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

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.

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Interaction of Mouse Pem Protein and Cell Division Cycle 37 Homolog

Acta Biochimica et Biophysica Sinica ◽

10.1111/j.1745-7270.2005.00102.x ◽

2005 ◽

Vol 37 (11) ◽

pp. 784-787 ◽

Cited By ~ 6

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.

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

Reproduction Fertility and Development ◽

10.1071/srb10abs326 ◽

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

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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

Journal of Bacteriology ◽

10.1128/jb.181.18.5855-5859.1999 ◽

1999 ◽

Vol 181 (18) ◽

pp. 5855-5859 ◽

Cited By ~ 19

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.

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Dimerization of the N-terminal Amphipathic α-Helix Domain of the Fungal Immunomodulatory Protein fromGanoderma tsugae(Fip-gts) Defined by a Yeast Two-hybrid System and Site-directed Mutagenesis

Journal of Biological Chemistry ◽

10.1074/jbc.272.32.20044 ◽

1997 ◽

Vol 272 (32) ◽

pp. 20044-20048 ◽

Cited By ~ 84

Author(s):

Wen-Huei Lin ◽

Chih-Hung Hung ◽

Chyong-Ing Hsu ◽

Jung-Yaw Lin

Keyword(s):

Hybrid System ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Fungal Immunomodulatory Protein ◽

Immunomodulatory Protein ◽

Α Helix ◽

Two Hybrid

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

ACTA AGRONOMICA SINICA ◽

10.3724/sp.j.1006.2012.01583 ◽

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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DETECTION OF MICROCYSTINS BASED ON YEAST TWO-HYBRID SYSTEM

Acta Hydrobiologica Sinica ◽

10.3724/sp.j.1035.2008.00201 ◽

2008 ◽

Vol 32 (2) ◽

pp. 201-206

Author(s):

Xiao PAN

Keyword(s):

Hybrid System ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Two Hybrid

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

Microorganisms ◽

10.3390/microorganisms9020304 ◽

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.

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