The peaT1 gene fragment was amplified from pGEM-6p-l-peaT1 by PCR, and recovered target gene was cloned into pLexA vector. After digestion and sequencing, the bait vector pLexA-peaT1 was transformed into yeast strain ...The peaT1 gene fragment was amplified from pGEM-6p-l-peaT1 by PCR, and recovered target gene was cloned into pLexA vector. After digestion and sequencing, the bait vector pLexA-peaT1 was transformed into yeast strain EGY48 [p8op-lacZ] by PEG/LiAC, and the transcriptional activity of bait vector was detected. The results showed that recombinant bait plasmid pLexA-PEMG1 was constructed, for the two bands of recombinant bait plasmid in agarose gel eleetrophoresis were expected after digesting by restriction endonuclease EcoR I and Xho I. Therefore, the recombinant bait plasmid could be used in yeast two-hybrid system to screen a cDNA library.展开更多
[Objective] The research aimed to find the extracellular binding proteins of CR4.[Method] The extracellular domain of OsCR4 was as the bait protein,and the yeast two-hybrid was used to screen cDNA library of seedling ...[Objective] The research aimed to find the extracellular binding proteins of CR4.[Method] The extracellular domain of OsCR4 was as the bait protein,and the yeast two-hybrid was used to screen cDNA library of seedling which was cultivated 14 d.[Result] A lot of proteins which included a peroxide B（D26484）,a methionine thioredoxin reductase（ABF96078）and an unknown function protein were gained.[Conclusion] It provided the theory basis for studying the signal transduction mechanism of CR4.展开更多
AIM: To identify the pre-X region in hepatitis B virus (HBV)genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we ...AIM: To identify the pre-X region in hepatitis B virus (HBV)genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we performed yeast two-hybrid to screen proteins in liver interacting with whole-X protein.METHODS: The pre-X region of HBV was amplified by polymerase chain reaction (PCR) method, and was cloned to pGEM Teasy vector. After the target region was sequenced, Vector 8.0 software was used to analyze the sequences. The whole-X bait plasmid was constructed by using yeast two-hybrid system 3. Yeast strain AH109 was transformed. After expression of the whole-X protein in AH109 yeast strains was proved, yeast two-hybrid screening was performed by mating AH109 with Y187 containing liver cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between whole-X protein and the protein obtained from positive colonies was further confirmed by repeating yeast two-hybrid. After extracting and sequencing of plasmid from blue colonies, we carried out analysis by bioinformatics. RESULTS: After sequencing, 27 of 45 clones (60%) were found encoding the pre-X peptide. Eighteen of twenty-seven clones (66.7%) of pre-X coding sequences were found from genotype C. Five positive colonies that interacted with whole-X protein were obtained and sequenced; namely, fetuin B, UDP glycosyltransferase 1 family-polypeptide A9, mannose-P-dolichol utilization defect 1, fibrinogen-B beta polypeptide, transmembrane 4 superfamily member 4CD81 (TM4SF4).CONCLUSION: The pre-X gene exists in HBV genome.Genes of proteins interacting with whole-X protein in hepatocytes were successfully cloned. These results brought some new clues for studying the biological functions of whole-X protein.展开更多
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 cloning the gene of p7 protein into p...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 cloning the gene of p7 protein into pGBKTT, 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 2×YPDA 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 nudeoporin 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 assodated protein.展开更多
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 tran...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. 2005 The WJG Press and Elsevier Inc. All rights reserved展开更多
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 ...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.展开更多
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 re...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...展开更多
Objective： To screen the proteins interacting with the Treg specification factor forkhead box protein P3 （FOXP3） by yeast two-hybrid system, Methods： Human FOXP3 gene was amplified by nest RT-PCR from peripheral b...Objective： To screen the proteins interacting with the Treg specification factor forkhead box protein P3 （FOXP3） by yeast two-hybrid system, Methods： Human FOXP3 gene was amplified by nest RT-PCR from peripheral blood mononuclear cells （PBMC） and inserted into plasmid pGBKT7 to construct the bait vector, then the self-activation and toxicity of the bait vector in host yeast strain AH109 were observed. Thereafter, a human liver cDNA library was screened by the bait vector. The positive clones were selected out by nutrient-deficient culture and back-hybridizing. The sequences from the candidate positive clones were blasted and analyzed by bioinformatics methods. Results： The constructed bait vector encoding FOXP3 was found no self-activation and toxicity in yeast AH109. Three proteins which interacted with FOXP3, including tumor protein D52, splicing factor 3b subunit 1 and hypothetical protein, were identified. Conclusion： Three new candidate proteins interacting with FOXP3 are selected out by this yeast two-hybrid system and library, which may facilitate the further study of FOXP3 in Treg.展开更多
The yeast two\|hybrid system is a molecular genetic approach for protein interaction and it is widely used to screen for proteins that interact with a protein of interest in recent years.This process includes,construc...The yeast two\|hybrid system is a molecular genetic approach for protein interaction and it is widely used to screen for proteins that interact with a protein of interest in recent years.This process includes,construction and testing of the bait plasmid,screening a plasmid library for interacting fusion protein,elimination of false positives and delection analysis of true positives.This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with a protein of interest.More and more studies have demonstrated that the two\|hybrid system is a powerful and sensitive technique for the identification of genes that code for proteins that interact in a biologically significant fashion with a protein of interest in higher plants.This method has been used to identify new interaction protein in many laboratories.The recently reported yeast tri\|brid system,should allow the investigation of more complex protein\|protein interactions.The aim of this review is to outline the recent progress made in protein interactions by using yeast two\|hybrid system.展开更多
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 ...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.展开更多
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 p...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.展开更多
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 inter...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.展开更多
To address the coarse texture and poor cooking quality of brown rice flour,we employed fermentation using lactobacillus and yeast in varying proportions.The fermented flour from early indica rice Pear 13 was then proc...To address the coarse texture and poor cooking quality of brown rice flour,we employed fermentation using lactobacillus and yeast in varying proportions.The fermented flour from early indica rice Pear 13 was then processed into semi-dried brown rice noodles.展开更多
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 re...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.展开更多
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 t...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.展开更多
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 inclu...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.展开更多
Using probiotics as animal feed additives instead of antibiotics is gaining momentum to avert adverse negative effects on human health. Liquid brewers yeast (LBY) is an industrial by-product containing probiotic micro...Using probiotics as animal feed additives instead of antibiotics is gaining momentum to avert adverse negative effects on human health. Liquid brewers yeast (LBY) is an industrial by-product containing probiotic microorganisms and is also used as a protein supplement for dairy animals. Nevertheless, value chain actors lack of appropriate handling practices compromises the by-products quality and safety. This study aimed to determine the effect of variation in temperature on microbial diversity and probiotic effects during the storage time of LBY sampled from distributors and farmers from Githunguri sub-county of Kenya. The samples were stored at 20C, 25C and 30C, then tested on 0, 5, 10, 15 and 20 days. The studys parameters involved determining the pH levels, lactic acid bacteria (LAB), total coliform count (TCC), mould, and yeast in LBY. The rate (k) of the reaction kinetics model was used to extrapolate the expected probiotic shelf life. The LAB and yeast populations were reduced in a first-order reaction at all storage temperatures. The rate of reduction in the numbers of LAB reduced with an increase in temperature (k = 0.019 and 0.023) at 20C and 30C, respectively. Yeasts highest rate of growth reduction was 25C (k = 0.009) and least at 30C (k = 0.043). The minimum effective concentration for probiotics of 106 CFU/mL needed to observe the beneficial physiological impact on farm animals was achieved between 34.9 and 35.5 days at the tested storage temperatures. The study provides insight into the unexploited low-cost probiotic potential of LBY in dairy production. Conversely, handling practices and environmental microbial contamination along the value chain can compromise product quality and safety. There is a need to advocate its use in dairy for improved productivity and sensitize farmers to appropriate hygienic measures along the LBY value chain.展开更多
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 equi...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.展开更多
Yeast immobilization is a process of physical entrapment of yeast cells using different techniques while maintaining their biological activity.Continuous fermentation systems have significant advantages over conventio...Yeast immobilization is a process of physical entrapment of yeast cells using different techniques while maintaining their biological activity.Continuous fermentation systems have significant advantages over conventional methods.Research highlights that immobilized yeast cell systems have several benefits as compared to free yeast cells.The immobilized yeast cell systems improve fermentation rates,especially when paired with continuous fermentation and appropriate immobilization techniques.Understanding various immobilization techniques,continuous fermentation processes,yeast metabolic activity related to beverage flavor production,and bioreactor designs is vital for optimizing the use of immobilized yeast cells systems on industrial scale.This review provides an overview of recent basic research on immobilized yeast cell systems,with a focus on continuous beverage fermentation.In this study,different reactor configurations and immobilization techniques are explored.The study focus on the impacts of immobilization on the yeast cells,and discuss the recent advancements in these techniques.The review concludes with a discussion on the practical applications of immobilized yeast cells and continuous fermentation in beverage production.展开更多
The aggregation of amyloid-beta （Aβ） peptide, has been demonstrated to be critical for the development of Alzheimer＇s disease （AD）. All aggregation inhibitors are thus considered to be drug candidates for AD the...The aggregation of amyloid-beta （Aβ） peptide, has been demonstrated to be critical for the development of Alzheimer＇s disease （AD）. All aggregation inhibitors are thus considered to be drug candidates for AD therapy. In the present study, we developed a novel screening tool based on the yeast two-hybrid system to screen Aβ aggregation inhibitors. The human Aβ42 peptide cDNA was cloned using assembly PCR and inserted into each of the yeast expression plasmids containing either the GAL4 activation domain （GAL4AD） or the DNA-binding domain （GAL4BD）. Co-transformation of the above plasmids led to the expression of the fusion proteins GAL4AD-Aβ42 and GAL4BD-Aβ42 in the AH 109 yeast strain. The self interaction of Aβ42 fragments reconstructed the GAL4 transcriptor and thus activated the GAL4 responsive transcription of four reporter genes including HIS3, ADE2, lacZ and MEL1. The expression of the reporter genes rendered the multiple auxotrophic yeast cells capable of growing on the synthetic SD media lacking adenine and histidine. Growth arrest was used as a marker for screening Aβ aggregation inhibitors in this system, and the evaluation of Rhodiola species revealed potential resources for the development of Aβ aggregation inhibitors.展开更多
基金Supported by the National“863”Program(2006AA10A210)~~
文摘The peaT1 gene fragment was amplified from pGEM-6p-l-peaT1 by PCR, and recovered target gene was cloned into pLexA vector. After digestion and sequencing, the bait vector pLexA-peaT1 was transformed into yeast strain EGY48 [p8op-lacZ] by PEG/LiAC, and the transcriptional activity of bait vector was detected. The results showed that recombinant bait plasmid pLexA-PEMG1 was constructed, for the two bands of recombinant bait plasmid in agarose gel eleetrophoresis were expected after digesting by restriction endonuclease EcoR I and Xho I. Therefore, the recombinant bait plasmid could be used in yeast two-hybrid system to screen a cDNA library.
基金Supported by Science Technology Research and Development Project in Shijiazhuang City in2010(10120803)Scientific Research Starting Fund Project of Shijiazhuang University in2007(2007012),Education Reform Research Item of Shijiazhuang University in2008(2008006)~~
文摘[Objective] The research aimed to find the extracellular binding proteins of CR4.[Method] The extracellular domain of OsCR4 was as the bait protein,and the yeast two-hybrid was used to screen cDNA library of seedling which was cultivated 14 d.[Result] A lot of proteins which included a peroxide B（D26484）,a methionine thioredoxin reductase（ABF96078）and an unknown function protein were gained.[Conclusion] It provided the theory basis for studying the signal transduction mechanism of CR4.
基金Supported by the grants from the National Natural Science Foundation, No. C03011402, No. C30070690the 9.5 Research and Technique Foundation of PLA, No. 98D063+1 种基金 the Launching Foundation for Student Studying Abroad of PLA, No. 98H038 the 10.5 Youth Research and Technique Foundation of PLA, No. 01Q138andNo. 01MB135
文摘AIM: To identify the pre-X region in hepatitis B virus (HBV)genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we performed yeast two-hybrid to screen proteins in liver interacting with whole-X protein.METHODS: The pre-X region of HBV was amplified by polymerase chain reaction (PCR) method, and was cloned to pGEM Teasy vector. After the target region was sequenced, Vector 8.0 software was used to analyze the sequences. The whole-X bait plasmid was constructed by using yeast two-hybrid system 3. Yeast strain AH109 was transformed. After expression of the whole-X protein in AH109 yeast strains was proved, yeast two-hybrid screening was performed by mating AH109 with Y187 containing liver cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between whole-X protein and the protein obtained from positive colonies was further confirmed by repeating yeast two-hybrid. After extracting and sequencing of plasmid from blue colonies, we carried out analysis by bioinformatics. RESULTS: After sequencing, 27 of 45 clones (60%) were found encoding the pre-X peptide. Eighteen of twenty-seven clones (66.7%) of pre-X coding sequences were found from genotype C. Five positive colonies that interacted with whole-X protein were obtained and sequenced; namely, fetuin B, UDP glycosyltransferase 1 family-polypeptide A9, mannose-P-dolichol utilization defect 1, fibrinogen-B beta polypeptide, transmembrane 4 superfamily member 4CD81 (TM4SF4).CONCLUSION: The pre-X gene exists in HBV genome.Genes of proteins interacting with whole-X protein in hepatocytes were successfully cloned. These results brought some new clues for studying the biological functions of whole-X protein.
基金Supported by the National Natural Scientific Foundation, No. C03011402, No. C30070690the Research and Technique Foundation of PLA during the 9th-five year plan period, No. 98D063the Launching Foundation for Student Studying Abroad of PLA, No. 98H038and the Youth Research and Technique Foundation of PLA during the 10th-five year plan period, No. 01Q138the Research and Technique Foundation of PLA during the 10th-five year plan period, No. 01MB135
文摘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 cloning the gene of p7 protein into pGBKTT, 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 2×YPDA 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 nudeoporin 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 assodated protein.
基金Supported by the National Natural Science Foundation of China, Nos. C03011402 and C30070690 and the Research and Technique Foundation of PLA during the 9~(th)-Five year plan period, No. 98D063 and the Launching Foundation for Student Studying Abroad of PLA, No. 98H038 and the Youth Research and Technique Foundation of PLA during the 10~(th)-Five Year Plan Period, No. 01Q138 and the Research and Technique Foundation of PLA during the 10~(th)-Five Year Plan Period, No. 01MB135
文摘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. 2005 The WJG Press and Elsevier Inc. All rights reserved
基金the National Natural Science Foundation of China, No. 30300116
文摘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.
基金supported by grants from National Natural Sciences Foundation of China(No.30672227,30600668)"973"Program of China(No.2009CB521800)Joint Research Fund for Overseas Chinese,Hong Kong and Macao Young Scholars(No.30628029)
文摘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...
基金the National Natural Science Foundation of China(30500447)
文摘Objective： To screen the proteins interacting with the Treg specification factor forkhead box protein P3 （FOXP3） by yeast two-hybrid system, Methods： Human FOXP3 gene was amplified by nest RT-PCR from peripheral blood mononuclear cells （PBMC） and inserted into plasmid pGBKT7 to construct the bait vector, then the self-activation and toxicity of the bait vector in host yeast strain AH109 were observed. Thereafter, a human liver cDNA library was screened by the bait vector. The positive clones were selected out by nutrient-deficient culture and back-hybridizing. The sequences from the candidate positive clones were blasted and analyzed by bioinformatics methods. Results： The constructed bait vector encoding FOXP3 was found no self-activation and toxicity in yeast AH109. Three proteins which interacted with FOXP3, including tumor protein D52, splicing factor 3b subunit 1 and hypothetical protein, were identified. Conclusion： Three new candidate proteins interacting with FOXP3 are selected out by this yeast two-hybrid system and library, which may facilitate the further study of FOXP3 in Treg.
文摘The yeast two\|hybrid system is a molecular genetic approach for protein interaction and it is widely used to screen for proteins that interact with a protein of interest in recent years.This process includes,construction and testing of the bait plasmid,screening a plasmid library for interacting fusion protein,elimination of false positives and delection analysis of true positives.This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with a protein of interest.More and more studies have demonstrated that the two\|hybrid system is a powerful and sensitive technique for the identification of genes that code for proteins that interact in a biologically significant fashion with a protein of interest in higher plants.This method has been used to identify new interaction protein in many laboratories.The recently reported yeast tri\|brid system,should allow the investigation of more complex protein\|protein interactions.The aim of this review is to outline the recent progress made in protein interactions by using yeast two\|hybrid system.
文摘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.
基金grants from "973" Program of China (No. 2002CB513100)National Natural Sci-ences Foundation of China (No. 30500596)
文摘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.
基金Supported by the National Nature Science Foundation of China(31372088)the "Academic Backbone" Project of Northeast Agricultural University(15XG05)China Agriculture Research System(CARS-26-02)
文摘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.
基金supported by the Natural Science Foundation of Hunan Province,China(Grant No.2023JJ30997)the National Key Research and Development Program of China(Grant No.2022YFD2101303)+3 种基金the Hunan Science and Technology Innovation Plan,China(Grant No.2023ZJ1070)the Local Fund for Science and Technology Development Guided by the Central Government,China(Grant No.2023ZYC008)the Hunan Forestry Science and Technology Research and Innovation Fund Project,China(Grant No.XLKY202326)the Scientific Research Project of Central South University of Forestry and Technology,China(Grant No.2022ZK3042)。
文摘To address the coarse texture and poor cooking quality of brown rice flour,we employed fermentation using lactobacillus and yeast in varying proportions.The fermented flour from early indica rice Pear 13 was then processed into semi-dried brown rice noodles.
文摘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.
文摘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.
基金supported by grants from the National Natural Science Foundation of China(32172340)。
文摘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.
文摘Using probiotics as animal feed additives instead of antibiotics is gaining momentum to avert adverse negative effects on human health. Liquid brewers yeast (LBY) is an industrial by-product containing probiotic microorganisms and is also used as a protein supplement for dairy animals. Nevertheless, value chain actors lack of appropriate handling practices compromises the by-products quality and safety. This study aimed to determine the effect of variation in temperature on microbial diversity and probiotic effects during the storage time of LBY sampled from distributors and farmers from Githunguri sub-county of Kenya. The samples were stored at 20C, 25C and 30C, then tested on 0, 5, 10, 15 and 20 days. The studys parameters involved determining the pH levels, lactic acid bacteria (LAB), total coliform count (TCC), mould, and yeast in LBY. The rate (k) of the reaction kinetics model was used to extrapolate the expected probiotic shelf life. The LAB and yeast populations were reduced in a first-order reaction at all storage temperatures. The rate of reduction in the numbers of LAB reduced with an increase in temperature (k = 0.019 and 0.023) at 20C and 30C, respectively. Yeasts highest rate of growth reduction was 25C (k = 0.009) and least at 30C (k = 0.043). The minimum effective concentration for probiotics of 106 CFU/mL needed to observe the beneficial physiological impact on farm animals was achieved between 34.9 and 35.5 days at the tested storage temperatures. The study provides insight into the unexploited low-cost probiotic potential of LBY in dairy production. Conversely, handling practices and environmental microbial contamination along the value chain can compromise product quality and safety. There is a need to advocate its use in dairy for improved productivity and sensitize farmers to appropriate hygienic measures along the LBY value chain.
文摘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.
文摘Yeast immobilization is a process of physical entrapment of yeast cells using different techniques while maintaining their biological activity.Continuous fermentation systems have significant advantages over conventional methods.Research highlights that immobilized yeast cell systems have several benefits as compared to free yeast cells.The immobilized yeast cell systems improve fermentation rates,especially when paired with continuous fermentation and appropriate immobilization techniques.Understanding various immobilization techniques,continuous fermentation processes,yeast metabolic activity related to beverage flavor production,and bioreactor designs is vital for optimizing the use of immobilized yeast cells systems on industrial scale.This review provides an overview of recent basic research on immobilized yeast cell systems,with a focus on continuous beverage fermentation.In this study,different reactor configurations and immobilization techniques are explored.The study focus on the impacts of immobilization on the yeast cells,and discuss the recent advancements in these techniques.The review concludes with a discussion on the practical applications of immobilized yeast cells and continuous fermentation in beverage production.
文摘The aggregation of amyloid-beta （Aβ） peptide, has been demonstrated to be critical for the development of Alzheimer＇s disease （AD）. All aggregation inhibitors are thus considered to be drug candidates for AD therapy. In the present study, we developed a novel screening tool based on the yeast two-hybrid system to screen Aβ aggregation inhibitors. The human Aβ42 peptide cDNA was cloned using assembly PCR and inserted into each of the yeast expression plasmids containing either the GAL4 activation domain （GAL4AD） or the DNA-binding domain （GAL4BD）. Co-transformation of the above plasmids led to the expression of the fusion proteins GAL4AD-Aβ42 and GAL4BD-Aβ42 in the AH 109 yeast strain. The self interaction of Aβ42 fragments reconstructed the GAL4 transcriptor and thus activated the GAL4 responsive transcription of four reporter genes including HIS3, ADE2, lacZ and MEL1. The expression of the reporter genes rendered the multiple auxotrophic yeast cells capable of growing on the synthetic SD media lacking adenine and histidine. Growth arrest was used as a marker for screening Aβ aggregation inhibitors in this system, and the evaluation of Rhodiola species revealed potential resources for the development of Aβ aggregation inhibitors.
