Recombination-Promoting Activity of the Bacteriophage λ Rap Protein in
            <i>Escherichia coli</i>
            K-12

Poteete, Anthony R.; Fenton, Anita C.; Wang, Hsinju R.

doi:10.1128/jb.184.16.4626-4629.2002

Cited by 12 publications

(16 citation statements)

References 18 publications

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“…Strain Y836 recombination, replication, and repair in E. coli. Orf (infected host drawn in Figure 1B) carries a cryptic prophage, suppresses the mutant phenotype not only of recF, recO, is SA500 F Ϫ his87 relA1 strA181 (bio275 cI [Ts]857 ⌬431), and and recR, but also of ruvAB and ruvC (Poteete and was made from Y832 (Hayes and Hayes 1986) derived from Fenton 2000; Poteete et al 2002;Poteete 2004). SA431 (Adhya et al 1968;Stevens et al 1971) The intercrossing between two phage genomes is rou--lysogen of SA500 F Ϫ his87 relA1 strA181 (Hayes 1991 (Daniels et al 1983) in Y832/SA431 with bio275 DNA ( Figure 1B) from in rec ϩ hosts, showing that the host could provide needed into pTP838 (Murphy et al 2000), and in pTP915 gfp replaces rap.…”

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confidence: 99%

“…The host requirements for the Red-or Ninand can substitute for the E. coli RuvC Holliday junction mediated imm rescue differed significantly. The two resolvase in Red recombination (Poteete et al 2002).…”

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“…SA431 (Adhya et al 1968;Stevens et al 1971) The intercrossing between two phage genomes is rou--lysogen of SA500 F Ϫ his87 relA1 strA181 (Hayes 1991 (Daniels et al 1983) in Y832/SA431 with bio275 DNA ( Figure 1B) from in rec ϩ hosts, showing that the host could provide needed into pTP838 (Murphy et al 2000), and in pTP915 gfp replaces rap. Poteete et al (2002) reported that the Rap ϩ phenotype Plasmids: pCH1 includes bases 34,449-41,732 (Hayes et in pTP914 was unaffected by the presence or absence of the or 8.0, 0.001 m Na 2 EDTA), and the DNA concentration was determined by spectrophotometer. Aliquots of the DNA estilac inducer IPTG, suggesting a basal level of expression of rap from p mac .…”

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NinR- and Red-Mediated Phage-Prophage Marker Rescue Recombination in Escherichia coli

et al. 2005

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We examined the requirement of recombination functions for marker rescue of cryptic prophage genes within the Escherichia coli chromosome. We infected lysogenic host cells with imm434 phages and selected for recombinant imm phages that had exchanged the imm434 region of the infecting phage for the heterologous 2.6-kb imm region from the prophage. Phage-encoded activity, provided by either Red or NinR functions, was required for the substitution. Red Ϫ phages with ⌬NinR, internal NinR deletions of rap-ninH, or orf-ninC were 117-, 12-, and 5-fold reduced for imm rescue in a Rec ϩ host, suggesting the participation of several NinR activities. RecA was essential for NinR-dependent imm rescue, but had slight influence on Red-dependent rescue. The host recombination activities RecBCD, RecJ, and RecQ participated in NinR-dependent recombination while they served to inhibit Red-mediated imm rescue. The opposite effects of several host functions toward NinR-and Red-dependent imm rescue explains why the independent pathways were not additive in a Rec ϩ host and why the NinR-dependent pathway appeared dominant. We measured the influence of the host recombination functions and DnaB on the appearance of ori -dependent replication initiation and whether ori replication initiation was required for imm marker rescue.

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NinR- and Red-Mediated Phage-Prophage Marker Rescue Recombination in Escherichia coli

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“…The orf gene was inserted into a plasmid vector and then crossed into the galK locus, essentially as previously described for the rap and gfp genes (31). The primers used for orf amplification had the sequences 5Ј-GGAGAGGGAACAT ATGAAAAAACTAACC-3Ј and 5Ј-ATATGCTGAGCTCCTTCAACCGGAG AA-3Ј.…”

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confidence: 99%

“…Its putative DNA sequence and details of its use will be provided upon request. The reduced-genome E. coli strain MDS12 (9) was used to stabilize the ⌬(recC ptr recB recD)::P tac gam bet exo pae cI822 ruvC genotype, which has a tendency to acquire suppressor mutations in the AB1157 background (31). The suppressor mutations presumably activate rusA (18,19).…”

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Modulation of DNA Repair and Recombination by the Bacteriophage λ Orf Function inEscherichia coliK-12

Poteete

2004

J Bacteriol

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The orf gene of bacteriophage , fused to a promoter, was placed in the galK locus of Escherichia coli K-12. Orf was found to suppress the recombination deficiency and sensitivity to UV radiation of mutants, in a ⌬(recC ptr recB recD)::P tac gam bet exo pae cI ⌬recG background, lacking recF, recO, recR, ruvAB, and ruvC functions. It also suppressed defects of these mutants in establishing replication of a pSC101-related plasmid. Compared to orf, the recA803 allele had only small effects on recF, recO, and recR mutant phenotypes and no effect on a ruvAB mutant. In a fully wild-type background with respect to known recombination and repair functions, orf partially suppressed the UV sensitivity of ruvAB and ruvC mutants.Conjugative recombination in a recBC sbcB sbcC mutant of Escherichia coli depends upon the recF, recO, and recR genes, as well as other rec genes and the ruv genes, and is said to proceed via the RecF pathway (for a review, see reference 14). In the same cell, however, homologous recombination between bacteriophage chromosomes does not depend on recF, recO, or recR, even when the phage lacks its own recombination system, Red, and is thus dependent upon the host's recombination system (33). Sawitzke and Stahl (33) showed that 's RecORF independence is due to its orf gene (previously called ninB). Further studies showed that orf encodes a protein and that plasmid-borne orf can act in trans to promote phage recombination, but not conjugative recombination, in the absence of RecORF (34).Recombination between phage chromosomes via the Red pathway requires neither RecORF nor Orf, but Orf nonetheless is a participant (39). The result of its action is to focus crossovers near the initiating double-strand break in both the RecF (35) and Red (39) pathways.An additional observation suggested that Orf can promote recombination events other than crossovers between phage chromosomes. In a cell in which the recC-ptr-recB-recD gene cluster is replaced by the Red system genes (gam, bet, and exo) and from which the recG gene is deleted, recombination between the bacterial chromosome and a 3.6-kbp linear double-stranded DNA (dsDNA) molecule is dependent upon recF, recO, and recR. However, in such a cell, ectopic expression from the bacterial chromosome of a segment of the chromosome, including the orf gene, partially decreases this dependence (29).For this study, the orf gene was installed by itself in the galK locus. The expression of chromosomally encoded orf was found to have surprisingly pleiotropic effects on recombination, replication, and repair in E. coli. Orf suppressed the mutant phenotypes of not only recF, recO, and recR, but ruvAB and ruvC as well. In contrast, the recA803 allele, which partially suppresses the phenotypes of recF, recO, and recR mutations affecting RecF pathway recombination and repair (44, 45), had no comparable effect on the Red pathway. MATERIALS AND METHODSBacterial strains. The bacterial strains used for this study are described in Table 1. Some of the constructions involved the repla...

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Orpheus Recombination

Woltjen

Ito

Tsuzuki

et al. 2008

Chromosomal Mutagenesis

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In recent years, methods to address the simplification of targeting vector (TV) construction have been developed and validated. Based on in vivo recombination in Escherichia coli, these protocols have reduced dependence on restriction endonucleases, allowing the fabrication of complex TV constructs with relative ease. Using a methodology based on phage-plasmid recombination, we have developed a comprehensive TV construction protocol dubbed Orpheus recombination (ORE). The ORE system addresses all necessary requirements for TV construction; from the isolation of genespecific regions of homology to the deposition of selection/disruption cassettes. ORE makes use of a small recombination plasmid, which bears positive and negative selection markers and a cloned homologous "probe" region. This probe plasmid may be introduced into and excised from phage-borne murine genomic clones by two rounds of single crossover recombination. In this way, desired clones can be specifically isolated from a heterogeneous library of phage. Furthermore, if the probe region contains a designed mutation, it may be deposited seamlessly into the genomic clone. The complete removal of operational sequences allows unlimited repetition of the procedure to customize and finalize TVs within a few weeks. Successful gene-specific clone isolation, point mutations, large deletions, cassette insertions, and finally coincident clone isolation and mutagenesis have all been demonstrated with this method.

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Recombination-Promoting Activity of the Bacteriophage λ Rap Protein in Escherichia coli K-12

Cited by 12 publications

References 18 publications

NinR- and Red-Mediated Phage-Prophage Marker Rescue Recombination in Escherichia coli

NinR- and Red-Mediated Phage-Prophage Marker Rescue Recombination in Escherichia coli

Modulation of DNA Repair and Recombination by the Bacteriophage λ Orf Function inEscherichia coliK-12

Orpheus Recombination

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