1887

Abstract

Excision of the transposon Tn from sites of insertion in plasmid DNA was shown to occur at high frequency in . Plasmids with Tn insertions conjugated poorly into and adversely affected growth compared to the respective parental plasmids. The kanamycin-resistance phenotype of Tn5 was expressed poorly in and kanamycin-sensitive strains were common during the manipulation of the transconjugants. Examination of plasmid DNA isolated from kanamycin-sensitive transconjugants revealed excision of Tn5 sequences. A plasmid containing a selectable marker (mercury resistance) inactivated by a Tn insertion was constructed, and Tn5 excised precisely, permitting the expression of the mercury-resistance marker at high frequency (10) in and at the expected low frequency (10) in . The recombinational mechanism that promotes frequent Tn excision in operated in the absence of the gene product. Fragments of Tn were also examined for excision and instability in . A plasmid containing the terminal 485 bp of inverted repeat sequences from Tn5, but lacking the transposase or kanamycin-resistance genes, also showed precise excision of Tn DNA at high frequency (10) in . Unlike plasmids containing a complete Tn insertion, this plasmid transferred to at normal frequencies and growth of the host was not severely impaired. In contrast, plasmids containing either IS50 element transferred to at greatly reduced frequencies, and transconjugants containing the IS50R element (which contains the active transposase gene) were small and especially difficult to maintain. transconjugants harbouring a plasmid containing only the DNA between the IS elements (which included the kanamycin-resistance gene) were of normal size and stably maintained. These observations suggest that frequent and precise excision of Tn in required the long inverted repeat sequences at the termini of Tn5. The adverse effects conferred by IS50 on the transconjugant formation and growth of were apparently not required to promote Tn excision.

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1990-05-01
2021-05-12
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