Advertisement

Use of Gene Fusions to Study Biological Problems

  • L. Guarente
Part of the Genetic Engineering book series (GEPM, volume 6)

Abstract

A fundamental practical problem in molecular biology is the detection and quantitation of particular proteins of interest. In those cases in which biological or immunological assays exist, problems of how the expression of particular genes is regulated, how proteins find their correct location inside or outside of a cell, or what features govern protein structure and function in vivo are within the realm of accessibility. In many cases, however, reliable assays are not available and the alternative method of gene fusion is appropriate. The principle of the approach is to fuse either a promoter region or a gene whose product is under study to another gene encoding a product which can be assayed conveniently, for example the lacZ gene of E. coli which encodes β-galactosidase. In promoter fusions, the promoter region will direct synthesis of wild-type β-galactosidase with transcription initiating in the promoter region, and translation starting at the amino-terminal AUG encoded by lacZ (Figure 1). In gene fusions, a hybrid protein will be encoded containing sequences from the protein under study at the amino terminus, and an active β-galactosidase moiety at the carboxy-terminus (Figure 1). These fusions are active because the amino-terminal sequences of β-galactosidase may be replaced with a wide variety of sequences without a loss in its enzymatic activity. The β-galactosidase moiety, in such fusions, serves as a tag which can be used to monitor amounts or cellular location of the protein to which it is fused. In promoter or gene fusions, the myriad of genetic methods developed to study the lac operon can now be adapted to a genetic analysis of the promoter or gene fused to lacZ. For studies of fusions in bacteria, this adaptation is complete, while studies of fusions in eukaryotic cells such as yeast can draw from only a subset of the prokaryotic methods.

Keywords

Gene Fusion Yeast Gene lacZ Gene Hybrid Protein lacZ Fusion 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Jacob, F., Ulmann, A. and Monod, J. (1961) J Mol. Biol. 31, 704–714.Google Scholar
  2. 2.
    Beckwith, J.R. and Signer, E.R. (1966) J. Mol. Biol. 19, 254–265.PubMedCrossRefGoogle Scholar
  3. 3.
    Beckwith, J.R., Signer, E.R. and Epstein, W. (1967) Cold Spring Harbor Symp. Quant. Biol. 31, 393–401.Google Scholar
  4. 4.
    Miller, J.H., Reznikoff, W.S., Silverstone, A.E., Ippan, K., Signer, E.R. and Beckwith, J.R. (1970) J. Bacteriol. 104, 1273–1279.PubMedGoogle Scholar
  5. 5.
    Mitchell, D.H., Reznikoff, W.S. and Beckwith, J. (1975) J. Mol. Biol. 93, 331–350.PubMedCrossRefGoogle Scholar
  6. 6.
    Mitchell, D.H., Reznikoff, W.S. and Beckwith, J. (1976) J. Mol. Biol. 101, 441–457.PubMedCrossRefGoogle Scholar
  7. 7.
    Casadaban, M.J. (1976) J. Mol. Biol. 104, 541–555.PubMedCrossRefGoogle Scholar
  8. 8.
    Muller-Hill, B. and Kania J. (1974) Nature 249, 561–563.PubMedCrossRefGoogle Scholar
  9. 9.
    Brickman, E., Silhavy, T.J., Bassford, P.J. Jr., Shuman, H.A. and Beckwith, J.R. (1979) J. Bacteriol. 139, 13–18.PubMedGoogle Scholar
  10. 10.
    Moreno, F., Fowler, A.V., Hall, M., Silhavy, T., Zabin, I. and Schwartz, M. (1980) Nature 286, 356–359.PubMedCrossRefGoogle Scholar
  11. 11.
    Casadaban, M. and Cohen, S.N. (1979) Proc. Nat. Acad. Sci. U.S.A. 76, 4530–4533.CrossRefGoogle Scholar
  12. 12.
    Kenyon, C. and Walker, G.C. (1980) Proc. Nat. Acad. Sci. U.S.A. 77, 2819–2823.CrossRefGoogle Scholar
  13. 13.
    Wanner, B., Wieder, S. and McSharry, R. (1981) J. Bacteriol. 146, 93–101.PubMedGoogle Scholar
  14. 14.
    Maurer, R., Meyer, B.J. and Ptashne, M. (1980) J. Mol. Biol. 139, 147–161.PubMedCrossRefGoogle Scholar
  15. 15.
    Meyer, B.J., Maurer, R. and Ptashne, M. (1980) J. Mol. Biol. 139, 163–194.PubMedCrossRefGoogle Scholar
  16. 16.
    Casadaban, M. and Cohen, S.N. (1980) J. Mol. Biol. 138, 179–207.PubMedCrossRefGoogle Scholar
  17. 17.
    Casadaban, M.J., Chou, J. and Cohen, S.N. (1980) J. Bacteriol. 143, 971–980.PubMedGoogle Scholar
  18. 18.
    Guarente, L., Lauer, G., Roberts, T.M. and Ptashne, M. (1980) Cell 20, 543–553.PubMedCrossRefGoogle Scholar
  19. 19.
    Guarente, L. and Ptashne, M. (1981) Proc. Nat. Acad. Sci. U.S.A. 78, 2199–2203.CrossRefGoogle Scholar
  20. 20.
    Rose, M., Casadaban, M.J. and Botstein, D. (1981) Proc. Nat. Acad. Sci. U.S.A. 78, 2460–2464.CrossRefGoogle Scholar
  21. 21.
    Beggs, J.D. (1978) Nature 275, 104–109.PubMedCrossRefGoogle Scholar
  22. 22.
    Stinchcomb, D., Mann, C. and Davis, R. (1982) J. Mol. Biol. 158, 157–180.PubMedCrossRefGoogle Scholar
  23. 23.
    Clarke, L. and Carbon, J. (1980) Nature 287, 504–509.PubMedCrossRefGoogle Scholar
  24. 24.
    Hinnen, A., Hicks, J.B. and Fink, G.R. (1978) Proc. Nat. Acad. Sci. U.S.A. 75, 1929–1933.CrossRefGoogle Scholar
  25. 25.
    Botstein, D., Falco, S.C., Stewart, S., Brennan, M., Scherer, S., Stinchcomb, D., Struhl, K. and Davis, R.W. (1979) Gene 8, 17–24.PubMedCrossRefGoogle Scholar
  26. 26.
    Rosenberg, M., McKenney, K. and Schumperli, D. (1982) in Promoters, Structure and Function (Rodriguez, R. and Chamberlin, M., eds.), pp. 387–406, Praeger Press, New York, NY.Google Scholar
  27. 27.
    McKenney, K., Shimatake, H., Court, D., Schmeissner, U., Brody, C. and Rosenberg, M. (1981) in Gene Amplification and Analysis II: Analysis of Nucleic Acids by Enzymatic Methods (Chirikjian, J.C. and Papas, T.S., eds.), pp. 383–415, Elsevier-North Holland, New York,NY.Google Scholar
  28. 28.
    West, R. and Rodriguez, R. (1982) Gene 20, 291–304.PubMedCrossRefGoogle Scholar
  29. 29.
    Close, T. and Rodriguez, R. (1982) Gene 26, 305–316.CrossRefGoogle Scholar
  30. 30.
    Schumperli, D., Howard, B. and Rosenberg, M. (1982) Proc. Nat. Acad. Sci. U.S.A. 79, 257–261.CrossRefGoogle Scholar
  31. 31.
    Gorman, C., Moffat, L. and Howard, B. (1982) Mol. Cell Biol. 2, 1044.PubMedGoogle Scholar
  32. 32.
    Mulligan, R. and Berg, P. (1980) Science 209, 1422–1427.PubMedCrossRefGoogle Scholar
  33. 33.
    Southern, P. and Berg, P. (1982) J. Mol. Appl. Gen. 1, 327.Google Scholar
  34. 34.
    Wigler, M., Silverstein, S., Lee, L., Pellicer, A., Cheng, Y. and Axel, R. (1977) Cell 11, 223–232.PubMedCrossRefGoogle Scholar
  35. 35.
    Mulligan, R., Howard, B. and Berg, P. (1979) Nature 277, 108–114.PubMedCrossRefGoogle Scholar
  36. 36.
    Dimaio, D., Treisman, R. and Maniatis, T. (1982) Proc. Nat. Acad. Sci. U.S.A. 79, 4030–4034.CrossRefGoogle Scholar
  37. Pellicer, A., Robins, D., Wold, B., Sweet, R., Jackson, J., Lowy, I., Roberts, J., Sim, G., Silverstein, S. and Axel, R. Science 209, 1414–1421.Google Scholar
  38. 38.
    Bassford, P., Beckwith, J., Berman, M., Brickman, E., Casadaban, M., Guarente, L., Saint-Girous, I., Sarthy, A., Schwartz, M. and Silhavy, T. (1978) in The Operon (Miller, J.H. and Reznikoff, W.S., eds.), pp. 245–262, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  39. 39.
    Ptashne, M., Jeffrey, A., Johnson, A., Maurer, R., Meyer, B., Pabo, C., Roberts, T. and Sauer, R. (1980) Cell, 19, 1–11.PubMedCrossRefGoogle Scholar
  40. 40.
    Johnson, A., Poteete, A., Lauer, G., Sauer, R., Ackers, G. and Ptashne, M. (1981) Nature 294, 217–233.PubMedCrossRefGoogle Scholar
  41. 41.
    Backman, K., Ptashne, M. and Gilbert, W. (1976) Proc. Nat. Acad. Sci. U.S.A. 73, 4174–4178.CrossRefGoogle Scholar
  42. 42.
    Backman, K. and Ptashne, M. (1978) Cell 13, 65–71.PubMedCrossRefGoogle Scholar
  43. 43.
    Casadaban, M.J. (1976) J. Mol. Biol. 104, 557–566.PubMedCrossRefGoogle Scholar
  44. 44.
    Hall, M. and Silhavy, T. (1981) J. Mol. Biol. 146, 23–44.PubMedCrossRefGoogle Scholar
  45. 45.
    Hall, M. and Silhavy, T. (1981) J. Mol. Biol. 151, 1–15.PubMedCrossRefGoogle Scholar
  46. 46.
    Laimins, L., Rhoads, D. and Epstein, W. (1981) Proc. Nat. Acad. Sci. U.S.A. 87, 464–468.CrossRefGoogle Scholar
  47. 47.
    Guarente, L. and Mason, T. (1983) Cell 32, 1279–1286.PubMedCrossRefGoogle Scholar
  48. 48.
    Débarbouillé, M., Shuman, H., Silhavy, T. and Schwartz, M. (1978) J. Mol. Biol. 124, 359–371.PubMedCrossRefGoogle Scholar
  49. 49.
    Berman, M. and Beckwith, J. (1979) J. Mol. Biol. 130, 285–301.PubMedCrossRefGoogle Scholar
  50. 50.
    Berman, M. and Beckwith, J. (1979) J. Mol. Biol. 130, 303–315.PubMedCrossRefGoogle Scholar
  51. 51.
    Ketner, G. and Campbell, A. (1975) J. Mol. Biol. 96, 13–27.PubMedCrossRefGoogle Scholar
  52. 51.
    Ketner, G. and Campbell, A. (1975) J. Mol. Biol. 96, 13–27.PubMedCrossRefGoogle Scholar
  53. 53.
    Guarente, L., Mitchell, D. and Beckwith, J. (1977) J. Mol. Biol. 112, 423–436.PubMedCrossRefGoogle Scholar
  54. 54.
    Guarente, L., Beckwith, J., Wu, A. and Platt, T. (1979) J. Mol. Biol. 133, 189–197.PubMedCrossRefGoogle Scholar
  55. 55.
    Wu, A., Chapman, A., Platt, T., Guarente, L. and Beckwith, J. (1980) Cell 19, 829–836.PubMedCrossRefGoogle Scholar
  56. 56.
    Wu, A. and Platt, T. (1978) Proc. Nat. Acad. Sci. U.S.A. 75, 5442–5446.CrossRefGoogle Scholar
  57. 57.
    Platt, T. (1983) in Gene Function in Prokaryotes (Beckwith, J., Davies, J. and Gallant, J., eds.), pp. 126–161, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  58. 58.
    Silverman, S., Rose, M., Botstein, D. and Fink, G. (1982) Mol. Cell Biol. 2, 1212–1219.PubMedGoogle Scholar
  59. 59.
    Martinez-Arias, A. and Casadaban, M. (1983) Mol. Cell Biol. 3, 580–586.PubMedGoogle Scholar
  60. 60.
    Osley, M. and Hereford, L. (1982) Proc. Nat. Acad. Sci. U.S.A. 79, 7689–7693.CrossRefGoogle Scholar
  61. 61.
    Faye, G., Leung, D.W., Tatchell, K., Hall, B. and Smith, M. (1981) Proc. Nat. Acad. Sci. U.S.A. 78, 2258–2262.CrossRefGoogle Scholar
  62. Guarente, L., Yocum, R. and Gifford, P. (182) Proc. Nat. Acad. Sci. U.S.A. 79, 7410–7414.Google Scholar
  63. 63.
    Guarente, L. (1983) Meth. Enzymol. 101, 181–191.PubMedCrossRefGoogle Scholar
  64. 64.
    Itakura, K., Hirose, T., Crea, R., Riggs, A.D., Heynecker, H.L., Boliver, F. and Boyer, H.W. (1977) Sicence 198, 1056–1063.CrossRefGoogle Scholar
  65. 65.
    Goeddel, D., Kleid, D., Bolivar, F., Heynecker, H.L., Yansura, D., Crea, R., Hírose, T., Kraszewski, A., Itakura, K. and Riggs, A.D. (1979) Proc. Nat. Acad. Sci. U.S.A. 76, 106–110.CrossRefGoogle Scholar
  66. 66.
    Fuller, F. (1981) Ph.D. Thesis, Harvard University.Google Scholar
  67. 67.
    Mercereau-Puijalon, O., Royal, A., Cami, B., Garapin, A., Krust, A., Gannon, F. and Kourilsky, P. (1978) Nature 275, 505–510.PubMedCrossRefGoogle Scholar
  68. 68.
    Hallewell, R. and Emtage, S. (1980) Gene 9, 27–47.PubMedCrossRefGoogle Scholar
  69. 69.
    Goeddel, D., Yelverton, E., Ullrich, A., Heynecker, H.L., Moizzari, G., Holmes, W., Seeburg, P., Dull, T., May, L., Stebbing, N., Crea, R., Maeda, S., McCandliss, R., Solma, A., Tabor, J.M., Gross, M., Familletti, P. and Petska, S. (1980) Nature 287, 411–416.PubMedCrossRefGoogle Scholar
  70. 70.
    Talmadge, K., Stahl, S. and Gilbert, W. (1980) Proc. Nat. Acad. Sci. U.S.A. 77, 3369–3373.CrossRefGoogle Scholar
  71. 71.
    Weinstock, G., Rhys, C., Berman, M., Hampar, B., Jackson, D., Silhavy, T., Weisemann, J. and Zweig, M. (1983) Proc. Nat. Acad. Sci. U.S.A. 80, 4432–4436.CrossRefGoogle Scholar
  72. 72.
    Roberts, T., Kacich, R. and Ptashne, M. (1979) Proc. Nat. Acad. Sci. U.S.A. 76, 760–764.CrossRefGoogle Scholar
  73. 73.
    Guarente, L., Roberts, T. and Ptashne, M. (1980) Science 209, 1428–1430.PubMedCrossRefGoogle Scholar
  74. 74.
    Taniguchi, T., Guarente, L., Roberts, T., Kimelman, D., Douhan, J. and Ptashne, M. (1980) Proc. Nat. Acad. Sci. U.S.A. 77, 5230–5233.CrossRefGoogle Scholar
  75. 75.
    Shuman, A., Silhavy, T. and Beckwith, J. (1980) J. Biol. Chem. 256, 168–174.Google Scholar
  76. 76.
    Gray, M., Colot, M., Guarente, L. and Rosbash, M. (1982) Proc. Nat. Acad. Sci. U.S.A. 79, 6598–6602.CrossRefGoogle Scholar
  77. 77.
    Zabeau, M. and Stanley, K. (1982) EMBO J. 1, 1217–1224.PubMedGoogle Scholar
  78. 78.
    Ruther, U., Koenen, M., Sippel, A. and Muller-Hill, B. (1982) Proc. Nat. Acad. Sci. U.S.A. 79, 6852–6855.CrossRefGoogle Scholar
  79. 79.
    Young, R. and Davis, R. (1983) Proc. Nat. Acad. Sci. U.S.A. 80, 1194–1198.CrossRefGoogle Scholar
  80. 79.
    Young, R. and Davis, R. (1983) Proc. Nat. Acad. Sci. U.S.A. 80, 1194–1198.CrossRefGoogle Scholar
  81. 79.
    Young, R. and Davis, R. (1983) Proc. Nat. Acad. Sci. U.S.A. 80, 1194–1198.CrossRefGoogle Scholar
  82. 82.
    Michaelis, S. and Beckwith, J. (1982) Ann. Rev. Microbiol. 36, 435–465.CrossRefGoogle Scholar
  83. 83.
    Silhavy, T., Shuman, H., Beckwith, J. and Scwhartz, M. (1977) Proc. Nat. Acad. Sci. U.S.A. 74, 5411–5415.CrossRefGoogle Scholar
  84. 84.
    Bassford, P., Silhavy, T. and Beckwith, J. (1979) J. Bacteriol. 139, 19–31.PubMedGoogle Scholar
  85. 85.
    Emr, S. and Silhavy, T. (1980) J. Mol. Biol. 141, 63–90.PubMedCrossRefGoogle Scholar
  86. 86.
    Bassford, P. and Beckwith, J. (1979) Nature 277, 538–541.PubMedCrossRefGoogle Scholar
  87. 87.
    Emr, S., Hanley-Way, S. and Silhavy, T. (1981) Cell 23, 79–88.PubMedCrossRefGoogle Scholar
  88. 88.
    Michaelis, S., Guarente, L. and Beckwith, J. (1983) J. Bacteriol. 154, 356–365.PubMedGoogle Scholar
  89. 89.
    Michaelis, S., Inouye, H., Oliver, D. and Beckwith, J. (1983) J. Bacteriol. 154, 366–374.PubMedGoogle Scholar
  90. 90.
    Oliver, D. and Beckwith J. (1981) Cell 25, 765–772.PubMedCrossRefGoogle Scholar
  91. 91.
    Oliver, D. and Beckwith, J. (1982) Cell 30, 311–319.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1984

Authors and Affiliations

  • L. Guarente
    • 1
  1. 1.Department of BiologyMassachusetts Institute of TechnologyUSA

Personalised recommendations