Quan ZHU ◽  
Yong-Jun YANG ◽  
He-Cui ZHANG ◽  
...  
Keyword(s):  
Hybrid System ◽  
Dna Sequences ◽  
Extracellular Domain ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

scholarly journals Structural Glycoprotein E2 of Classical Swine Fever Virus Interacts with Host Protein Dynactin Subunit 6 (DCTN6) during the Virus Infectious Cycle

2019 ◽  
Vol 94 (1) ◽  
Author(s):  
M. V. Borca ◽  
E. A. Vuono ◽  
E. Ramirez-Medina ◽  
P. Azzinaro ◽  
K. A. Berggren ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Interaction ◽  
Hybrid Approach ◽  
Classical Swine Fever ◽  
Host Protein ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Viral Virulence ◽  
Glycoprotein E2 ◽  
Two Hybrid

ABSTRACT The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence. IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.

scholarly journals Implication of the Whitefly Protein Vps Twenty Associated 1 (Vta1) in the Transmission of Cotton Leaf Curl Multan Virus

2021 ◽  
Vol 9 (2) ◽  
pp. 304
Author(s):  
Yao Chi ◽  
Li-Long Pan ◽  
Shu-Sheng Liu ◽  
Shahid Mansoor ◽  
Xiao-Wei Wang
Keyword(s):  
Coat Protein ◽  
Molecular Mechanisms ◽  
Cotton Leaf ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Vacuolar Protein ◽  
Leaf Curl Disease ◽  
Two Hybrid ◽  
Asia Ii 1 ◽  
Leaf Curl

Cotton leaf curl Multan virus (CLCuMuV) is one of the major casual agents of cotton leaf curl disease. Previous studies show that two indigenous whitefly species of the Bemisia tabaci complex, Asia II 1 and Asia II 7, are able to transmit CLCuMuV, but the molecular mechanisms underlying the transmission are poorly known. In this study, we attempted to identify the whitefly proteins involved in CLCuMuV transmission. First, using a yeast two-hybrid system, we identified 54 candidate proteins of Asia II 1 that putatively can interact with the coat protein of CLCuMuV. Second, we examined interactions between the CLCuMuV coat protein and several whitefly proteins, including vacuolar protein sorting-associated protein (Vps) twenty associated 1 (Vta1). Third, using RNA interference, we found that Vta1 positively regulated CLCuMuV acquisition and transmission by the Asia II 1 whitefly. In addition, we showed that the interaction between the CLCuMuV coat protein and Vta1 from the whitefly Middle East-Asia Minor (MEAM1), a poor vector of CLCuMuV, was much weaker than that between Asia II 1 Vta1 and the CLCuMuV coat protein. Silencing of Vta1 in MEAM1 did not affect the quantity of CLCuMuV acquired by the whitefly. Taken together, our results suggest that Vta1 may play an important role in the transmission of CLCuMuV by the whitefly.

scholarly journals The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins

2001 ◽  
Vol 355 (3) ◽  
pp. 663-670 ◽  
Author(s):  
Claudia TROST ◽  
Christiane BERGS ◽  
Nina HIMMERKUS ◽  
Veit FLOCKERZI
Keyword(s):  
Amino Acid ◽  
Ion Channels ◽  
Transient Receptor Potential ◽  
Direct Interaction ◽  
Receptor Potential ◽  
Amino Acid Residues ◽  
Glutathione S Transferase ◽  
Calmodulin Binding ◽  
Transient Receptor

The mammalian gene products, transient receptor potential (trp)1 to trp7, are related to the Drosophila TRP and TRP-like ion channels, and are candidate proteins underlying agonist-activated Ca2+-permeable ion channels. Recently, the TRP4 protein has been shown to be part of native store-operated Ca2+-permeable channels. These channels, most likely, are composed of other proteins in addition to TRP4. In the present paper we report the direct interaction of TRP4 and calmodulin (CaM) by: (1) retention of in vitro translated TRP4 and of TRP4 protein solubilized from bovine adrenal cortex by CaM–Sepharose in the presence of Ca2+, and (2) TRP4–glutathione S-transferase pull-down experiments. Two domains of TRP4, amino acid residues 688–759 and 786–848, were identified as being able to interact with CaM. The binding of CaM to both domains occurred only in the presence of Ca2+ concentrations above 10µM, with half maximal binding occurring at 16.6µM (domain 1) and 27.9µM Ca2+ (domain 2). Synthetic peptides, encompassing the two putative CaM binding sites within these domains and covering amino acid residues 694–728 and 829–853, interacted directly with dansyl–CaM with apparent Kd values of 94–189nM. These results indicate that TRP4/Ca2+-CaM are parts of a signalling complex involved in agonist-induced Ca2+ entry.

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