A New Generalizable Test for Detection of Mutations Affecting Tn10 Transposition (original) (raw)

Abstract

We describe here a new rapid screen that allows easy detection of transposon or host mutations that affect Tn_10_ transposition in Escherichia coli. This test involves a new Tn_10_ derivative called the "mini-lacZ-kanR fusion hopper" or mini-Tn_10_-LK for short. This element does not direct expression of β-galactosidase when present at its original starting location on a suitably engineered plasmid or phage genome because it lacks appropriate transcription and translation start signals. However, transposition of this element into the chromosome of E. coli lacZ- bacteria leads to productive fusions in which the lacZ gene within the transposon is expressed from external chromosomal signals. Such fusions are readily detectable on MacConkey lactose indicator plates as red (Lac+) papillae inside of white (LacZ- ) colonies. The length of time required to see red papillae appearing in a white colony sensitively and accurately reflects the transposition frequency of the mini-transposon within the colonies. Differences in times for color formation are sensitive enough that 10-fold differences in transposition frequency can readily be detected. This papillation assay can be used to identify mutant clones in which the frequency of Tn_10_ transposition is either increased or decreased. We have successfully used the assay to identify mutations in the terminal sequences of Tn_10_; mutations in the Tn_10_ transposase gene or the bacterial host can be isolated just as easily. This screen should be readily adaptable to transposable elements other than Tn_10_.

Full Text

The Full Text of this article is available as a PDF (560.3 KB).

Selected References

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

  1. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  4. Foster T. J., Davis M. A., Roberts D. E., Takeshita K., Kleckner N. Genetic organization of transposon Tn10. Cell. 1981 Jan;23(1):201–213. doi: 10.1016/0092-8674(81)90285-3. [DOI] [PubMed] [Google Scholar]
  5. Huisman O., Raymond W., Froehlich K. U., Errada P., Kleckner N., Botstein D., Hoyt M. A. A Tn10-lacZ-kanR-URA3 gene fusion transposon for insertion mutagenesis and fusion analysis of yeast and bacterial genes. Genetics. 1987 Jun;116(2):191–199. doi: 10.1093/genetics/116.2.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kleckner N. Transposable elements in prokaryotes. Annu Rev Genet. 1981;15:341–404. doi: 10.1146/annurev.ge.15.120181.002013. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Maurer R., Meyer B., Ptashne M. Gene regulation at the right operator (OR) bacteriophage lambda. I. OR3 and autogenous negative control by repressor. J Mol Biol. 1980 May 15;139(2):147–161. doi: 10.1016/0022-2836(80)90302-2. [DOI] [PubMed] [Google Scholar]
  9. Morisato D., Way J. C., Kim H. J., Kleckner N. Tn10 transposase acts preferentially on nearby transposon ends in vivo. Cell. 1983 Mar;32(3):799–807. doi: 10.1016/0092-8674(83)90066-1. [DOI] [PubMed] [Google Scholar]
  10. Roberts D., Hoopes B. C., McClure W. R., Kleckner N. IS10 transposition is regulated by DNA adenine methylation. Cell. 1985 Nov;43(1):117–130. doi: 10.1016/0092-8674(85)90017-0. [DOI] [PubMed] [Google Scholar]
  11. Simons R. W., Hoopes B. C., McClure W. R., Kleckner N. Three promoters near the termini of IS10: pIN, pOUT, and pIII. Cell. 1983 Sep;34(2):673–682. doi: 10.1016/0092-8674(83)90400-2. [DOI] [PubMed] [Google Scholar]
  12. Way J. C., Davis M. A., Morisato D., Roberts D. E., Kleckner N. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene. 1984 Dec;32(3):369–379. doi: 10.1016/0378-1119(84)90012-x. [DOI] [PubMed] [Google Scholar]
  13. Way J. C., Kleckner N. Essential sites at transposon Tn 10 termini. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3452–3456. doi: 10.1073/pnas.81.11.3452. [DOI] [PMC free article] [PubMed] [Google Scholar]