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. 1984 Jan;81(2):535–539. doi: 10.1073/pnas.81.2.535

In vivo formation of gene fusions encoding hybrid beta-galactosidase proteins in one step with a transposable Mu-lac transducing phage.

M J Casadaban, J Chou
PMCID: PMC344713  PMID: 6320194

Abstract

A Mu-lac bacteriophage transposon, MudII301 (Ap, lac), was constructed to form hybrid protein gene fusions. When it integrates into structural genes in the appropriate direction and reading phase, transcription and translation from outside gene controlling regions can proceed across 116 nucleotides from the right end of Mu into lacZ codons to form hybrid proteins that are enzymatically active for beta-galactosidase. Integration can be obtained either by infection to form lysogens or by transposition during growth of a lysogen. The size of the hybrid protein product either corresponds to or, in the cases of translation restart or protein degradation, is a minimal estimate of the distance of the Mu insertion from the translation initiation site of the gene. Hybrid proteins formed by insertions in randomly selected genes and in the araB and A genes were examined by polyacrylamide gel electrophoresis.

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Selected References

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  1. Bachmann B. J., Low K. B. Linkage map of Escherichia coli K-12, edition 6. Microbiol Rev. 1980 Mar;44(1):1–56. doi: 10.1128/mr.44.1.1-56.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beny G., Boyen A., Charlier D., Lissens W., Feller A., Glansdorff N. Promoter mapping and selection of operator mutants by using insertion of bacteriophage Mu in the argECBH divergent operon of Escherichia coli K-12. J Bacteriol. 1982 Jul;151(1):62–67. doi: 10.1128/jb.151.1.62-67.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brickman E., Silhavy T. J., Bassford P. J., Jr, Shuman H. A., Beckwith J. R. Sites within gene lacZ of Escherichia coli for formation of active hybrid beta-galactosidase molecules. J Bacteriol. 1979 Jul;139(1):13–18. doi: 10.1128/jb.139.1.13-18.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Casadaban M. J., Chou J., Cohen S. N. Overproduction of the Tn3 transposition protein and its role in DNA transposition. Cell. 1982 Feb;28(2):345–354. doi: 10.1016/0092-8674(82)90352-x. [DOI] [PubMed] [Google Scholar]
  6. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  7. Casadaban M. J., Cohen S. N. Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4530–4533. doi: 10.1073/pnas.76.9.4530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Casadaban M. J. Fusion of the Escherichia coli lac genes to the ara promoter: a general technique using bacteriophage Mu-1 insertions. Proc Natl Acad Sci U S A. 1975 Mar;72(3):809–813. doi: 10.1073/pnas.72.3.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  10. Chaconas G., de Bruijn F. J., Casadaban M. J., Lupski J. R., Kwoh T. J., Harshey R. M., DuBow M. S., Bukhari A. I. In vitro and in vivo manipulations of bacteriophage Mu DNA: cloning of Mu ends and construction of mini-Mu's carrying selectable markers. Gene. 1981 Jan-Feb;13(1):37–46. doi: 10.1016/0378-1119(81)90041-x. [DOI] [PubMed] [Google Scholar]
  11. Chou J., Casadaban M. J., Lemaux P. G., Cohen S. N. Identification and characterization of a self-regulated repressor of translocation of the Tn3 element. Proc Natl Acad Sci U S A. 1979 Aug;76(8):4020–4024. doi: 10.1073/pnas.76.8.4020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chou J., Lemaux P. G., Casadaban M. J., Cohen S. N. Transposition protein of Tn3: identification and characterisation of an essential repressor-controlled gene product. Nature. 1979 Dec 20;282(5741):801–806. doi: 10.1038/282801a0. [DOI] [PubMed] [Google Scholar]
  13. Csonka L. N., Howe M. M., Ingraham J. L., Pierson L. S., 3rd, Turnbough C. L., Jr Infection of Salmonella typhimurium with coliphage Mu d1 (Apr lac): construction of pyr::lac gene fusions. J Bacteriol. 1981 Jan;145(1):299–305. doi: 10.1128/jb.145.1.299-305.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ditto M. D., Chou J., Hunkapiller M. W., Fennewald M. A., Gerrard S. P., Hood L. E., Cohen S. N., Casadaban M. J. Amino-terminal sequence of the Tn3 transposase protein. J Bacteriol. 1982 Jan;149(1):407–410. doi: 10.1128/jb.149.1.407-410.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dixon R., Eady R. R., Espin G., Hill S., Iaccarino M., Kahn D., Merrick M. Analysis of regulation of Klebsiella pneumoniae nitrogen fixation (nif) gene cluster with gene fusions. Nature. 1980 Jul 10;286(5769):128–132. doi: 10.1038/286128a0. [DOI] [PubMed] [Google Scholar]
  16. Engebrecht J., Nealson K., Silverman M. Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri. Cell. 1983 Mar;32(3):773–781. doi: 10.1016/0092-8674(83)90063-6. [DOI] [PubMed] [Google Scholar]
  17. Faelen M., Toussaint A. Bacteriophage Mu-1: a tool to transpose and to localize bacterial genes. J Mol Biol. 1976 Jul 5;104(3):525–539. doi: 10.1016/0022-2836(76)90118-2. [DOI] [PubMed] [Google Scholar]
  18. Fowler A. V., Zabin I. The amino acid sequence of beta-galactosidase of Escherichia coli. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1507–1510. doi: 10.1073/pnas.74.4.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Guarente L., Lauer G., Roberts T. M., Ptashne M. Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit beta-globin. Cell. 1980 Jun;20(2):543–553. doi: 10.1016/0092-8674(80)90640-6. [DOI] [PubMed] [Google Scholar]
  20. Hall M. N., Silhavy T. J. Genetic analysis of the major outer membrane proteins of Escherichia coli. Annu Rev Genet. 1981;15:91–142. doi: 10.1146/annurev.ge.15.120181.000515. [DOI] [PubMed] [Google Scholar]
  21. Howe M. M. Prophage deletion mapping of bacteriophage Mu-1. Virology. 1973 Jul;54(1):93–101. doi: 10.1016/0042-6822(73)90118-9. [DOI] [PubMed] [Google Scholar]
  22. Howe M. M. Transduction by bacteriophage MU-1. Virology. 1973 Sep;55(1):103–117. doi: 10.1016/s0042-6822(73)81012-8. [DOI] [PubMed] [Google Scholar]
  23. Kahmann R., Kamp D. Nucleotide sequences of the attachment sites of bacteriophage Mu DNA. Nature. 1979 Jul 19;280(5719):247–250. doi: 10.1038/280247a0. [DOI] [PubMed] [Google Scholar]
  24. Kenyon C. J., Walker G. C. DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc Natl Acad Sci U S A. 1980 May;77(5):2819–2823. doi: 10.1073/pnas.77.5.2819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kolodrubetz D., Schleif R. L-arabinose transport systems in Escherichia coli K-12. J Bacteriol. 1981 Nov;148(2):472–479. doi: 10.1128/jb.148.2.472-479.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Komeda Y., Iino T. Regulation of expression of the flagellin gene (hag) in Escherichia coli K-12: analysis of hag-lac gene fusions. J Bacteriol. 1979 Sep;139(3):721–729. doi: 10.1128/jb.139.3.721-729.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Lee J. H., Heffernan L., Wilcox G. Isolation of ara-lac gene fusions in Salmonella typhimurium LT2 by using transducing bacteriophage Mu d (Apr lac). J Bacteriol. 1980 Sep;143(3):1325–1331. doi: 10.1128/jb.143.3.1325-1331.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meyer B. J., Maurer R., Ptashne M. Gene regulation at the right operator (OR) of bacteriophage lambda. II. OR1, OR2, and OR3: their roles in mediating the effects of repressor and cro. J Mol Biol. 1980 May 15;139(2):163–194. doi: 10.1016/0022-2836(80)90303-4. [DOI] [PubMed] [Google Scholar]
  30. Moreno F., Fowler A. V., Hall M., Silhavy T. J., Zabin I., Schwartz M. A signal sequence is not sufficient to lead beta-galactosidase out of the cytoplasm. Nature. 1980 Jul 24;286(5771):356–359. doi: 10.1038/286356a0. [DOI] [PubMed] [Google Scholar]
  31. Müller-Hill B., Kania J. Lac repressor can be fused to beta-galactosidase. Nature. 1974 Jun 7;249(457):561–563. doi: 10.1038/249561a0. [DOI] [PubMed] [Google Scholar]
  32. Müller-Hill B. Lac repressor and lac operator. Prog Biophys Mol Biol. 1975;30(2-3):227–252. doi: 10.1016/0079-6107(76)90011-0. [DOI] [PubMed] [Google Scholar]
  33. Ptashne M., Jeffrey A., Johnson A. D., Maurer R., Meyer B. J., Pabo C. O., Roberts T. M., Sauer R. T. How the lambda repressor and cro work. Cell. 1980 Jan;19(1):1–11. doi: 10.1016/0092-8674(80)90383-9. [DOI] [PubMed] [Google Scholar]
  34. Rosner J. L. Formation, induction, and curing of bacteriophage P1 lysogens. Virology. 1972 Jun;48(3):679–689. doi: 10.1016/0042-6822(72)90152-3. [DOI] [PubMed] [Google Scholar]
  35. Sarthy A., Fowler A., Zabin I., Beckwith J. Use of gene fusions to determine a partial signal sequence of alkaline phosphatase. J Bacteriol. 1979 Sep;139(3):932–939. doi: 10.1128/jb.139.3.932-939.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shuman H. A., Silhavy T. J., Beckwith J. R. Labeling of proteins with beta-galactosidase by gene fusion. Identification of a cytoplasmic membrane component of the Escherichia coli maltose transport system. J Biol Chem. 1980 Jan 10;255(1):168–174. [PubMed] [Google Scholar]
  37. Silhavy T. J., Casadaban M. J., Shuman H. A., Beckwith J. R. Conversion of beta-galactosidase to a membrane-bound state by gene fusion. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3423–3427. doi: 10.1073/pnas.73.10.3423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wanner B. L., Wieder S., McSharry R. Use of bacteriophage transposon Mu d1 to determine the orientation for three proC-linked phosphate-starvation-inducible (psi) genes in Escherichia coli K-12. J Bacteriol. 1981 Apr;146(1):93–101. doi: 10.1128/jb.146.1.93-101.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Welply J. K., Fowler A. V., Beckwith J. R., Zabin I. Positions of early nonsense and deletion mutations in lacZ. J Bacteriol. 1980 May;142(2):732–734. doi: 10.1128/jb.142.2.732-734.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]

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