Screening proteins that interact with mutant superoxide dismutase 1 from familial amyotrophic lateral sclerosis using a yeast two-hybrid system

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.

Screening of genes of proteins interacting with p7 protein of hepatitis C virus from human liver cDNA library by yeast two-hybrid system

AIM: To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes.METHODS: We constructed p7 protein bait plasmid by doning the gene of p7 protein into pGBKT7, then transformed it into yeast AH109 (a type). The transformed yeast was mated with yeast Y187 (α type) containing liver cDNA library plasmid, pACT2 in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal for selection and screening. After extracting and sequencing of plasmids from blue colonies, we performed sequence analysis by bioinformatics.RESULTS: Fifty colonies were selected and sequenced.Among them, one colony was Homo sapiens signal sequence receptor, seven colonies were Homo sapiens H19, seven colonies were immunoglobulin superfamily containing leucine-rich repeat, three colonies were spermatid peri-nuclear RNA binding proteins, two colonies were membrane-spanning 4-domains, 24 colonies were cancer-associated antigens, four colonies were nucleoporin 214 ku and two colonies were CLL-associated antigens.CONCLUSION: The successful cloning of gene of protein interacting with p7 protein paves a way for the study of the physiological function of p7 protein and its associated protein.

Screening for Novel Binding Proteins Interacting with Human Papillomavirus Type 18 E6 Oncogene in the Hela cDNA Library by Yeast Two-Hybrid System

To screen for novel binding proteins interacting with high-risk HPV 18 E6 oncogene, the strain AH 109 was transformed with pGBKT7-HPV 18 E6 plasmid, and subsequent transference was utilized to screen for interacting proteins with HPV 18 E6 in human Hela cDNA library. HPV 18 E6 mRNA was expressed in yeast and there was no self-activation and toxicity in AH109. Seven proteins that interacted with HPV18 E6, including transmembrane protein 87B, phosphonoformate immuno-associated protein 5, vimentin, KM-HN-1 protein, dedicator of cytokinesis 7, vaccinia related kinase 2 and a hypothetical protein, were identified. It was suggested that yeast two-hybrid system is an efficient for screening interacting proteins. The high-risk HPV 18 E6 oncogene may interact with the proteins, which may be associated with signal transduction and transcriptional control, epithelial cell invasion and migration, as well as humoral and cellular immune etc. This investigation provides functional clues for further exploration of potential oncogenesis targets for cancer biotherapy.

Screening of hepatocyte proteins binding to F protein of hepatitis C virus by yeast two-hybrid system

AIM: To investigate the biological function of F protein by yeast two-hybrid system.METHODS: We constructed F protein bait plasmid by cloning the gene of F protein into pGBKT7, then recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-HisAde) containing X-α-gal for selection and screening.After extracting and sequencing plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics.RESULTS: Thirty-six colonies were selected and sequenced.Among them, 11 colonies were zymogen granule protein,5 colonies were zinc finger protein, 4 colonies were zinc-α-2-glycoprotein, 1 colony was sialyltransferase, 1 colony was complement control protein factor Ⅰ, 1 colony was vitronectin, and 2 colonies were new genes with unknown function.CONCLUSION: The yeast two-hybrid system is an effective method for identifying hepatocyte proteins interacting with F protein of hepatitis C virus. F protein may bind to different proteins.

Effect of β-Glucan (Angel Yeast) Compared to a Placebo on Cold and Flu Incidence and Symptoms in an Adult Population—A Double Blind, Randomised Controlled Trial

Background: 1-3, 1-6 β-glucan derived from Baker’s yeast (Saccharomyces cerevisiae) has been widely studied for its immune stimulatory capabilities and safety. Previous studies found β-glucan to have efficacy at reducing incidence of URTIs as well as being a low risk for negative side effects. The current study aimed to examine the effects of yeast β-glucan (Angel Yeast) on cold and flu incidences and symptoms in healthy adults. Methods: Two hundred and thirty-one males and females aged 18 to 65 years old supplemented with either β-glucan or a placebo for 3-months. Participants completed a general health questionnaire every 4 weeks and in addition, if participants experienced any cold or flu symptoms, these were recorded daily (along with severity) until resolved or up to 2 weeks. Results: Supplementation with β-glucan reduced the self-reported severity of sore throats and improved sleep quality compared to the placebo group. Conclusions: Yeast β-glucan supplementation appears to be able to help reduce certain symptoms experienced during a cold or flu episode and is safe and well tolerated.

Influence of nitrogen status on fermentation performances of non-Saccharomyces yeasts:a review

Nitrogen,one of the most crucial nutrients present in grapes and musts,plays a key role in yeast activities during alcoholic fermentation.Such influences are imposed on yeast growth and fermentation performances including the formation of secondary metabolites.Saccharomyces cerevisiae,the main yeast responsible for fermentation,has been studied extensively regarding nitrogen impacts.On the other hand,a similar study for non-Saccharomyces yeasts,whose contributions to winemaking have gradually been acknowledged,remains to be fully explored,with a few studies being reported.This review starts by discussing nitrogen impacts on non-Saccharomyces yeast growth and fermentation kinetics in different case scenarios,then proceeds to summarize the nitrogen preferences of individual yeast strains with regulation mechanisms elucidated by recent studies.Detailed discussions on the influences on the production of volatile compounds and proposed pathways therein are made,followed by future work suggested as the final section.In summarizing the nitrogen impacts on non-Saccharomyces yeasts throughout alcoholic fermentation,this review will be helpful in obtaining a more comprehensive view on these non-conventional wine yeasts in terms of nutrient requirements and corresponding volatile production.Research gaps will therefore be elucidated for future research.

Mathematical Modeling of Cell Polarity Establishment of Budding Yeast

The budding yeast Saccharomyces cerevisiae is a powerful model system for studying the cell polarity establishment.The cell polarization process is regulated by signaling molecules,which are initially distributed in the cytoplasm and then recruited to a proper location on the cell membrane in response to spatial cues or spontaneously.Polarization of these signaling molecules involves complex regulation,so the mathematical models become a useful tool to investigate the mechanism behind the process.In this review,we discuss how mathematical modeling has shed light on different regulations in the cell polarization.We also propose future applications for the mathematical modeling of cell polarization and morphogenesis.

Microbiological Quality of “Rabilé”, a Yeast Used for Fermentation of Dolo, a Local Beer in Dédougou, Burkina Faso

Sorghum beer or dolo is part of the eating habits of part of the population of Dédougou because of its low price compared with industrial beers. Its production is an ancestral tradition that uses traditional equipment and gives dolo organoleptic properties that are not found in industrial beers. The production process involves several stages, including fermentation, which itself comprises natural lactic fermentation followed by alcoholic fermentation using traditional yeasts, which are not controlled in any way. The general aim of this study is to assess the microbiological quality of these fermentative yeasts in the town of Dédougou, in order to contribute to the health safety of the population and the promotion of these local beers. Twenty samples of fermenting yeast were analyzed according to ISO standards, to isolate enterobacteria, total and faecal coliforms according to standard procedures for isolating these micro-organisms. The isolated strains were identified using the API20E gallery. Microbiological analyses revealed the presence of 51.17% enterobacteria, 45.38% total coliforms and 3.45% thermotolerant coliforms. We counted 40% Escherichia coli, 20% Enterobacter cloacae, 20% Klebsiella pneumoniae and 20% Klebsiella spp. All the strains detected are capable of surviving in hostile conditions and could harm the quality of the dolo, consumer health and cause real collective food poisoning in the town of Dédougou. This enabled us to assess the microbial quality of these yeasts and to propose more suitable measures for producing and preserving dolo under hygienic conditions to protect consumer health.

Screening of genes for proteins interacting with the PS1TP5 protein of hepatitis B virus:probing a human leukocyte cDNA library using the yeast two-hybrid system

Background The hepatitis B virus （HBV） genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 （human gene 5 transactivated by pre-S1 protein of HBV） is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory （GenBank accession： AY427953）. In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS1TP5 protein. Methods The reverse transcription polymerase chain reaction （RT-PCR） was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then ＂bait＂ plasmid pGBKT7-PS1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blotting hybridization. After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH109 with Y187 containing a leukocyte cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis. Results Forty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes with known functions were obtained, including Homo sapien leukocyte adhesion protein p150, 95, interleukin 2 receptor gamma chain, PALM2-AKAP2 protein （PALM2-AKAP2）, eukaryotic translation initiation factor 4A, beta-2-microglobin, solute carrier family 9 （sodium/hydrogen exchanger）, calreticulin, asialoglycoprotein receptor 1 （ASGR1）, MHC class Ⅱ lymphocyte antigen, cytochrome c oxidase subunit 1, lymphocyte antigen 86 （LY86） and lymphocyte cytosolic protein 1. One novel gene with unknown function was found and named as PS1TP5BP1. After being electronically spliced, it was deposited in GenBank （accession number： DQ471327）. Conclusions Genes of proteins interacting with PS1TP5 were successfully screened from leukocyte cDNA library. These results suggested that PS1TP5 was closely correlated with immunoregulation, carbohydrate metabolism, signal transduction, the formation of hepatic fibrosis and initiation and development of tumors and also brought some new clues for further studying the biological functions of the pre-S 1 protein.

Construction and Identification of a Yeast Two-Hybrid Bait Vector and Its Effect on the Growth of Yeast Cells and the Self-Activating Function of Reporter Genes for Screening of HPV18 E6-Interacting Protein

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...

Isolation and characterization of a human apoptosis-inducing gene with yeast two-hybrid system

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

Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding a, p, y, 8 and e subunits of Spinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding p-galactosidase was detected. Of all the combinations, that of y and e subunit genes showed the highest level of reporter gene expression, while those of a and p, a and e, p and e and p and 8 induced stable and significant reporter gene expression. The combination of 8 and e as well as that of 8 and y induced weak and unstable reporter gene expression. However, combinations of a and y, p and y and a and 8 did not induce reporter gene expression. These results suggested that specific and strong interactions between y and e, a and p, a and e, p and e and p and 8 subunits, and weak and transient interactions between 8 and e and 8 and y subunits occurred in the

A LexA-based yeast two-hybrid system for studying light- switchable interactions of phytochromes with their interacting partners

Phytochromes are a family of photoreceptors in plants that perceive the red(R)and far-red(FR)components of their light environment.Phytochromes exist in vivo in two forms,the inactive Pr form and the active Pfr form,that are interconvertible by treatments with R or FR light.It is believed that phytochromes transduce light signals by interacting with their signaling partners.A GAL4-based lightswitchable yeast two-hybrid(Y2H)system was developed two decades ago and has been successfully employed in many studies to determine phytochrome interactions with their signaling components.However,several pairs of interactions between phytochromes and their interactors,such as the phyACOP1 and phyA-TZP interactions,were demonstrated by other assay systems but were not detected by this GAL4 Y2H system.Here,we report a modified LexA Y2H system,in which the LexA DNA-binding domain is fused to the C-terminus of a phytochrome protein.The conformational changes of phytochromes in response to R and FR light are achieved in yeast cells by exogenously supplying phycocyanobilin(PCB)extracted from Spirulina.The well-defined interaction pairs,including phyA-FHY1 and phyB-PIFs,are well reproducible in this system.Moreover,we show that our system is successful in detecting the phyA-COP1 and phyA-TZP interactions.Together,our study provides an alternative Y2H system that is highly sensitive and reproducible for detecting light-switchable interactions of phytochromes with their interacting partners.

Screening of the interacting proteins with NifA in Azospirillum brasilense Sp7 by the yeast two-hybrid system

NifA in Azospirillum brasilense plays a key role in regulating the synthesis and activity of nitrogenase in re- sponse to ammonia and oxygen available. In this work we used the yeast two-hybrid system to identify the proteins that interact with NifA. The nifA gene was fused to the yeast two-hybrid vector pGBD-C2, and three A. brasilense Sp7 genomic libraries for use in yeast two-hybrid studies were constructed. Screening of the libraries identified four clones encoding proteins that interact with NifA. The confirmation of the interactions of each gene product of the four clones and NifA were carried out by exchanging the vectors for nifA and the four clones and by mutageneses of the four clones with shift reading frame experiments in yeast two-hybrid studies. DNA sequence analyses showed that two clones en- code proteins containing PAS domains that play an impor- tant role in signal transduction. One clone has high similarity with the fhuE gene of Escherichia coli, whose gene product is involved in iron uptake and transportation, and the other clone encodes an unknown protein.

Interaction of hTCF4 by yeast two-hybrid system

To investigate the interaction of hTCF4, the yeast two-hybrid system has been used for testing the interaction of mutants of hTCF4 with themselves. Mutants of hTCF4 (hTCF4 Ⅰ ) and hTCF4 Ⅱ) have been obtained by polymerase chain reaction (PCR). Bait (hTCF4 Ⅰ -pDBLeu and hTCF4Ⅱ-pDBLeu) and prey (hTCF4 Ⅰ -pPC86 and hTCF4Ⅱ-pPC86) have been constructed by DNA recombination for yeast two-hybrid. Interaction of hTCF4Ⅱ with itself is found in the reverse yeast two-hybrid system (GIBCOBRL Co.). However, no interactions are found in hTCF4Ⅱ with hTCF4 Ⅰ and in hTCF4 Ⅰ with hTCF4 Ⅰ . These results suggest that hTCF4 could interact with itself to form homodimer or homocopolymer and perform the transcriptional activating function through LZ or HLH motifs in nucleus of renal cell carcinoma.

Shared and discrete interacting partners of ELL1 and ELL2 by yeast two-hybrid assay

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.

Study on the interaction between Jak3 and IL-2R γ using the yeast two-hybrid system

Both interleukin-2 (IL-2) receptor y subunit and non-receptor tyrosine kinase Jak3 play important roles in IL-2 physiological functions. Jak3 has been known to bind IL-2Ry and the interaction is very important for IL-2 signaling. In order to find the domains directly involved in the interaction between IL-2Ry and Jak3, various deletion mutants have been constructed

Screening of proteins that interact with human thrombopoietin receptor c-Mpl using yeast two-hybrid system

Thrombopoietin (TPO) is the major cytokine involved in platelet production and exerts its effects via the receptor c-Mpl. The yeast two-hybrid system has been used to screen the proteins interacting with c-Mpl. First, the cDNA fragment of c-Mpl intracellular domain was cloned into two-hybrid vector pAS2, and the resulting plasmid is designated as pASMM. Then a human placenta cDNA library was screened using the pASMM as a target plasmid. Seven positive clones were isolated from 150 000 independent transformants. Sequence analysis of one of the positive clones demonstrates that a part of coding sequence of vimentin from 611 bp to 3’ end and flanking non-translation region was obtained. Therefore, there is an interaction between vimentin and TPO receptor. The results suggest that cytoskeletal protein may play an important role in TPO signal transduction pathway.

Screening of Host Proteins Interacting with PorcineEpidemic Diarrhea Virus (PEDV) N Protein by YeastTwo-hybrid System

[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.

Construction of a Three-frame Yeast Two-hybrid cDNA Library of Fusarium oxysporum

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.