Formation of the tandem repeat (IS30)2 and its role in IS30-mediated transpositional DNA rearrangements

Olasz, Ferenc; Stalder, Rolf; Arber, Werner

doi:10.1007/bf00281616

Cited by 69 publications

(95 citation statements)

References 35 publications

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“…1. Site-specific dimerization designates the reaction which leads to the formation of an (IS30)2 intermediate structure [2]. The structure of the transpositional products was verified by restriction analysis.…”

Section: The Effect Of Transcription Level On the Transpositional Frementioning

confidence: 98%

“…l); the compatible substrate replicons (Fig. 1C) carried different transpositional target sequences and produced no functional tranposase, (2) To obtain a higher level of expression and transpositional activity, and to be able to investigate the correlation between them, the original promoter of the ORF-A was replaced with the inducible tac promoter in the transposase producer plasmid pJKIll0 and pJKI132 (Fig. 1B).…”

Section: The Construction Of the Systemmentioning

confidence: 99%

“…1A) is a low copy number mobile genetic element residential in the K-12 and C strains of Escherichia coli [1]. The transposition of IS30 proceeds in a conservative pathway, via an intermediate that consists of two elements joining to each other with their left and right terminal inverted repeats (IR) [2]. The highly unstable (IS30)2 intermediate is formed by means of site-specific dimerization and mediates further DNA rearrangements characteristic of the element.…”

Section: Introductionmentioning

confidence: 99%

“…IS30 appears to be bifunctional as it catalyzes site-specific reactions (dimerization) and 'classical' transpositional events: simple insertion, deletion, inversion and transpositional fusion (cointegration). IS30 has a defined target specificity: natural hot spot sequences were described from lambda and P1 bacteriophages [2], but its own inverted repeats are also preferred targets. The 1221 bp long element contains one large open reading frame (ORF-A, bp 63-1211) coding for the 44.3 kDa transposase protein ( Fig.…”

Section: Introductionmentioning

confidence: 99%

“…It was previously demonstrated that the genomic copies of IS30 were unable to complement the mutations in the transposase gene (Olasz et al, manuscript submitted) [2]. The lack of detectable complementation can be explained in two ways: (1) the amount of available transposase protein (synthesized *Corresponding author.…”

Section: Introductionmentioning

confidence: 99%

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The construction and characterization of an effective transpositional system based on IS30

1996

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We constructed an in vivo system to detect transpositional rearrangements induced by the insertion sequence IS30. The transposase protein expressed from the transposase producer plasmids catalyzed rearrangements on different target sequences presented in trans. High yields, up to 83%, of transpositional frequencies were observed. The frequency of rearrangements correlated with the amount of transposase protein produced and the attractivity of the target sequences. Alteration in the frequency of transposition was observed in the recA-E. coli strains JM109 and TG2. Remarkable structural and functional analogy was found with site-specific recombination systems.Key words: IS30; Overproduction of IS30 transposase; In vivo assay; Transpositional frequency; Escherichia coli by the genomic copies of the element) is too low to carry out transpositional reactions effectively; (2) the transposase protein can act only in cis, i.e. on the same replicon from which it was produced -as was shown in the case of Tn5 [7][8][9].To clarify this question we studied the in trans activity of the IS30 transposase in reactions characteristic of the element, and investigated the correlation between the expression level of IS30 transposase and the transpositional frequency. In our experiments the IS30 transposase was able to perform sitespecific and 'classical' transpositional reactions in trans (dimerization and deletion, respectively), at very high frequencies, up to 82.7%. The system described here represents a simple and useful tool in the isolation and characterization of mutants and in the development of an in vitro system.

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Section: The Effect Of Transcription Level On the Transpositional Frementioning

confidence: 98%

Section: The Construction Of the Systemmentioning

confidence: 99%