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Volume 147, Issue 2

A novel rolling-circle-replicating plasmid from P8: molecular characterization and use as vector

The EMBL and GenBank accession number for the nucleotide sequence of pPP8–1 is AJ289784.

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Abstract

In P8, three cryptic circular plasmids were detected, i.e. pPP8-1 (25 kbp), pPP8-2 (42 kbp) and pPP8-3 (100 kbp). Cloning and complete sequencing of pPP8-1 revealed a 2534 bp element harbouring four open reading frames (ORFs A, B, C and D). No function could be attributed to the latter three ORFs, whereas the predicted ORF A gene product is homologous to replication proteins known from small multicopy plasmids of Gram-positive bacteria and single-stranded (ss) phages, genetic elements replicating via a rolling circle (RC) mechanism involving characteristic ssDNA intermediates. Consistently, a double-strand origin of replication, highly conserved in rolling-circle-replicating (RCR) elements, was identified in pPP8-1, along with a putative single-strand origin. Beyond this, ss replication intermediates were confirmed by Southern analysis and mungbean-nuclease digestion. This being the first element of this type known in pseudomonads, a kanamycin-resistance gene was ligated into pPP8-1 and the resulting vector was successfully used for the transformation of both and

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Keyword(s): cloning , cryptic plasmid , ds, double-stranded; , DSO, double-strand origin; , extrachromosomal element , IR, imperfect repeat; , KmR, kanamycin resistance; , pP81–1 , RC, rolling circle; , RCR, rolling circle replicating; , repA , S/D, Shine–Dalgarno; , ss, single-stranded; , SSB, single-strand DNA-binding protein; and SSO, single-strand origin.
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2001-02-01
2025-11-14

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/content/journal/micro/10.1099/00221287-147-2-337
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A novel rolling-circle-replicating plasmid from Pseudomonas putida P8: molecular characterization and use as vectorThe EMBL and GenBank accession number for the nucleotide sequence of pPP8–1 is AJ289784.
Microbiology 147, 337 (2001); https://doi.org/10.1099/00221287-147-2-337
/content/journal/micro/10.1099/00221287-147-2-337
/content/journal/micro/10.1099/00221287-147-2-337
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