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. 1994 Mar;176(5):1332–1338. doi: 10.1128/jb.176.5.1332-1338.1994

Inversions and deletions generated by a mini-gamma delta (Tn1000) transposon.

G Wang 1, X Xu 1, J M Chen 1, D E Berg 1, C M Berg 1
PMCID: PMC205197  PMID: 8113172

Abstract

Intramolecular transposition by an engineered derivative of the transposon gamma delta (Tn1000) is described. This 1-kb element contains inverted repeats of the 40 bp of the delta end of gamma delta, bracketing a kan gene, but it contains no resolution site. Transposition was analyzed in two plasmids; one contained two contraselectable (conditional lethal) genes (thyA and sacB) adjacent to the mini-gamma delta element in a 13.0-kb pBR322/pUC-based two-component plasmid (a heterodimer), and the other contained a different contraselectable gene (strA [rpsL]) in a 13.2-kb three-component plasmid (a heterotrimer). Selection for loss of function of a single contraselectable gene yielded inversions and deletions. Each inversion plasmid was 1 kb larger than the parent plasmid: it had a second copy of mini-gamma delta inserted in the contraselected gene, with that copy plus the intervening segment inverted, and the 5-bp target site duplicated. Each deletion plasmid was smaller than the parent plasmid and had a deletion that extended from one transposon end into or through the contraselected gene for distances of up to 9.4 kb. The frequencies of deletions versus inversions ending in a single target gene were similar, although overall, deletions outnumbered inversions because deletions, but not inversions, into sites beyond the contraselected gene inactivate it. This work also demonstrates that thyA (which encodes thymidylate synthetase) is a useful contraselectable marker.

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Selected References

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  1. Berg C. M., Vartak N. B., Wang G., Xu X., Liu L., MacNeil D. J., Gewain K. M., Wiater L. A., Berg D. E. The m gamma delta-1 element, a small gamma delta (Tn1000) derivative useful for plasmid mutagenesis, allele replacement and DNA sequencing. Gene. 1992 Apr 1;113(1):9–16. doi: 10.1016/0378-1119(92)90664-b. [DOI] [PubMed] [Google Scholar]
  2. Berg C. M., Wang G., Strausbaugh L. D., Berg D. E. Transposon-facilitated sequencing of DNAs cloned in plasmids. Methods Enzymol. 1993;218:279–306. doi: 10.1016/0076-6879(93)18022-5. [DOI] [PubMed] [Google Scholar]
  3. Bishop R., Sherratt D. Transposon Tn1 intra-molecular transposition. Mol Gen Genet. 1984;196(1):117–122. doi: 10.1007/BF00334102. [DOI] [PubMed] [Google Scholar]
  4. Dean D. A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids. Gene. 1981 Oct;15(1):99–102. doi: 10.1016/0378-1119(81)90108-6. [DOI] [PubMed] [Google Scholar]
  5. Deonier R. C., Yun K., Kuppermann M. Gamma delta-mediated deletions of chromosomal segments on F-prime plasmids. Mol Gen Genet. 1983;190(1):42–50. doi: 10.1007/BF00330322. [DOI] [PubMed] [Google Scholar]
  6. Gay P., Le Coq D., Steinmetz M., Berkelman T., Kado C. I. Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol. 1985 Nov;164(2):918–921. doi: 10.1128/jb.164.2.918-921.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Grant S. G., Jessee J., Bloom F. R., Hanahan D. Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4645–4649. doi: 10.1073/pnas.87.12.4645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Guyer M. S. Uses of the transposon gamma delta in the analysis of cloned genes. Methods Enzymol. 1983;101:362–369. doi: 10.1016/0076-6879(83)01027-7. [DOI] [PubMed] [Google Scholar]
  9. Klaer R., Kühn S., Tillmann E., Fritz H. J., Starlinger P. The sequence of IS4. Mol Gen Genet. 1981;181(2):169–175. doi: 10.1007/BF00268423. [DOI] [PubMed] [Google Scholar]
  10. Krishnan B. R., Blakesley R. W., Berg D. E. Linear amplification DNA sequencing directly from single phage plaques and bacterial colonies. Nucleic Acids Res. 1991 Mar 11;19(5):1153–1153. doi: 10.1093/nar/19.5.1153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Oka A., Sugisaki H., Takanami M. Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol. 1981 Apr 5;147(2):217–226. doi: 10.1016/0022-2836(81)90438-1. [DOI] [PubMed] [Google Scholar]
  12. Pinter K., Davisson V. J., Santi D. V. Cloning, sequencing, and expression of the Lactobacillus casei thymidylate synthase gene. DNA. 1988 May;7(4):235–241. doi: 10.1089/dna.1988.7.235. [DOI] [PubMed] [Google Scholar]
  13. Prentki P., Teter B., Chandler M., Galas D. J. Functional promoters created by the insertion of transposable element IS1. J Mol Biol. 1986 Oct 5;191(3):383–393. doi: 10.1016/0022-2836(86)90134-8. [DOI] [PubMed] [Google Scholar]
  14. STACEY K. A., SIMSON E. IMPROVED METHOD FOR THE ISOLATION OF THYMINE-REQUIRING MUTANTS OF ESCHERICHIA COLI. J Bacteriol. 1965 Aug;90:554–555. doi: 10.1128/jb.90.2.554-555.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Tomcsanyi T., Berg C. M., Phadnis S. H., Berg D. E. Intramolecular transposition by a synthetic IS50 (Tn5) derivative. J Bacteriol. 1990 Nov;172(11):6348–6354. doi: 10.1128/jb.172.11.6348-6354.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Tsai M. M., Wong R. Y., Hoang A. T., Deonier R. C. Transposition of Tn1000: in vivo properties. J Bacteriol. 1987 Dec;169(12):5556–5562. doi: 10.1128/jb.169.12.5556-5562.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wang G., Blakesley R. W., Berg D. E., Berg C. M. pDUAL: a transposon-based cosmid cloning vector for generating nested deletions and DNA sequencing templates in vivo. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7874–7878. doi: 10.1073/pnas.90.16.7874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Weinert T. A., Schaus N. A., Grindley N. D. Insertion sequence duplication in transpositional recombination. Science. 1983 Nov 18;222(4625):755–765. doi: 10.1126/science.6314502. [DOI] [PubMed] [Google Scholar]
  19. Wiater L. A., Grindley N. D. Integration host factor increases the transpositional immunity conferred by gamma delta ends. J Bacteriol. 1990 Sep;172(9):4951–4958. doi: 10.1128/jb.172.9.4951-4958.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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