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. 1984 May;158(2):488–495. doi: 10.1128/jb.158.2.488-495.1984

Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons.

B A Castilho, P Olfson, M J Casadaban
PMCID: PMC215454  PMID: 6327606

Abstract

Small bacteriophage Mu transposable elements containing the lac operon structural genes were constructed to facilitate the isolation and use of Mu insertions and lac gene fusions. These mini-Mu elements have selectable genes for either ampicillin or kanamycin resistance and can be used to form both transcriptional and translational lac gene fusions. Some of the mini-Mu-lac elements constructed are deleted for the Mu A and B transposition genes and form stable insertions that cannot undergo transposition unless complemented for these functions. A procedure was developed for selecting mini-Mu insertions specifically into plasmids, including commonly used high-copy-number cloning vectors such as pBR322. Mu insertions in pBR322 were found to be distributed around the plasmid, but insertions in certain regions occurred more frequently than in others.

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Selected References

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  1. Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
  2. Berg D. E., Weiss A., Crossland L. Polarity of Tn5 insertion mutations in Escherichia coli. J Bacteriol. 1980 May;142(2):439–446. doi: 10.1128/jb.142.2.439-446.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bossi L., Ciampi M. S. DNA sequences at the sites of three insertions of the transposable element Tn5 in the histidine operon of Salmonella. Mol Gen Genet. 1981;183(2):406–408. doi: 10.1007/BF00270649. [DOI] [PubMed] [Google Scholar]
  4. Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
  5. Bukhari A. I. Bacteriophage mu as a transposition element. Annu Rev Genet. 1976;10:389–412. doi: 10.1146/annurev.ge.10.120176.002133. [DOI] [PubMed] [Google Scholar]
  6. Bukhari A. I., Taylor A. L. Influence of insertions on packaging of host sequences covalently linked to bacteriophage Mu DNA. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4399–4403. doi: 10.1073/pnas.72.11.4399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Büchel D. E., Gronenborn B., Müller-Hill B. Sequence of the lactose permease gene. Nature. 1980 Feb 7;283(5747):541–545. doi: 10.1038/283541a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Casadaban M. J., Chou J. In vivo formation of gene fusions encoding hybrid beta-galactosidase proteins in one step with a transposable Mu-lac transducing phage. Proc Natl Acad Sci U S A. 1984 Jan;81(2):535–539. doi: 10.1073/pnas.81.2.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. Casadaban M. J. Regulation of the regulatory gene for the arabinose pathway, araC. J Mol Biol. 1976 Jul 5;104(3):557–566. doi: 10.1016/0022-2836(76)90120-0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Cesareni G., Muesing M. A., Polisky B. Control of ColE1 DNA replication: the rop gene product negatively affects transcription from the replication primer promoter. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6313–6317. doi: 10.1073/pnas.79.20.6313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Cohen S. N., Chang A. C. Revised interpretation of the origin of the pSC101 plasmid. J Bacteriol. 1977 Nov;132(2):734–737. doi: 10.1128/jb.132.2.734-737.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Csonka L. N., Clark A. J. Deletions generated by the transposon Tn10 in the srl recA region of the Escherichia coli K-12 chromosome. Genetics. 1979 Oct;93(2):321–343. doi: 10.1093/genetics/93.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Daniell E., Roberts R., Abelson J. Mutations in the lactose operon caused by bacteriophage Mu. J Mol Biol. 1972 Aug 14;69(1):1–8. doi: 10.1016/0022-2836(72)90019-8. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Emr S. D., Silhavy T. J. Mutations affecting localization of an Escherichia coli outer membrane protein, the bacteriophage lambda receptor. J Mol Biol. 1980 Jul 25;141(1):63–90. doi: 10.1016/s0022-2836(80)80029-5. [DOI] [PubMed] [Google Scholar]
  24. Galas D. J., Calos M. P., Miller J. H. Sequence analysis of Tn9 insertions in the lacZ gene. J Mol Biol. 1980 Nov 25;144(1):19–41. doi: 10.1016/0022-2836(80)90213-2. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Halling S. M., Kleckner N. A symmetrical six-base-pair target site sequence determines Tn10 insertion specificity. Cell. 1982 Jan;28(1):155–163. doi: 10.1016/0092-8674(82)90385-3. [DOI] [PubMed] [Google Scholar]
  27. Heffron F., McCarthy B. J., Ohtsubo H., Ohtsubo E. DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3. Cell. 1979 Dec;18(4):1153–1163. doi: 10.1016/0092-8674(79)90228-9. [DOI] [PubMed] [Google Scholar]
  28. Howe M. M., Bade E. G. Molecular biology of bacteriophage mu. Science. 1975 Nov 14;190(4215):624–632. doi: 10.1126/science.1103291. [DOI] [PubMed] [Google Scholar]
  29. Inselburg J. Isolation and characterization of mutants of colicin plasmids E1 and E2 after Mu bacteriophage infection. J Bacteriol. 1974 Aug;119(2):469–477. doi: 10.1128/jb.119.2.469-477.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Kalnins A., Otto K., Rüther U., Müller-Hill B. Sequence of the lacZ gene of Escherichia coli. EMBO J. 1983;2(4):593–597. doi: 10.1002/j.1460-2075.1983.tb01468.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kania J., Müller-Hill B. Construction, isolation and implications of repressor-galactosidase - beta-galactosidase hybrid molecules. Eur J Biochem. 1977 Oct 3;79(2):381–386. doi: 10.1111/j.1432-1033.1977.tb11819.x. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Liebart J. C., Ghelardini P., Paolozzi L. Conservative integration of bacteriophage Mu DNA into pBR322 plasmid. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4362–4366. doi: 10.1073/pnas.79.14.4362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Maynard-Smith S., Leach D., Coelho A., Carey J., Symonds N. The isolation and characteristics of plasmids derived from the insertion of MupAp1 into pML2: their behavior during transposition. Plasmid. 1980 Jul;4(1):34–50. doi: 10.1016/0147-619x(80)90081-5. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. O'Connor M. B., Malamy M. H. A new insertion sequence, IS121, is found on the Mu dI1 (Ap lac) bacteriophage and the Escherichia coli K-12 chromosome. J Bacteriol. 1983 Nov;156(2):669–679. doi: 10.1128/jb.156.2.669-679.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Priess H., Kamp D., Kahmann R., Bräuer B., Delius H. Nucleotide sequence of the immunity region of bacteriophage Mu. Mol Gen Genet. 1982;186(3):315–321. doi: 10.1007/BF00729448. [DOI] [PubMed] [Google Scholar]
  40. Raibaud O., Roa M., Braun-Breton C., Schwartz M. Structure of the malB region in Escherichia coli K12. I. Genetic map of the malK-lamB operon. Mol Gen Genet. 1979 Jul 24;174(3):241–248. doi: 10.1007/BF00267796. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Rothstein S. J., Jorgensen R. A., Postle K., Reznikoff W. S. The inverted repeats of Tn5 are functionally different. Cell. 1980 Mar;19(3):795–805. doi: 10.1016/s0092-8674(80)80055-9. [DOI] [PubMed] [Google Scholar]
  43. Schumann W., Bade E. G. In vitro constructed plasmids containing both ends of bacteriophage Mu DNA express phage functions. Mol Gen Genet. 1979 Jan 16;169(1):97–105. doi: 10.1007/BF00267550. [DOI] [PubMed] [Google Scholar]
  44. Stüber D., Bujard H. Organization of transcriptional signals in plasmids pBR322 and pACYC184. Proc Natl Acad Sci U S A. 1981 Jan;78(1):167–171. doi: 10.1073/pnas.78.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  46. Tait R. C., Boyer H. W. On the nature of tetracycline resistance controlled by the plasmid pSC101. Cell. 1978 Jan;13(1):73–81. doi: 10.1016/0092-8674(78)90139-3. [DOI] [PubMed] [Google Scholar]
  47. Toussaint A., Faelen M., Résibois A. Chromosomal rearrangements induced by mini-Mu and mini-D108: mini review and new data. Gene. 1981 Jun-Jul;14(1-2):115–119. doi: 10.1016/0378-1119(81)90153-0. [DOI] [PubMed] [Google Scholar]
  48. Tu C. P., Cohen S. N. Translocation specificity of the Tn3 element: characterization of sites of multiple insertions. Cell. 1980 Jan;19(1):151–160. doi: 10.1016/0092-8674(80)90396-7. [DOI] [PubMed] [Google Scholar]
  49. de Mendoza D., Clark D., Cronan J. E., Jr One-step gene amplification by Mu-mediated transposition of E. coli genes to a multicopy plasmid. Gene. 1981 Oct;15(1):27–32. doi: 10.1016/0378-1119(81)90101-3. [DOI] [PubMed] [Google Scholar]

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