Formation of the tandem repeat (IS 30)2 and its role in IS 30-mediated transpositional DNA rearrangements

F. Olasz, Rolf Stalder, Werner Arber

Research output: Article

52 Citations (Scopus)

Abstract

Plasmids carrying two IS 30 elements in the same orientation, as in the composite transposon Tn 2706, are structurally unstable in Escherichia coli. A primary segregation product is formed by site-specific deletion of the sequences carried between the two IS 30 elements. The resulting covalently closed replicon carries the two IS 30 elements as tandem repeats separated by only 2 bp. This (IS 30)2 structure is extremely unstable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transformation. Among the descendants of transformants of recA- bacteria, replicated copies of the introduced (IS 30)2 structure are still present, together with various kinds of segregation products which provide evidence for the efficient generation of DNA rearrangements. Most abundant is the product of another site-specific recombination between two identical ends of the IS 30 elements involved, which results in the presence of just one intact IS 30 on the plasmid. Apart from this, and depending on the presence of appropriate targets for IS 30 transposition, various transposition products of (IS 30)2 are also seen. Intramolecular reactions lead to DNA inversions and deletions with breakpoints other than IS 30 ends. In intermolecular reactions inverse transposition occurs at high frequency and one also obtains simple transposition and cointegration. A mutational study revealed the requirement in cis of one intact IS 30 transposase gene and of both proximal ends of the two IS 30 elements concerned not only for the formation of (IS 30)2, but also for its further rearrangement reactions, including the efficient formation of site-specific deletions. A model is proposed, which postulates that (IS 30)2 intermediates play a key role in IS 30 transposition pathways in which the formation of (IS 30)2 may be rate-limiting. Once this structure is formed, it gives rise to a burst of transpositional rearrangements in the subclone carrying (IS 30)2. Evolutionary implications of these findings are discussed.

Original languageEnglish
Pages (from-to)177-187
Number of pages11
JournalMGG Molecular & General Genetics
Volume239
Issue number1-2
DOIs
Publication statusPublished - máj. 1993

Fingerprint

Tandem Repeat Sequences
DNA Transposable Elements
Gene Rearrangement
Plasmids
Transposases
Replicon
Sequence Deletion
Genetic Recombination
Escherichia coli
Bacteria
DNA
Genes

ASJC Scopus subject areas

  • Genetics

Cite this

Formation of the tandem repeat (IS 30)2 and its role in IS 30-mediated transpositional DNA rearrangements. / Olasz, F.; Stalder, Rolf; Arber, Werner.

In: MGG Molecular & General Genetics, Vol. 239, No. 1-2, 05.1993, p. 177-187.

Research output: Article

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AB - Plasmids carrying two IS 30 elements in the same orientation, as in the composite transposon Tn 2706, are structurally unstable in Escherichia coli. A primary segregation product is formed by site-specific deletion of the sequences carried between the two IS 30 elements. The resulting covalently closed replicon carries the two IS 30 elements as tandem repeats separated by only 2 bp. This (IS 30)2 structure is extremely unstable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transformation. Among the descendants of transformants of recA- bacteria, replicated copies of the introduced (IS 30)2 structure are still present, together with various kinds of segregation products which provide evidence for the efficient generation of DNA rearrangements. Most abundant is the product of another site-specific recombination between two identical ends of the IS 30 elements involved, which results in the presence of just one intact IS 30 on the plasmid. Apart from this, and depending on the presence of appropriate targets for IS 30 transposition, various transposition products of (IS 30)2 are also seen. Intramolecular reactions lead to DNA inversions and deletions with breakpoints other than IS 30 ends. In intermolecular reactions inverse transposition occurs at high frequency and one also obtains simple transposition and cointegration. A mutational study revealed the requirement in cis of one intact IS 30 transposase gene and of both proximal ends of the two IS 30 elements concerned not only for the formation of (IS 30)2, but also for its further rearrangement reactions, including the efficient formation of site-specific deletions. A model is proposed, which postulates that (IS 30)2 intermediates play a key role in IS 30 transposition pathways in which the formation of (IS 30)2 may be rate-limiting. Once this structure is formed, it gives rise to a burst of transpositional rearrangements in the subclone carrying (IS 30)2. Evolutionary implications of these findings are discussed.

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KW - Transposition mechanism

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