1887

Abstract

Derivatives of IS the A3(2) 2·6 kb minicircle, transpose efficiently in targeting chromosomal sites resembling translation start signals. Two IS derivatives, plJ4696 and plJ4697, containing a hygromycin-resistance gene in opposite orientations were introduced into by electroporation and found to integrate into one of three specific sites. Integrations at sites A and B were frequent while integration at site C was observed only once. Only one site was occupied in each transformant. Sites A and B had either single or tandem integrations. PFGE analysis located these sites on different genomic fragments. The sequences of the chromosome-IS junctions confirmed that integration was via the same IS attachment site as in that there was no target site duplication, and that the orientation of IS at each site was fixed. In contrast to the situation in no deletions were created by the transposition and no circular forms could be detected. Comparison of the three chromosomal IS target sites ( ) with known primary and secondary sites showed that only a 2 bp ‘AG’ sequence at the crossover point was conserved. Dividing the sites into two groups produced two longer consensus target sites, GtcAAGg and gCCGATAGg. Most of the IS target sites resemble translational start sites, and site C resembles strongly the amino-terminal sequence of a aminopeptidase. The level of hygromycin resistance in the transformants was high and independent of the site of integration, the number of copies integrated, or the orientation of the gene. plJ4696 at all three sites was stable in in the absence of selection for at least 60 cell divisions. plJ4696, plJ4697 and other IS derivatives are promising vectors for the stable, integrative cloning of genes in

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/content/journal/micro/10.1099/13500872-145-5-1201
1999-05-01
2019-10-16
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http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-145-5-1201
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