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Isolation and Characterization of a Rolling-Circle-Type Plasmid from Rhodococcus erythropolis and Application of the Plasmid to Multiple-Recombinant-Protein Expression. Nobutaka Nakashima, 2004.We isolated, sequenced, and characterized the cryptic plasmid pRE8424 from Rhodococcus erythropolis DSM8424 . Plasmid pRE8424 is a 5,987-bp circular plasmid; it carries six open reading frames and also contains cis-acting elements, specifically a single-stranded origin and a double-stranded origin, which are characteristic of rolling-circle-replication plasmids . Experiments with pRE8424 derivatives carrying a mutated single-stranded origin sequence showed that single-stranded DNA intermediates accumulated in the cells because of inefficient conversion from single-stranded DNA to double-stranded DNA . This result indicates that pRE8424 belongs to the pIJ101/pJV1 family of rolling-circle-replication plasmids . Expression vectors that are functional in several Rhodococcus species were constructed by use of the replication origin from pRE8424 . We previously reported a cryptic plasmid, pRE2895, from R . erythropolis, which may replicate by a  -type mechanism, like ColE2 plasmids . The new expression vectors originating from pRE8424 were compatible with those derived from pRE2895 . Coexpression experiments with these compatible expression vectors indicated that the plasmids are suitable for the simultaneous expression of multiple recombinant proteins .
Analysis of Genome Plasticity in Pathogenic and Commensal Escherichia coli Isolates by Use of DNA Arrays. Ulrich Dobrindt, 2003.Genomes of prokaryotes differ significantly in size and DNA composition . Escherichia coli is considered a model organism to analyze the processes involved in bacterial genome evolution, as the species comprises numerous pathogenic and commensal variants . Pathogenic and nonpathogenic E . coli strains differ in the presence and absence of additional DNA elements contributing to specific virulence traits and also in the presence and absence of additional genetic information . To analyze the genetic diversity of pathogenic and commensal E . coli isolates, a whole-genome approach was applied . Using DNA arrays, the presence of all translatable open reading frames (ORFs) of nonpathogenic E . coli K-12 strain MG1655 was investigated in 26 E . coli isolates, including various extraintestinal and intestinal pathogenic E . coli isolates, 3 pathogenicity island deletion mutants, and commensal and laboratory strains . Additionally, the presence of virulence-associated genes of E . coli was determined using a DNA "pathoarray" developed in our laboratory . The frequency and distributional pattern of genomic variations vary widely in different E . coli strains . Up to 10% of the E . coli K-12-specific ORFs were not detectable in the genomes of the different strains . DNA sequences described for extraintestinal or intestinal pathogenic E . coli are more frequently detectable in isolates of the same origin than in other pathotypes . Several genes coding for virulence or fitness factors are also present in commensal E . coli isolates . Based on these results, the conserved E . coli core genome is estimated to consist of at least 3,100 translatable ORFs . The absence of K-12-specific ORFs was detectable in all chromosomal regions . These data demonstrate the great genome heterogeneity and genetic diversity among E . coli strains and underline the fact that both the acquisition and deletion of DNA elements are important processes involved in the evolution of prokaryotes .
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