I recount the history of how I became interested in the use of gene fusions for studying biological problems. Initially, selections for mutations that would restore function to an inactivated operon unexpectedly yielded fusions in which was expressed from the controlling elements of upstream genes. Subsequently, by chance, I generated strains in which the operon was transposed from its normal position on the chromosome to a position close to the operon, thus facilitating sets of useful fusions of the two operons. The development of a more generalized technique for obtaining fusions by my student Malcolm Casadaban opened up a much broader set of biological problems that could be approached with fusions. Work on these problems included the study of protein translocation across membranes, the analysis of membrane protein topology, and the discovery of the pathway of electron transfer that leads to disulfide bond formation in proteins.


Article metrics loading...

Loading full text...

Full text loading...


Literature Cited

  1. Ames BN, Hartman PE, Jacob F. 1.  1963. Chromosomal alterations affecting the regulation of histidine biosynthetic enzymes in Salmonella. J. Mol. Biol. 7:23–42 [Google Scholar]
  2. Anfinsen CB, Haber E, Sela M, White FH Jr. 2.  1961. The kinetics of formation of native ribonuclease during oxidation of the reduced polypeptide chain. Proc. Natl. Acad. Sci. USA 47:1309–14 [Google Scholar]
  3. Baglioni C. 3.  1962. The fusion of two peptide chains in hemoglobin Lepore and its interpretation as a genetic deletion. Proc. Natl. Acad. Sci. USA 48:1880–86 [Google Scholar]
  4. Bardwell JC, McGovern K, Beckwith J. 4.  1991. Identification of a protein required for disulfide bond formation in vivo. Cell 67:581–89 [Google Scholar]
  5. Bassford P, Beckwith J. 5.  1979. Escherichia coli mutants accumulating the precursor of a secreted protein in the cytoplasm. Nature 277:538–41 [Google Scholar]
  6. Beckwith J. 6.  1963. Restoration of operon activity by suppressors. Biochim. Biophys. Acta 76:162–64 [Google Scholar]
  7. Beckwith JR. 7.  1964. A deletion analysis of the Lac operator region in Escherichia coli. J. Mol. Biol. 8:427–30 [Google Scholar]
  8. Beckwith JR, Signer ER. 8.  1966. Transposition of the lac region of Escherichia coli. I. Inversion of the lac operon and transduction of lac by phi80. J. Mol. Biol. 19:254–65 [Google Scholar]
  9. Beckwith JR, Signer ER, Epstein W. 9.  1966. Transposition of the lac region of E. coli. Cold Spring Harb. Symp. Quant. Biol. 31:393–401 [Google Scholar]
  10. Bedouelle H, Bassford PJ Jr, Fowler AV, Zabin I, Beckwith J, Hofnung M. 10.  1980. The nature of mutational alterations in the signal sequence of the maltose binding protein of Escherichia coli. Nature 285:78–81 [Google Scholar]
  11. Benzer S, Champe SP. 11.  1962. A change from nonsense to sense in the genetic code. Proc. Natl. Acad. Sci. USA 48:1114–21 [Google Scholar]
  12. Berman ML, Beckwith J. 12.  1979. Use of gene fusions to isolate promoter mutants in the transfer RNA gene tyrT of Escherichia coli. J. Mol. Biol. 130:303–15 [Google Scholar]
  13. Bernstein HD, Hyndman JB. 13.  2001. Physiological basis for conservation of the signal recognition particle targeting pathway in Escherichia coli. J. Bacteriol. 183:2187–97 [Google Scholar]
  14. Bissell MJ, Tosi R, Gorini L. 14.  1971. Mechanism of excretion of a bacterial proteinase: factors controlling accumulation of the extracellular proteinase of a Sarcina strain (Coccus P). J. Bacteriol. 105:1099–109 [Google Scholar]
  15. Blobel G, Dobberstein B. 15.  1975. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J. Cell Biol. 67:835–51 [Google Scholar]
  16. Bonner DM, Yanofsky C. 16.  1949. Quinolinic acid accumulation in the conversion of 3-hydroxyanthranilic acid to niacin in Neurospora. Proc. Natl. Acad. Sci. USA 1949 35:576–81 [Google Scholar]
  17. Brenner S, Beckwith JR. 17.  1965. Ochre mutants, a new class of suppressible nonsense mutants. J. Mol. Biol. 13:629–37 [Google Scholar]
  18. Casadaban MJ. 18.  1976. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and mu. J. Mol. Biol. 104:541–55 [Google Scholar]
  19. Cuzin F, Jacob F. 19.  1964. Deletions chromosomiques et integration d'un episome sexuel chez Escherichia coli K12. C. R. Acad. Sci. Paris 258:1350–52 [Google Scholar]
  20. Debarbouillé M, Shuman HA, Silhavy TJ, Schwartz M. 20.  1978. Dominant constitutive mutations in malT, the positive regulator gene of the maltose regulon in Escherichia coli. J. Mol. Biol. 124:359–71 [Google Scholar]
  21. Derman AI, Prinz WA, Belin D, Beckwith J. 21.  1993. Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science 262:1744–47 [Google Scholar]
  22. Dutton RJ, Wayman A, Wei JR, Rubin EJ, Beckwith J, Boyd D. 22.  2010. Inhibition of bacterial disulfide bond formation by the anticoagulant warfarin. Proc. Natl. Acad. Sci. USA 107:297–301 [Google Scholar]
  23. Emr SD, Hanley-Way S, Silhavy TJ. 23.  1981. Suppressor mutations that restore export of a protein with a defective signal sequence. Cell 23:79–88 [Google Scholar]
  24. Emr SD, Hedgpeth J, Clement J-M, Silhavy TJ, Hofnung M. 24.  1980. Sequence analysis of mutations that prevent export of lambda receptor, an Escherichia coli outer membrane protein. Nature 285:82–85 [Google Scholar]
  25. Emr SD, Schwartz M, Silhavy TJ. 25.  1978. Mutations altering the cellular localization of the phage lambda receptor, an Escherichia coli outer membrane protein. Proc. Natl. Acad. Sci. USA 75:5802–6 [Google Scholar]
  26. Englesberg E, Sheppard D, Squires C, Meronk F Jr. 26.  1969. An analysis of “revertants” of a deletion mutant in the C gene of the l-arabinose gene complex in Escherichia coli B-r: isolation of initiator constitutive mutants (Ic). J. Mol. Biol. 43:281–98 [Google Scholar]
  27. Franklin NC, Dove WF, Yanofsky C. 27.  1965. The linear insertion of a prophage into the chromosome of E. coli shown by deletion mapping. Biochem. Biophys. Res. Comm. 18:891–99 [Google Scholar]
  28. Froshauer S, Green GN, Boyd D, McGovern K, Beckwith J. 28.  1988. Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli. J. Mol. Biol. 200:501–11 [Google Scholar]
  29. Gardel C, Benson SA, Hunt J, Michaelis S, Beckwith J. 29.  1987. secD, a new gene involved in protein export in Escherichia coli. J. Bacteriol. 169:1286–90 [Google Scholar]
  30. Gottesman S, Beckwith JR. 30.  1969. Directed transposition of the arabinose operon: a technique for the isolation of specialized transducing bacteriophages for any Escherichia coli gene. J. Mol. Biol. 44:117–27 [Google Scholar]
  31. Guarente LP, Mitchell DH, Beckwith J. 31.  1977. Transcription termination at the end of the tryptophan operon of Escherichia coli. J. Mol. Biol. 112:423–36 [Google Scholar]
  32. Inouye H, Beckwith J. 32.  1977. Synthesis and processing of an E. coli alkaline phosphatase precursor in vitro. Proc. Natl. Acad. Sci. USA 74:1440–44 [Google Scholar]
  33. Ippen K, Miller JH, Scaife J, Beckwith J. 33.  1968. New controlling element in the Lac operon of E. coli. Nature 217:825–27 [Google Scholar]
  34. Ippen K, Shapiro JA, Beckwith JR. 34.  1971. Transposition of the lac region to the gal region of the Escherichia coli chromosome: isolation of lambda-lac transducing bacteriophages. J. Bacteriol. 108:5–9 [Google Scholar]
  35. Ito K, Wittekind M, Nomura M, Shiba K, Yura T. 35.  et al. 1983. A temperature-sensitive mutant of E. coli exhibiting slow processing of exported proteins. Cell 32:789–97 [Google Scholar]
  36. Jacob F, Perrin D, Sanchez C, Monod J. 36.  1960. L'opéron: groupe des gènes à expression coordonnée par un opérateur. C. R. Acad. Sci. Paris 250:1727–29 [Google Scholar]
  37. Kadokura H, Beckwith J. 37.  2010. Mechanisms of oxidative protein folding in the bacterial cell envelope. Antioxid. Redox Signal. 13:1231–46 [Google Scholar]
  38. Kumamoto CA, Beckwith J. 38.  1983. Mutations in a new gene, secB, cause defective protein localization in Escherichia coli. J. Bacteriol. 154:253–60 [Google Scholar]
  39. Manoil C, Beckwith J. 39.  1986. A genetic approach to analyzing membrane protein topology. Science 233:1403–8 [Google Scholar]
  40. Michaelis S, Hunt JF, Beckwith J. 40.  1986. Effects of signal sequence mutations on the kinetics of alkaline phosphatase export to the periplasm in Escherichia coli. J. Bacteriol. 167:160–67 [Google Scholar]
  41. Michaelis S, Inouye H, Oliver D, Beckwith J. 41.  1983. Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli. J. Bacteriol. 154:366–74 [Google Scholar]
  42. Miller JH, Ippen K, Scaife JG, Beckwith JR. 42.  1968. The promoter-operator region of the lac operon of Escherichia coli. J. Mol. Biol. 38:413–20 [Google Scholar]
  43. Miller JH, Reznikoff WS, Silverstone AE, Ippen K, Signer ER, Beckwith JR. 43.  1970. Fusions of the lac and trp regions of the Escherichia coli chromosome. J. Bacteriol. 104:1273–79 [Google Scholar]
  44. Mitchell DH, Reznikoff WS, Beckwith J. 44.  1976. Genetic fusions that help define a transcription termination region in Escherichia coli. J. Mol. Biol. 101:441–57 [Google Scholar]
  45. Müller-Hill B, Kania J. 45.  1974. lac repressor can be fused to β-galactosidase. Nature 249:561–63 [Google Scholar]
  46. Nishiyama K, Mizushima S, Tokuda H. 46.  1993. A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli. EMBO J. 12:3409–15 [Google Scholar]
  47. Oliver DB, Beckwith J. 47.  1981. E. coli mutant pleiotropically defective in the export of secreted proteins. Cell 25:2765–72 [Google Scholar]
  48. Pardee AB, Beckwith JR. 48.  1962. Genetic determination of constitutive enzyme levels. Biochim. Biophys. Acta 60:452–54 [Google Scholar]
  49. Saint-Girons I. 49.  1978. New regulatory mutations affecting the expression of the threonine operon in Escherichia coli K-12. Mol. Gen. Genet. 162:95–100 [Google Scholar]
  50. Scaife J, Beckwith JR. 50.  1966. Mutational alteration of the maximal level of Lac operon expression. Cold Spring Harb. Symp. Quant. Biol. 31:403–8 [Google Scholar]
  51. Shuman HA, Silhavy TJ, Beckwith JR. 51.  1980. Labeling of proteins with β-galactosidase by gene fusion: identification of a cytoplasmic membrane protein component of the Escherichia coli maltose transport system. J. Biol. Chem. 255:168–74 [Google Scholar]
  52. Silhavy TJ, Beckwith JR. 52.  1985. Uses of lac fusions for the study of biological problems. Microbiol. Rev. 49:398–418 [Google Scholar]
  53. Tian H, Beckwith J. 53.  2002. Genetic screen yields mutations in genes encoding all known components of the Escherichia coli signal recognition particle pathway. J. Bacteriol. 184:111–18 [Google Scholar]
  54. Wickner W, Driessen AJ, Hartl FU. 54.  1991. The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu. Rev. Biochem. 60:101–24 [Google Scholar]
  55. Wu AM, Chapman AB, Platt T, Guarente LP, Beckwith J. 55.  1980. Deletions of distal sequence after termination of transcription at the end of the tryptophan operon in E. coli. Cell 19:829–36 [Google Scholar]
  56. Yanofsky C. 56.  2001. Advancing our knowledge in biochemistry, genetics, and microbiology through studies on tryptophan metabolism. Annu. Rev. Biochem. 70:1–37 [Google Scholar]
  57. Bardwell JCA, Lee J-O, Jander G, Martin N, Belin, Beckwith JD. 57.  1993. A pathway for disulfide bond formation in vivo. Proc. Natl. Acad. Sci. USA 90:1038–42 [Google Scholar]

Data & Media loading...

  • Article Type: Review Article
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error