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. 1987 Oct;169(10):4426–4430. doi: 10.1128/jb.169.10.4426-4430.1987

Instability of Tn5 inserts in cyanobacterial cloning vectors.

S M Gendel 1
PMCID: PMC213803  PMID: 2820923

Abstract

Transposon Tn5 was used to produce random insertions in two hybrid cloning vectors for the unicellular cyanobacterium Anacystis nidulans. The transposon-containing plasmids were used to localize essential replication functions and to characterize the stability of large inserts in these vectors. The effect of the insertions on plasmid function was tested by transformation into a derivative of A. nidulans that had been cured of the endogenous plasmid used to construct the vectors. A region of approximately 4 kilobases was essential for successful plasmid transformation and replication. This region has also been shown to be involved in plasmid replication by deletion analysis. High rates of excision of Tn5 inserts within this region and restoration of normal replication function were observed when transformants were selected by using a resistance marker outside the replication region in the absence of selection for the transposon-coded kanamycin resistance. Transposon inserts outside this region were not deleted.

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Selected References

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

  1. Berg D. E., Egner C., Hirschel B. J., Howard J., Johnsrud L., Jorgensen R. A., Tlsty T. D. Insertion, excision, and inversion of Tn5. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):115–123. doi: 10.1101/sqb.1981.045.01.020. [DOI] [PubMed] [Google Scholar]
  2. Buzby J. S., Porter R. D., Stevens S. E., Jr Plasmid transformation in Agmenellum quadruplicatum PR-6: construction of biphasic plasmids and characterization of their transformation properties. J Bacteriol. 1983 Jun;154(3):1446–1450. doi: 10.1128/jb.154.3.1446-1450.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chauvat F., Astier C., Vedel F., Joset-Espardellier F. Transformation in the cyanobacterium Synechococcus R2: improvement of efficiency; role of the pUH24 plasmid. Mol Gen Genet. 1983;191(1):39–45. doi: 10.1007/BF00330887. [DOI] [PubMed] [Google Scholar]
  4. Dzelzkalns V. A., Bogorad L. Stable transformation of the cyanobacterium Synechocystis sp. PCC 6803 induced by UV irradiation. J Bacteriol. 1986 Mar;165(3):964–971. doi: 10.1128/jb.165.3.964-971.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gendel S., Straus N., Pulleyblank D., Williams J. Shuttle cloning vectors for the cyanobacterium Anacystis nidulans. J Bacteriol. 1983 Oct;156(1):148–154. doi: 10.1128/jb.156.1.148-154.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Golden S. S., Sherman L. A. Optimal conditions for genetic transformation of the cyanobacterium Anacystis nidulans R2. J Bacteriol. 1984 Apr;158(1):36–42. doi: 10.1128/jb.158.1.36-42.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Kuhlemeier C. J., Borrias W. E., van den Hondel C. A., van Arkel G. A. Vectors for cloning in cyanobacteria: construction and characterization of two recombinant plasmids capable of transformation of Escherichia coli K12 and Anacystis nidulans R2. Mol Gen Genet. 1981;184(2):249–254. doi: 10.1007/BF00272912. [DOI] [PubMed] [Google Scholar]
  9. Kuhlemeier C. J., Hardon E. M., van Arkel G. A., van de Vate C. Self-cloning in the cyanobacterium Anacystis nidulans R2: fate of a cloned gene after reintroduction. Plasmid. 1985 Nov;14(3):200–208. doi: 10.1016/0147-619x(85)90003-4. [DOI] [PubMed] [Google Scholar]
  10. Kuhlemeier C. J., Thomas A. A., van der Ende A., van Leen R. W., Borrias W. E., van den Hondel C. A., van Arkel G. A. A host-vector system for gene cloning in the cyanobacterium Anacystis nidulans R2. Plasmid. 1983 Sep;10(2):156–163. doi: 10.1016/0147-619x(83)90068-9. [DOI] [PubMed] [Google Scholar]
  11. O'Neill E. A., Kiely G. M., Bender R. A. Transposon Tn5 encodes streptomycin resistance in nonenteric bacteria. J Bacteriol. 1984 Jul;159(1):388–389. doi: 10.1128/jb.159.1.388-389.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ruvkun G. B., Ausubel F. M. A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85–88. doi: 10.1038/289085a0. [DOI] [PubMed] [Google Scholar]
  13. Sherman L. A., van de Putte P. Construction of a hybrid plasmid capable of replication in the bacterium Escherichia coli and the cyanobacterium Anacystis nidulans. J Bacteriol. 1982 Apr;150(1):410–413. doi: 10.1128/jb.150.1.410-413.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Wolk C. P., Vonshak A., Kehoe P., Elhai J. Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1561–1565. doi: 10.1073/pnas.81.5.1561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. van den Hondel C. A., Verbeek S., van der Ende A., Weisbeek P. J., Borrias W. E., van Arkel G. A. Introduction of transposon Tn901 into a plasmid of Anacystis nidulans: preparation for cloning in cyanobacteria. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1570–1574. doi: 10.1073/pnas.77.3.1570. [DOI] [PMC free article] [PubMed] [Google Scholar]

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