A Method to Generate Full-Length cDNA Molecules from Yeast Two-Hybrid Clones and RACE Products

1999 ◽  
Vol 268 (1) ◽  
pp. 161-162 ◽  
Author(s):  
Charles S. Hemenway ◽  
Benjamin W. Halligan ◽  
Grahame C.D. Gould
Keyword(s):  
Full Length ◽  
Yeast Two Hybrid ◽  
Full Length Cdna ◽  
Hybrid Clones ◽  
Two Hybrid

scholarly journals Definition of a minimal munc18c domain that interacts with syntaxin 4

2000 ◽  
Vol 350 (3) ◽  
pp. 741-746 ◽  
Author(s):  
Julian GRUSOVIN ◽  
Violet STOICHEVSKA ◽  
Keith H. GOUGH ◽  
Katrina NUNAN ◽  
Colin W. WARD ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Full Length ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Interaction Studies ◽  
Syntaxin 4 ◽  
Definition Of ◽  
Two Hybrid

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein–protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c1–592, munc18c1–139 and munc18c1–225, but not munc18c226–592, munc18c1–100, munc18c43–139 or munc18c66–139, interacted with the cytoplasmic portion of syntaxin 4, Stx42–273, as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by β-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx429–157, failed to interact with full-length munc18c1–592, indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein–protein interaction studies between Stx42–273 and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c1–592, munc18c1–139 and munc18c1–225 interacted with Stx42–273 whereas munc18c1–100 did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.

326. SPIF - A NOVEL TESTIS-SPECIFIC GENE AND ITS INTERACTION WITH PKA

2010 ◽  
Vol 22 (9) ◽  
pp. 126
Author(s):  
S. J. Tannock ◽  
E. A. McLaughlin ◽  
R. J. Aitken ◽  
S. D. Roman
Keyword(s):  
Northern Blotting ◽  
Full Length ◽  
Specific Gene ◽  
Binding Motif ◽  
Yeast Two Hybrid ◽  
Truncated Form ◽  
Binding Partners ◽  
Testis Cdna Library ◽  
Testis Cdna ◽  
Two Hybrid

The activation of protein kinase A (PKA) is strongly implicated in capacitation and sperm motility. However, the full pathway is yet to be elucidated. To identify potential PKA binding partners in sperm, a yeast two-hybrid assay was performed using the testis specific catalytic subunit (Cs) of PKA as the ‘bait’ to screen a mouse testis cDNA library. A novel cDNA clone termed Sperm PKA Interacting Factor (SPIF) was identified from the screen on three separate occasions. The interaction was confirmed by a protein pull-down using a C-terminal recombinant protein to SPIF and a PKACs antibody. During cloning and sequence analysis, SPIF was found to contain two isoforms; a full length (4770 bp) and a truncated form (2784 bp) with alternate start sites and an identical 3′ end, with only the full length isoform containing the PKA binding motif. SPIF was found to be testis specific using PCR and Northern Blotting with high expression levels in round spermatids and adult testis. The interaction between SPIF and PKA was further demonstrated with protein co-localisation in round spermatids and in the midpiece and flagellum of mouse sperm. In summary, we have identified a novel testis specific gene that in concert with PKA could prove to be an essential link in the incomplete capacitation pathway

Neurofilament triplet protein interactions: evidence for the preferred formation of NF-L-containing dimers and a putative function for the end domains

1996 ◽  
Vol 109 (10) ◽  
pp. 2493-2498 ◽  
Author(s):  
D.A. Carpenter ◽  
W. Ip
Keyword(s):  
Intermediate Filaments ◽  
Hybrid System ◽  
Weak Interaction ◽  
Molecular Interactions ◽  
Protein Interactions ◽  
Building Blocks ◽  
Full Length ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

In this report we examine the molecular interactions that lead to formation of neurofilaments, the intermediate filaments in neurons. Using the yeast two-hybrid system, we found that the rod domains of all three NF triplet proteins interacted strongly with one another and with rod domains of the Type III IF proteins, vimentin and desmin. A slight preference toward NF-L-containing dimers was observed over ones not containing NF-L. Interactions among the full length NF triplet proteins exhibited more specificity. Full length NF-L had only a relatively weak interaction with another full length NF-L molecule, but reacted more robustly with full length NF-M or NF-H lacking only part of the head domain. No homologous or heterologous dimerization of NF-M and NF-H was detectable. These results support the hypothesis that neurofilaments are obligate heteropolymers and that heterodimeric subunits are the preferred building blocks. They further suggest that the mechanism that specifies heterodimeric interaction among the NF triplet proteins resides in the end domains.

scholarly journals Direct binding of a fragment of the Wiskott-Aldrich syndrome protein to the C-terminal end of the anaphylatoxin C5a receptor

2003 ◽  
Vol 372 (2) ◽  
pp. 453-463 ◽  
Author(s):  
Marianne TARDIF ◽  
Laurence BROUCHON ◽  
Marie-Josèphe RABIET ◽  
François BOULAY
Keyword(s):  
Active Form ◽  
Hybrid Approach ◽  
Full Length ◽  
Actin Dynamics ◽  
Yeast Two Hybrid ◽  
C5a Receptor ◽  
Wiskott Aldrich Syndrome ◽  
C5a Anaphylatoxin ◽  
Syndrome Protein ◽  
Two Hybrid

Migration of myeloid cells towards a source of chemoattractant, such as the C5a anaphylatoxin, is triggered by the activation of a G-protein-coupled receptor. In the present study, we have used a yeast two-hybrid approach to find unknown partners of the C5a receptor (C5aR). Using the cytosolic C-terminal region of C5aR as bait to screen a human leucocyte cDNA library, we identified the Wiskott–Aldrich syndrome protein (WASP) as a potential partner of C5aR. WASP is known to have an essential function in regulating actin dynamics at the cell leading edge. The interaction was detected with both the fragment of WASP containing amino acids 1–321 (WASP.321) and WASP with its actin-nucleation-promoting domain [verprolin-like, central and acidic (VCA) domain] deleted. The interaction between C5aR and the WASP.321 was supported further by an in vitro binding assay between a radiolabelled WASP.321 fragment and a receptor C-terminus glutathione S-transferase (GST) fusion protein, as well as by GST pull-down, co-immunoprecipitation and immunofluorescence experiments. In the yeast two-hybrid assay, full-length WASP showed no ability to interact with the C-terminal domain of C5aR. This is most probably due to an auto-inhibited conformation imposed by the VCA domain. In HEK-293T cells co-transfected with full-length WASP and C5aR, only a small amount of WASP was co-precipitated with the receptor. However, in the presence of the active form of the GTPase Cdc42 (Cdc42V12), which is thought to switch WASP to an active ‘open conformation’, the amount of WASP associated with the receptor was markedly increased. We hypothesize that a transient interaction between C5aR and WASP occurs following the stimulation of C5aR and Cdc42 activation. This might be one mechanism by which WASP is targeted to the plasma membrane and by which actin assembly is spatially controlled in cells moving in a gradient of C5a.

scholarly journals A comparative approach expands the protein–protein interaction node of the immune receptor XA21 in wheat and rice

Genome ◽  
2013 ◽  
Vol 56 (6) ◽  
pp. 315-326 ◽  
Author(s):  
Baoju Yang ◽  
Randy Ruan ◽  
Dario Cantu ◽  
Xiaodong Wang ◽  
Wanquan Ji ◽  
...  
Keyword(s):  
Signal Transduction Pathway ◽  
Full Length ◽  
Bimolecular Fluorescence Complementation ◽  
Comparative Approach ◽  
The Novel ◽  
Yeast Two Hybrid ◽  
Immune Receptor ◽  
Protein Protein Interaction ◽  
Novel Interactions ◽  
Two Hybrid

The rice (Oryza sativa) OsXA21 receptor kinase is a well-studied immune receptor that initiates a signal transduction pathway leading to resistance to Xanthomonas oryzae pv. oryzae. Two homologs of OsXA21 were identified in wheat (Triticum aestivum): TaXA21-like1 located in a syntenic region with OsXA21, and TaXA21-like2 located in a nonsyntenic region. Proteins encoded by these two wheat genes interact with four wheat orthologs of known OsXA21 interactors. In this study, we screened a wheat yeast-two-hybrid (Y2H) library using the cytosolic portion of TaXA21-like1 as bait to identify additional interactors. Using full-length T. aestivum and T. monococcum proteins and Y2H assays we identified three novel TaXA21-like1 interactors (TaARG, TaPR2, TmSKL1) plus one previously known in rice (TaSGT1). An additional full-length wheat protein (TaCIPK14) interacted with TaXA21-like2 and OsXA21 but not with TaXA21-like1. The interactions of TaXA21-like1 with TmSKL1 and TaSGT1 were also observed in rice protoplasts using bimolecular fluorescence complementation assays. We then cloned the rice homologs of the novel wheat interactors and confirmed that they all interact with OsXA21. This last result suggests that interspecific comparative interactome analyses can be used not only to transfer known interactions from rice to wheat, but also to identify novel interactions in rice.

scholarly journals Identification of an N-terminal domain of the plum pox potyvirus CI RNA helicase involved in self-interaction in a yeast two-hybrid system

2001 ◽  
Vol 82 (3) ◽  
pp. 677-686 ◽  
Author(s):  
Lissett López ◽  
Ana Urzainqui ◽  
Elvira Domínguez ◽  
Juan Antonio García
Keyword(s):  
Hybrid System ◽  
Rna Helicase ◽  
Full Length ◽  
Genome Replication ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Cylindrical Inclusions ◽  
Infected Cells ◽  
Two Hybrid

Potyvirus CI RNA helicase is a protein involved in RNA genome replication and virus movement. The protein aggregates in the cytoplasm of infected cells to form typical cylindrical inclusions. A yeast two-hybrid system was used to analyse interactions of the CI RNA helicase from plum pox potyvirus (PPV) with itself and with other viral proteins. No interactions could be detected of full-length CI protein with itself or with PPV P3/6K1, NIa, NIb or CP proteins. However, positive self-interactions were detected for N-terminal fragments of the CI protein, allowing the mapping of a CI–CI binding domain to the N-terminal 177 aa of the protein. Further deletion analysis suggested that several regions of this domain contribute to the interaction. Moreover, pull-down experiments demonstrate that, at least in vitro, full-length PPV CI protein is able to self-interact in the absence of other virus or plant factors.

Establishment and Identification of a Normalized Full-Length cDNA Library of CCRI 36

2009 ◽  
Vol 35 (4) ◽  
pp. 602-607 ◽  
Author(s):  
Dong WU ◽  
Jun-Jie LIU ◽  
Shu-Xun YU ◽  
Shu-Li FAN ◽  
Mei-Zhen SONG
Keyword(s):  
Cdna Library ◽  
Full Length ◽  
Full Length Cdna

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
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