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. 1987 May;169(5):2026–2030. doi: 10.1128/jb.169.5.2026-2030.1987

Molecular cloning of Mu d(bla lacZ) transcriptional and translational fusions.

B L Wanner
PMCID: PMC212079  PMID: 3032905

Abstract

The vector pBW2 was made to selectively clone chimeric plasmids with chromosomal Mu d(bla lacZ) transcriptional or translational fusions. It was tetracycline resistant and had the carboxyl-terminal end of bla distal to its PstI site. Because ligation of PstI-digested chromosomal DNA of a Mu d(bla lacZ) insertion with pBW2 restored bla, ampicillin-resistant chimeric plasmids were selectable. These plasmids had the Mu d bla amino terminus and simultaneously acquired other Mu d sequences including lacZ, the chromosomal fusion joint, and the DNA adjacent to the nearest chromosomal PstI site. The plasmid pBW2 was useful in the molecular cloning of several psi and pho::lacZ(Mu d) fusions, as well as chromosomal genes located near Mu d insertions.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baker T. A., Howe M. M., Gross C. A. Mu dX, a derivative of Mu d1 (lac Apr) which makes stable lacZ fusions at high temperature. J Bacteriol. 1983 Nov;156(2):970–974. doi: 10.1128/jb.156.2.970-974.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bremer E., Silhavy T. J., Weisemann J. M., Weinstock G. M. Lambda placMu: a transposable derivative of bacteriophage lambda for creating lacZ protein fusions in a single step. J Bacteriol. 1984 Jun;158(3):1084–1093. doi: 10.1128/jb.158.3.1084-1093.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Castilho B. A., Olfson P., Casadaban M. J. Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons. J Bacteriol. 1984 May;158(2):488–495. doi: 10.1128/jb.158.2.488-495.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Close T. J., Rodriguez R. L. Construction and characterization of the chloramphenicol-resistance gene cartridge: a new approach to the transcriptional mapping of extrachromosomal elements. Gene. 1982 Dec;20(2):305–316. doi: 10.1016/0378-1119(82)90048-8. [DOI] [PubMed] [Google Scholar]
  9. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  10. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  11. Froehlich B., Epstein W. Escherichia coli mutants in which transcription is dependent on recA function. J Bacteriol. 1981 Sep;147(3):1117–1120. doi: 10.1128/jb.147.3.1117-1120.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Groisman E. A., Castilho B. A., Casadaban M. J. In vivo DNA cloning and adjacent gene fusing with a mini-Mu-lac bacteriophage containing a plasmid replicon. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1480–1483. doi: 10.1073/pnas.81.5.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Guan C. D., Wanner B., Inouye H. Analysis of regulation of phoB expression using a phoB-cat fusion. J Bacteriol. 1983 Nov;156(2):710–717. doi: 10.1128/jb.156.2.710-717.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harshey R. M., Getzoff E. D., Baldwin D. L., Miller J. L., Chaconas G. Primary structure of phage mu transposase: homology to mu repressor. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7676–7680. doi: 10.1073/pnas.82.22.7676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hediger M. A., Johnson D. F., Nierlich D. P., Zabin I. DNA sequence of the lactose operon: the lacA gene and the transcriptional termination region. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6414–6418. doi: 10.1073/pnas.82.19.6414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Higgins N. P., Moncecchi D., Manlapaz-Ramos P., Olivera B. M. Bacteriophage Mu DNA replication in vitro. J Biol Chem. 1983 Apr 10;258(7):4293–4297. [PubMed] [Google Scholar]
  18. Hughes K. T., Roth J. R. Conditionally transposition-defective derivative of Mu d1(Amp Lac). J Bacteriol. 1984 Jul;159(1):130–137. doi: 10.1128/jb.159.1.130-137.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Kieser T. Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid. 1984 Jul;12(1):19–36. doi: 10.1016/0147-619x(84)90063-5. [DOI] [PubMed] [Google Scholar]
  21. Kroos L., Kaiser D. Construction of Tn5 lac, a transposon that fuses lacZ expression to exogenous promoters, and its introduction into Myxococcus xanthus. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5816–5820. doi: 10.1073/pnas.81.18.5816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Linn T., Ralling G. A versatile multiple- and single-copy vector system for the in vitro construction of transcriptional fusions to lacZ. Plasmid. 1985 Sep;14(2):134–142. doi: 10.1016/0147-619x(85)90073-3. [DOI] [PubMed] [Google Scholar]
  23. Manoil C., Beckwith J. A genetic approach to analyzing membrane protein topology. Science. 1986 Sep 26;233(4771):1403–1408. doi: 10.1126/science.3529391. [DOI] [PubMed] [Google Scholar]
  24. McKenney K., Shimatake H., Court D., Schmeissner U., Brady C., Rosenberg M. A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase. Gene Amplif Anal. 1981;2:383–415. [PubMed] [Google Scholar]
  25. Miller J. L., Anderson S. K., Fujita D. J., Chaconas G., Baldwin D. L., Harshey R. M. The nucleotide sequence of the B gene of bacteriophage Mu. Nucleic Acids Res. 1984 Nov 26;12(22):8627–8638. doi: 10.1093/nar/12.22.8627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nakai C., Kagamiyama H., Nozaki M., Nakazawa T., Inouye S., Ebina Y., Nakazawa A. Complete nucleotide sequence of the metapyrocatechase gene on the TOI plasmid of Pseudomonas putida mt-2. J Biol Chem. 1983 Mar 10;258(5):2923–2928. [PubMed] [Google Scholar]
  27. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Peden K. W. Revised sequence of the tetracycline-resistance gene of pBR322. Gene. 1983 May-Jun;22(2-3):277–280. doi: 10.1016/0378-1119(83)90112-9. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Shapira S. K., Chou J., Richaud F. V., Casadaban M. J. New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase. Gene. 1983 Nov;25(1):71–82. doi: 10.1016/0378-1119(83)90169-5. [DOI] [PubMed] [Google Scholar]
  32. Stachel S. E., An G., Flores C., Nester E. W. A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J. 1985 Apr;4(4):891–898. doi: 10.1002/j.1460-2075.1985.tb03715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Wanner B. L. Bacterial alkaline phosphatase clonal variation in some Escherichia coli K-12 phoR mutant strains. J Bacteriol. 1986 Dec;168(3):1366–1371. doi: 10.1128/jb.168.3.1366-1371.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wanner B. L. Control of phoR-dependent bacterial alkaline phosphatase clonal variation by the phoM region. J Bacteriol. 1987 Feb;169(2):900–903. doi: 10.1128/jb.169.2.900-903.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wanner B. L., McSharry R. Phosphate-controlled gene expression in Escherichia coli K12 using Mudl-directed lacZ fusions. J Mol Biol. 1982 Jul 5;158(3):347–363. doi: 10.1016/0022-2836(82)90202-9. [DOI] [PubMed] [Google Scholar]
  37. Wanner B. L. Novel regulatory mutants of the phosphate regulon in Escherichia coli K-12. J Mol Biol. 1986 Sep 5;191(1):39–58. doi: 10.1016/0022-2836(86)90421-3. [DOI] [PubMed] [Google Scholar]
  38. Wanner B. L. Overlapping and separate controls on the phosphate regulon in Escherichia coli K12. J Mol Biol. 1983 May 25;166(3):283–308. doi: 10.1016/s0022-2836(83)80086-2. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Weinstock G. M., Berman M. L., Silhavy T. J. Chimeric genetics with beta-galactosidase. Gene Amplif Anal. 1983;3:27–64. [PubMed] [Google Scholar]
  41. Yanofsky C., Platt T., Crawford I. P., Nichols B. P., Christie G. E., Horowitz H., VanCleemput M., Wu A. M. The complete nucleotide sequence of the tryptophan operon of Escherichia coli. Nucleic Acids Res. 1981 Dec 21;9(24):6647–6668. doi: 10.1093/nar/9.24.6647. [DOI] [PMC free article] [PubMed] [Google Scholar]

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