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Transposition of IS 117 , the 2.5 kb Streptomyces coelicolor A3(2) ‘minicircle’: roles of open reading frames and origin of tandem insertions
Author(s) -
Smokvina Tamara,
Henderson Duncan J.,
Melton Rachel E.,
Brolle DirkF,
Kieser Tobias,
Hopwood David A.
Publication year - 1994
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1994.tb01034.x
Subject(s) - biology , transposase , streptomyces coelicolor , tn10 , transposable element , transposition (logic) , plasmid , minicircle , genetics , insertion sequence , mutant , actinorhodin , transformation (genetics) , insert (composites) , insertional mutagenesis , dna , gene , linguistics , philosophy , mechanical engineering , engineering
Summary IS 117 is a 2527 bp transposable element from Streptomyces coelicolor A3(2) with a circular transposition intermediate. Disruption of 0RF1 of IS 117 , presumed to encode a transposase, abolished transposition. Deletion or mutation of 0RF2 and 0RF3, which overlap each other on opposite strands of IS 117, caused a c. 20‐fold reduction in integration frequency of the circular form of IS 117 into the Streptomyces lividans chromosome or into the preferred chromosomal target site cloned on a plasmid in transformation experiments. In contrast, inactivation of ORF2/3 did not significantly influence transposition of IS 117 derivatives from an already integrated state in the chromosome to the preferred target site cloned on a plasmid. 0RF2 mutants apparently excised readily from the S. lividans chromosome, whereas excision of integrated wild‐type IS 117 derivatives to yield the unoccupied site was not detected; presumably, therefore, the circular transposition intermediate normally arises replicatively. Attempts to promote integration of a plasmid carrying the attachment site of IS 117 by providing the ORF1 product in trans were unsuccessful. Most transformation of S. lividans with circular IS 117 derivatives yielded tandem chromosomal insertions, which arose by co‐transformation rather than dimerization of a monomeric insert. Typically, two to three transforming elements gave a transformed strain, suggesting a local concentration of transposase as a limit on integration.