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Yeast two-hybrid: State of the art

Biological Procedures Online volume 2, pages 1–38 (1999)Cite this article

Abstract

Genome projects are approaching completion and are saturating sequence databases. This paper discusses the role of the two-hybrid system as a generator of hypotheses. Apart from this rather exhaustive, financially and labour intensive procedure, more refined functional studies can be undertaken. Indeed, by making hybrids of two-hybrid systems, customised approaches can be developed in order to attack specific function-related problems. For example, one could set-up a “differential” screen by combining a forward and a reverse approach in a three-hybrid set-up. Another very interesting project is the use of peptide libraries in two-hybrid approaches. This could enable the identification of peptides with very high specificity comparable to “real” antibodies. With the technology available, the only limitation is imagination.

References

  1. Choi K.Y., Satterberg B., Lyons D.M., Elion E.A. 1994. Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae. Cell 78, 499–512.

    Article  PubMed  CAS  Google Scholar 

  2. Kischkel F.C., Hellbardt S., Behrmann I., Germer M., Pawlita M., Krammer P.H., Peter M.E. 1995. Cytotoxicity-dependent APO-1 Fas/CD95-associated proteins form a death-inducing signaling complex DISC with the receptor. EMBO J. 14, 5579–5588.

    PubMed  CAS  Google Scholar 

  3. Phizicky E.M., Fields S. 1995. Protein-protein interactions: methods for detection and analysis. Microbiol.Rev. 59, 94–123.

    PubMed  CAS  Google Scholar 

  4. Hope I.A., Struhl K. 1986. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell 46, 885–894.

    Article  PubMed  CAS  Google Scholar 

  5. Keegan L., Gill G., Ptashne M. 1986. Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. Science 231, 699–704.

    Article  PubMed  CAS  Google Scholar 

  6. Brent R., Ptashne M. 1985. A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell 43, 729–736.

    Article  PubMed  CAS  Google Scholar 

  7. Ma J., Ptashne M. 1988. Converting a eukaryotic transcriptional inhibitor into an activator. Cell 55, 443–446.

    Article  PubMed  CAS  Google Scholar 

  8. Fields S., Song O.K. 1989. A novel genetic system to detect protein-protein interactions. Nature 340, 245–246.

    Article  PubMed  CAS  Google Scholar 

  9. Fields S., Sternglanz R. 1994. The two-hybrid system: an assay for protein-protein interactions. Trends Genet. 10, 286–292.

    Article  PubMed  CAS  Google Scholar 

  10. Young K.H., Ozenberger B.A. 1995. Investigation of ligand binding to members of the cytokine receptor family within a microbial system. Ann.N.Y.Acad.Sci. 766, 279–281.

    Article  PubMed  CAS  Google Scholar 

  11. Kajkowski E.M., Price L.A., Pausch M.H., Young K.H., Ozenberger B.A. 1997. Investigation of growth hormone releasing hormone receptor structure and activity using yeast expression technologies. J.Recept.Signal.Transduct.Res. 17, 293–303.

    Article  PubMed  CAS  Google Scholar 

  12. Denis C.L., Ferguson J., Young E.T. 1983. mRNA levels for the fermentative alcohol dehydrogenase of Saccharomyces cerevisiae decrease upon growth on a nonfermentable carbon source. J.Biol.Chem. 258, 1165–1171.

    PubMed  CAS  Google Scholar 

  13. Tornow J., Santangelo G.G. 1990. Efficient expression of Saccharomyces cerevisiae glycolytic gene ADH1 is dependent upon a cis-acting regulatory element UAS-PRG found initially in genes encoding ribosomal proteins. Gene 90, 79–85.

    Article  PubMed  CAS  Google Scholar 

  14. Ruohonen L., Penttila M., Keranen S. 1991. Optimization of Bacillus amylase Production by Saccharomyces cerevisiae. Yeast 7, 337–346.

    Article  PubMed  CAS  Google Scholar 

  15. Legrain P., Dokhelar M.C., Transy C. 1994. Detection of protein-protein interactions using different vectors in the two-hybrid system. Nucleic Acids Res. 22, 3241–3242.

    Article  PubMed  CAS  Google Scholar 

  16. Fradet Y., Tardif M., Parent-Vaugeois C. 1985. The use of multiparameter flow cytometry in the detection and evaluation of human bladder tumors. Union.Med.Can. 114, 778–780.

    PubMed  CAS  Google Scholar 

  17. Parent S.A., Fenimore C.M., Bostian K.A. 1985. Vector systems for the expression, analysis and cloning of DNA sequences in S. cerevisiae. Yeast 1, 83–138.

    Article  PubMed  CAS  Google Scholar 

  18. Green N., Alexander H., Olson A., Alexander S., Shinick T.M., Sutcliffe J.G., Lerner R.A. 1982. Immunogenic structure of the influenza virus hemagglutinin. Cell 28, 477–487.

    Article  PubMed  CAS  Google Scholar 

  19. Kaufer A.F., Fried H.M., Schwindinger W.F., Jasin M., Warner J.R. 1983. Cycloheximide resistance in yeast: the gene and its protein. Nucleic Acids Res. 11, 3123

    Article  PubMed  CAS  Google Scholar 

  20. Langlands K., Prochownik E.V. 1997. A rapid method for the preparation of yeast lysates that facilitates the immunodetection of proteins generated by the yeast two-hybrid system. Anal.Biochem. 249, 250–252.

    Article  PubMed  CAS  Google Scholar 

  21. Beranger F., Aresta S., de Gunzburg J., Camonis J. 1997. Getting more from the two-hybrid system: N-terminal fusions to LexA are efficient and sensitive baits for two-hybrid studies. Nucleic Acids Res. 25, 2035–2036.

    Article  PubMed  CAS  Google Scholar 

  22. Johnston S.A., Zavortink M.J., Debouck C., Hopper J.E. 1986. Functional domains of the yeast regulatory protein GAL4. Proc.Natl.Acad.Sci.U.S.A. 83, 6553–6557.

    Article  PubMed  CAS  Google Scholar 

  23. Marmorstein R., Carey M., Ptashne M., Harrison S.C. 1992. DNA recognition by GAL4: structure of a protein-DNA complex. Nature 356, 408–414.

    Article  PubMed  CAS  Google Scholar 

  24. Carey M., Kakidani H., Leatherwood J., Mostashari F., Ptashne M. 1989. An amino-terminal fragment of GAL4 binds DNA as a dimer. J.Mol.Biol. 209, 423–432.

    Article  PubMed  CAS  Google Scholar 

  25. Silver P.A., Keegan L.P., Ptashne M. 1984. Amino terminus of the yeast GAL4 gene product is sufficient for nuclear localization. Proc.Natl.Acad.Sci.U.S.A. 81, 5951–5955.

    Article  PubMed  CAS  Google Scholar 

  26. Silver P.A., Chiang A., Sadler I. 1988. Mutations that alter both localization and production of a yeast nuclear protein. Genes and Dev. 2, 707–717.

    Article  PubMed  CAS  Google Scholar 

  27. Silver P.A., Brent R., Ptashne M. 1986. DNA binding is not sufficient for nuclear localization of regulatory proteins in Saccharomyces cerevisiae. Mol.Cell.Biol. 6, 4763–4766.

    PubMed  CAS  Google Scholar 

  28. Brent R., Ptashne M. 1984. A bacterial repressor protein or a yeast transcriptional terminator can block upstream activation of a yeast gene. Nature 612–615.

  29. Dagher M.C., Filhol Cochet O. 1997. Making hybrids of two-hybrid systems. Biotechniques 22, 916–8,920–2.

    PubMed  CAS  Google Scholar 

  30. Guthrie C., Fink G.R. 1991. Guide to yeast genetics and molecular biology. Methods Enzym. 194, 1–932.

    Google Scholar 

  31. Giniger E., Ptashne M. 1988. Cooperative DNA binding of the yeast transcriptional activator GAL4. Proc.Natl.Acad.Sci.U.S.A. 85, 382–386.

    Article  PubMed  CAS  Google Scholar 

  32. Tirode F., Malaguti C., Romero F., Attar R., Camonis J., Egly J.M. 1997. A conditionally expressed third partner stabilizes or prevents the formation of a transcriptional activator in a three-hybrid system. J.Biol.Chem. 272, 22995–22999.

    Article  PubMed  CAS  Google Scholar 

  33. Ebina Y., Takahara Y., Kishi F., Nakazawa A. 1983. LexA protein is a repressor of colicin E1 gene. J.Biol.Chem. 258, 13258–13261.

    PubMed  CAS  Google Scholar 

  34. Estojak J., Brent R., Golemis E.A. 1995. Correlation of two-hybrid affinity data with in vitro measurements. Mol.Cell.Biol. 15, 5820–5829.

    PubMed  CAS  Google Scholar 

  35. West R.W.Jr., Yoccum R.R., Ptashne M. 1984. Saccharomyces cerevisiae GAL1-GAL10 divergent promotor region: location and function of the upstream activating sequence UASG. Mol.Cell.Biol. 2467–2478.

  36. Gietz R.D., Schiestl R.H. 1991. Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier. Yeast 7, 253–263.

    Article  PubMed  CAS  Google Scholar 

  37. Gietz R.D., St.Jean A., Woods R.A., Schiestl R.H. 1992. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20, 1425

    Article  PubMed  CAS  Google Scholar 

  38. Gietz R.D., Schiestl R.H., Willems A.R., Woods R.A. 1995. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355–360.

    Article  PubMed  CAS  Google Scholar 

  39. Gietz R.D. 1997. The Gietz Lab Yeast Transformation Home Page. http://www.umanitoba.ca/faculties/medicine/human_genetics/gietz/Trafo. html

  40. Schenk J.A., Heymann S., Peters L.E., Micheel B. 1996. Screening for recombinant plasmids in yeast colonies of the two-hybrid system using PCR. Biotechniques 20, 812–4, 816.

    PubMed  CAS  Google Scholar 

  41. Herskowitz I. 1988. Life cycle of the budding yeast Saccharomyces cerevisiae. Microbiol.Rev. 52, 536

    PubMed  CAS  Google Scholar 

  42. Bartel P., Chien C.T., Sternglanz R., Fields S. 1993. Elimination of false positives that arise in using the two-hybrid system. Biotechniques 14, 920–924.

    PubMed  CAS  Google Scholar 

  43. Golemis E.A., Khazak V. 1997. Alternative yeast two-hybrid systems. The interaction trap and interaction mating. Methods Mol.Biol. 63, 197–218.

    PubMed  CAS  Google Scholar 

  44. Hengen P.N. 1997. False positives from the yeast two-hybrid system. Trends Biochem.Sci. 22, 33–34.

    Article  PubMed  CAS  Google Scholar 

  45. Golemis E. 1997. Lab of Erica Golemis. http://chaos.fccc.edu/research/labs/ golemis

  46. Yavuzer U., Goding C.R. 1995. pWITCH: a versatile two-hybrid assay vector for the production of epitope/activation domain-tagged proteins both in vitro and in yeast. Gene 165, 93–96.

    Article  PubMed  CAS  Google Scholar 

  47. Wong C., Naumovski L. 1997. Method to screen for relevant yeast two-hybrid-derived clones by coimmunoprecipitation and colocalization of epitope-tagged fragments, application to Bcl-XL. Anal.Biochem. 252, 33–39.

    Article  PubMed  CAS  Google Scholar 

  48. Roder K.H., Wolf S.S., Schweizer M. 1996. Refinement of vectors for use in the yeast two-hybrid system. Anal.Biochem. 241, 260–262.

    Article  PubMed  CAS  Google Scholar 

  49. Le Douarin B., Pierrat B., vom Baur E., Chambon P., Losson R. 1995. A new version of the two-hybrid assay for detection of protein-protein interactions. Nucleic Acids Res. 23, 876–878.

    Article  PubMed  Google Scholar 

  50. Drees BL. 1999. Progress and variations in two-hybrid and three-hybrid technologies. Curr Opin Chem Biol. 3, 64–70.

    Article  PubMed  CAS  Google Scholar 

  51. Leanna C.A., Hannink M. 1996. The reverse two-hybrid system: a genetic scheme for selection against specific protein/protein interactions. Nucleic Acids Res. 24, 3341–3347.

    Article  PubMed  CAS  Google Scholar 

  52. Boeke J.D., LaCroute F., Fink G.R. 1984. A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol.Gen.Genet. 197, 345–346.

    Article  PubMed  CAS  Google Scholar 

  53. Shih H.M., Goldman P.S., DeMaggio A.J., Hollenberg S.M., Goodman R.H., Hoekstra M.F. 1996. A positive genetic selection for disrupting protein-protein interactions: identification of CREB mutations that prevent association with the coactivator CBP. Proc.Natl.Acad.Sci.U.S.A. 93, 13896–13901.

    Article  PubMed  CAS  Google Scholar 

  54. Vidal M., Brachmann R.K., Fattaey A., Harlow E., Boeke J.D. 1996a. Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions. Proc.Natl.Acad.Sci.U.S.A. 93, 10315–10320.

    Article  PubMed  CAS  Google Scholar 

  55. Vidal M., Braun P., Chen E., Boeke J.D., Harlow E. 1996b. Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system. Proc.Natl.Acad.Sci.U.S.A. 93, 10321–10326.

    Article  PubMed  CAS  Google Scholar 

  56. Aronheim A. 551997a. Improved efficiency Sos recruitment system: expression of the mammalian GAP reduces isolation of Ras GTPase false positives. Nucleic Acids Res. 25, 3373–3374.

  57. Aronheim A., Zandi E., Hennemann H., Elledge S.J., Karin M. 1997b. Isolation of an AP-1 Repressor by a Novel Method for Detecting Protein-Protein Interactions. Mol.Cell.Biol. 17, 3094–3102.

    PubMed  CAS  Google Scholar 

  58. Johnsson N., Varhavsky A. 1994. Split ubiquitin as a sensor of protein interactions in vivo. Proc.Natl.Acad.Sci.U.S.A. 91, 10340–10344.

    Article  PubMed  CAS  Google Scholar 

  59. Buchert M., Schneider S., Adams M.T., Hefti H.P., Moelling K., Hovens C.M. 1997. Useful vectors for the two-hybrid system in mammalian cells. Biotechniques 23, 396–8, 400, 402.

    PubMed  CAS  Google Scholar 

  60. Dixon E.P., Johnstone E.M., Liu X., Little S.P. 1997. An inverse mammalian two-hybrid system for secretase and other proteases. Anal.Biochem. 249, 239–241.

    Article  PubMed  CAS  Google Scholar 

  61. Osborne M.A., Dalton D., Kochan J.P. 1995. The yeast tribrid system-genetic detection of tansphosphorylated ITAM-SH2-interactions. Biotechnology 13, 1474–1478.

    Article  PubMed  CAS  Google Scholar 

  62. Zhang J., Lautar S. 1996. A yeast three-hybrid method to clone ternary protein complex components. Anal.Biochem. 242, 68–72.

    Article  PubMed  CAS  Google Scholar 

  63. Ozenberger B.A., Young K.H. 1995. Functional interaction of ligands and receptors of the hematopoietic superfamily in yeast. Mol.Endocrinol. 9, 1321–1329.

    Article  PubMed  CAS  Google Scholar 

  64. Licitra E.J., Liu J.O. 1996. A three-hybrid system for detecting small ligand-protein receptor interactions. Proc.Natl.Acad.Sci.U.S.A. 93, 12817–12821.

    Article  PubMed  CAS  Google Scholar 

  65. Putz U., Skehel P., Kuhl D. 1996. A tri-hybrid system for the analysis and detection of RNA-protein interactions. Nucleic Acids Res. 24, 4838–4040.

    Article  PubMed  CAS  Google Scholar 

  66. Sengupta D.J. 1996. Application of two-hybrid and three-hybrid technologies. EMBO Two-Hybrid Course, October 12, Munich.

  67. Brachmann R.K., Boeke J.D. 1997. Tag games in yeast: the two-hybrid system and beyond. Curr.Op.Biotech. 561–568.

  68. Van Criekinge W, van Gurp M, Decoster E, Schotte P, Van de Craen M, Fiers W, Vandenabeele P, Beyaert R 1998 Use of the yeast three-hybrid system as a tool to study caspases. Anal Biochem. 263, 62–64.

    Article  PubMed  Google Scholar 

  69. Van Aelst L., White M.A., Wigler M.H. 1994. Ras partners. Cold.Spring.Harb.Symp.Quant. Biol. 59, 181–186.

    PubMed  Google Scholar 

  70. White M.A., Nicolette C., Minden A., Polverino A., Van Aelst L., Karin M., Wigler M.H. 1995. Multiple Ras functions can contribute to mammalian cell transformation. Cell 80, 533–541.

    Article  PubMed  CAS  Google Scholar 

  71. Khosravi-Far R., White M.A., Westwick J.K., Solski P.A., Chrzanowska-Wodnicka M., Van Aelst L., Wigler M.H., Der C.J. 1996. Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation. Mol.Cell.Biol. 16, 3923–3933.

    PubMed  CAS  Google Scholar 

  72. Jessen T. 1996. Pharmaceutical applications of the interaction trap. EMBO Two-Hybrid Course, October 10, Munich.

  73. Van Criekinge W., Cornelis S., Van de Craen M., Vandenabeele P., Fiers W. & Beyaert R. 1999. GAL4 is a substrate for caspases: implications for two-hybrid screening and other GAL4-based assays. Mol. Cell Biol. Res. Commun. 1, 158–161.

    Article  PubMed  Google Scholar 

  74. Fromont-Racine M., Rain J.C., Legrain P. 1997. Towards a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nature Gen. 277–281.

  75. Robzyk K, Kassir Y. 1992. A simple and highly efficient procedure for rescuing autonomous plasmids from yeast. Nucleic Acids Res 20, 3790.

    Article  PubMed  CAS  Google Scholar 

  76. James P, Halladay J, Craig EA 1996. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425–36.

    PubMed  CAS  Google Scholar 

  77. Bartel PL, Roecklein JA, SenGupta D, Fields S 1996. A protein linkage map of Escherichia coli bacteriophage T7. Nat Genet 12, 72–77.

    Article  PubMed  CAS  Google Scholar 

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  1. DEVGEN N.V. Technologiepark 9, 9052, Zwijnaarde-Gent

    Wim Van Criekinge

  2. Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, B-9000, Ghent, Belgium

    Rudi Beyaert

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Van Criekinge, W., Beyaert, R. Yeast two-hybrid: State of the art. Biol Proced Online 2, 1–38 (1999). https://doi.org/10.1251/bpo16

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