Molecular cloning and yeast two‐hybrid provide new evidence for unique sporophytic self‐incompatibility system of Corylus

Plant Biology ◽  
2021 ◽  
Author(s):  
S. Hou ◽  
T. Zhao ◽  
Z. Yang ◽  
D. Yang ◽  
Q. Li ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Self Incompatibility ◽  
Yeast Two Hybrid ◽  
Incompatibility System ◽  
New Evidence ◽  
Two Hybrid

scholarly journals SLFL Genes Participate in the Ubiquitination and Degradation ofS-RNase in Self-Compatible Chinese Peach

2017 ◽  
Author(s):  
Qiuju Chen ◽  
Dong Meng ◽  
Wei Li ◽  
Zhaoyu Gu ◽  
Hui Yuan ◽  
...  
Keyword(s):  
Prunus Persica ◽  
Hypervariable Region ◽  
Self Incompatibility ◽  
S Locus ◽  
Yeast Two Hybrid ◽  
Peach Genome ◽  
General Inhibitor ◽  
Two Hybrid

AbstractThe gametophytic self-incompatibility (SI) mediated by S-RNase of Rosaceae, Solanaceae and Plantaginaceae, is controlled by two tightly linked genes located at highly polymorphic S-locus: the S-RNase for pistil specificity and the F-box gene (SFB/SLF) for pollen specificity, respectively. The F-box gene of peach (Prunus persica) isShaplotype-specific F-box (SFB). In this study, we selected 37 representative varieties according to the evolution route of peach and identified their S genotypes. We cloned pollen determinant genes mutantPperSFB1m, PperSFB2m, PperSFB4mand normalPperSFB2, and style determinant genesS1-RNase, S2-RNase, S2m-RNaseandS4-RNase.MutantPperSFBswere translated terminated prematurely because of fragment insertion. Yeast two-hybrid showed that mutant PperSFBs and normal PperSFB2 interacted with all S-RNases. NormalPperSFB2was divided into four parts: box, box-V1, V1-V2 and HVa-HVb. Protein interaction analyses showed that the box portion did not interact with S-RNases, both of the box-V1 and V1-V2 had interactions with S-RNases, while the hypervariable region ofPperSFB2HVa-HVb only interacted with S2-RNase. Bioinformatics analysis of peach genome revealed that there were other F-box genes located at S-locus, and of which three F-box genes were specifically expressed in pollen, namelyPperSLFL1, PperSLFL2andPperSLFL3, respectively. Phylogenetic analysis showed that PperSFBs and PperSLFLs were classified into two different clades. Yeast two-hybrid analysis revealed that as with PperSFBs, the three F-box proteins interacted with PperSSK1. Yeast two-hybrid and BiFC showed that PperSLFLs interacted with S-RNases with no allelic specificity. In vitro ubiquitination assay showed that PperSLFLs could tag ubiquitin molecules to PperS-RNases. In all, the above results suggest that threePperSLFLsare the appropriate candidates for the ‘general inhibitor’, which would inactivate the S-RNases in pollen tubes, and the role of three PperSLFL proteins is redundant, as S-RNase repressors involved in the self-incompatibility of peach.

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.

Determination of estrogenic activity in landfill leachate by simplified yeast two-hybrid assay

2002 ◽  
Vol 4 (6) ◽  
pp. 1040-1046 ◽  
Author(s):  
Yasunori Kawagoshi ◽  
Yukiko Tsukagoshi ◽  
Isao Fukunaga
Keyword(s):  
Landfill Leachate ◽  
Estrogenic Activity ◽  
Yeast Two Hybrid ◽  
Two Hybrid

scholarly journals The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5

2021 ◽  
Author(s):  
Orsolya Frittmann ◽  
Vamsi K Gali ◽  
Miklos Halmai ◽  
Robert Toth ◽  
Zsuzsanna Gyorfy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Dna Lesions ◽  
Template Switching ◽  
Adjacent Region ◽  
Yeast Two Hybrid ◽  
Replication Complex ◽  
Two Hybrid

Abstract DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

scholarly journals Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

2007 ◽  
Vol 18 (11) ◽  
pp. 4317-4326 ◽  
Author(s):  
Hiroshi Qadota ◽  
Kristina B. Mercer ◽  
Rachel K. Miller ◽  
Kozo Kaibuchi ◽  
Guy M. Benian
Keyword(s):  
Caenorhabditis Elegans ◽  
Binding Assays ◽  
Lim Domain ◽  
Yeast Two Hybrid ◽  
Lim Domains ◽  
Thick Filaments ◽  
Vitro Binding ◽  
Two Hybrid ◽  
Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

Sign in / Sign up

Export Citation Format

Share Document