Genetic analysis and molecular cloning of the Escherichia coli ruv gene

Shurvinton, Claire E.; Lloyd, Robert G.; Benson, Fiona E.; Attfield, Paul V.

doi:10.1007/bf00383535

Cited by 63 publications

(28 citation statements)

References 32 publications

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“…CS85 was originally isolated as a ruv mutant by Shurvinton et al, and the ruv-53 mutation was mapped to the ruv locus (30). Introduction of pHS501, a low-copynumber plasmid carrying the 2.7-kb PvuII fragment containing the ruvA and ruvB genes, did not complement the ruv-53 mutation in CS85.…”

Section: Resultsmentioning

confidence: 99%

“…coli CS85 (ruv-53) (30) was used as the host strain for cloning and subcloning of the ruvC gene. CS40a (11) was used as a ruv+ control strain.…”

Section: Methodsmentioning

confidence: 99%

“…Sharples et al (25) have identified the approximate location of this mutation on the physical map of this region and designated it ruvC. The ruvC mutants, as with the other ruv mutants, are defective in DNA repair and recombination of the RecE and the RecF pathways (1,12,19,25,30). In this study we analyzed the nucleotide sequence of the ruvC gene and its flanking regions and identified the proteins encoded by the ruvC region.…”

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confidence: 98%

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Molecular analysis of the Escherichia coli ruvC gene, which encodes a Holliday junction-specific endonuclease

et al. 1991

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The Escherichia coli ruvC gene is involved in DNA repair and recombination and encodes an endonuclease that resolves Holliday structure in vitro. The 2.8-kb chromosomal DNA fragment that encompasses the ruvC gene and its flanking regions was cloned and sequenced. Four open reading frames were identified in the order orfl7-orJ26-ruvC-orfJ3 immediately upstream of the ruvAB operon, and their orientations are the same as the ruvAB operon, except for orf23. Proteins encoded by orfl7, or426, and ruvC (orfl9) were identified by the maxicell method, and their sizes agreed with those predicted from the DNA sequences. Among the open reading frames in this region, only ruvC is involved in the repair of UV-damaged DNA. ruvC appeared to be regulated by at least two promoters, but, in contrast to the ruvAB operon, ruvC is not regulated by the SOS system as demonstrated by operon fusions.Escherichia coli strains carrying mutations in the ruv locus were isolated initially as mutants which were sensitive to mitomycin C (19) or which showed reduced ability of recombination between duplicated gal genes (32). These mutants are also sensitive to various DNA-damaging agents such as UV, ionizing radiation, and some chemical mutagens, and they form multinucleated cells after treatment with low doses of such agents (18)(19)(20)32). Although ruv mutations have little effect on conjugal recombination in otherwise wild-type strains, they reduce the ability of recombination in recBC sbcA and recBC sbcBC strains (11)(12)(13). This evidence suggests that the ruv locus may be involved in recombinational repair by the RecE and the RecF pathways.The ruv locus contains two genes, ruvA and ruvB, that constitute an operon regulated by the SOS system (3,27,29). Recently these genes were also shown to be involved in mutagenesis induced by UV and X irradiation and by some chemicals (9, 24). The purified RuvB protein possesses weak ATPase activity (7), which is stimulated by the RuvA protein in the presence of DNA (26). The RuvA-RuvB complex in the presence of ATP renatures cruciform structure in supercoiled DNA with a palindromic sequence, indicating that it may promote strand exchange reactions in homologous recombination (26).While we were cloning the chromosomal DNA fragment that complemented the repair defect in the ruv mutants, we noticed that the phenotype of one of the ruv mutants we analyzed, CS85 (ruv-53), was not complemented by the plasmid carrying the ruvAB operon, but was complemented by the chromosomal DNA region upstream of the ruvAB operon. Sharples et al. (25) have identified the approximate location of this mutation on the physical map of this region and designated it ruvC. The ruvC mutants, as with the other ruv mutants, are defective in DNA repair and recombination of the RecE and the RecF pathways (1,12,19,25,30). In this study we analyzed the nucleotide sequence of the ruvC gene and its flanking regions and identified the proteins encoded by the ruvC region.RuvC protein has been highly purified recently and has an * Correspondin...

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Section: Resultsmentioning

confidence: 99%

“…coli CS85 (ruv-53) (30) was used as the host strain for cloning and subcloning of the ruvC gene. CS40a (11) was used as a ruv+ control strain.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 98%

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Molecular analysis of the Escherichia coli ruvC gene, which encodes a Holliday junction-specific endonuclease

et al. 1991

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“…The thermosensitivity of the prc mutant was tested on salt-free 1/2L agar medium, in which the content of Bacto-Tryptone (Difco) and yeast extract of L-agar medium was reduced by half and neither NaCl nor glucose was included. Eda and FadD phenotypes were tested on minimal agar media (65) with 20 mM glucuronic acid (57) and 0.1% oleic acid (dissolved with 0.4% Brij 35) (48), respectively, as the sole carbon sources. The Pel phenotype was tested by cross-streaking against phage Avir on EMB agar medium (41) containing 1% mannose.…”

Section: Methodsmentioning

confidence: 99%

Cloning, mapping, and characterization of the Escherichia coli prc gene, which is involved in C-terminal processing of penicillin-binding protein 3

et al. 1991

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The prc gene, which is involved in cleavage of the C-terminal peptide from the precursor form of penicillin-binding protein 3 (PBP 3) of Escherichia coli, was cloned and mapped at 40.4 min on the chromosome. The gene product was identified as a protein of about 80 kDa in maxicell and in vitro systems. Fractionation of the maxicells producing the product suggested that the product was associated with the periplasmic side of the cytoplasmic membrane. This was consistent with the notion that the C-terminal processing of PBP 3 probably occurs outside the cytoplasmic membrane: the processing was found to be dependent on the secY and secA functions, indicating that the prc product or PBP 3 or both share the translocation machinery with other extracytoplasmic proteins. DNA sequencing analysis of the prc gene region identified an open reading frame, with two possible translational starts 6 bp apart from each other, that could code for a product with a calculated molecular weight of 76,667 or 76,432. The prc mutant was sensitive to thermal and osmotic stresses. Southern analysis of the chromosomal DNA of the mutant unexpectedly revealed that the mutation was a deletion of the entire prc gene and thus that the prc gene is conditionally dispensable. The mutation resulted in greatly reduced heat shock response at low osmolarity and in leakage of periplasmic proteins.

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“…Some years ago, a mutant of E. coli K-12, a tls-1 mutant, that exhibited a temperature-sensitive growth phenotype in low-salt media was identified (16,17). DNA fragments alleviating the temperature sensitivity of a tls-1 strain (CS143) were cloned and sequenced.…”

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confidence: 99%

Characterization of a thermosensitive Escherichia coli aspartyl-tRNA synthetase mutant

et al. 1997

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The Escherichia coli tls-1 strain carrying a mutated aspS gene (coding for aspartyl-tRNA synthetase), which causes a temperature-sensitive growth phenotype, was cloned by PCR, sequenced, and shown to contain a single mutation resulting in substitution by serine of the highly conserved proline 555, which is located in motif 3. When an aspS fragment spanning the codon for proline 555 was transformed into the tls-1 strain, it was shown to restore the wild-type phenotype via homologous recombination with the chromosomal tls-1 allele. The mutated AspRS purified from an overproducing strain displayed marked temperature sensitivity, with half-life values of 22 and 68 min (at 42°C), respectively, for tRNA aminoacylation and ATP/PP i exchange activities. K m values for aspartic acid, ATP, and tRNA Asp did not significantly differ from those of the native enzyme; thus, mutation Pro555Ser lowers the stability of the functional configuration of both the acylation and the amino acid activation sites but has no significant effect on substrate binding. This decrease in stability appears to be related to a conformational change, as shown by gel filtration analysis. Structural data strongly suggest that the Pro555Ser mutation lowers the stability of the Lys556 and Thr557 positions, since these two residues, as shown by the crystallographic structure of the enzyme, are involved in the active site and in contacts with the tRNA acceptor arm, respectively.

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Genetic analysis and molecular cloning of the Escherichia coli ruv gene

Cited by 63 publications

References 32 publications

Molecular analysis of the Escherichia coli ruvC gene, which encodes a Holliday junction-specific endonuclease

Molecular analysis of the Escherichia coli ruvC gene, which encodes a Holliday junction-specific endonuclease

Cloning, mapping, and characterization of the Escherichia coli prc gene, which is involved in C-terminal processing of penicillin-binding protein 3

Characterization of a thermosensitive Escherichia coli aspartyl-tRNA synthetase mutant

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