Homologous Recombination and Mutagenesis of γ-Irradiated Plasmid DNA in Escherichia coli Host Cells

Repair of UV-irradiated plasmid DNA microinjected into frog oocytes was measured by two techniques: transformation of repair-deficient (delta uvrB delta recA delta phr) bacteria, and removal of UV endonuclease-sensitive sites (ESS). Transformation efficiencies relative to unirradiated plasmids were used to estimate the number of lethal lesions; the latter were assumed to be Poisson distributed. These estimates were in good agreement with measurements of ESS. By both criteria, plasmid DNA was efficiently repaired, mostly during the first 2 h, when as many as 2 x 10(10) lethal lesions were removed per oocyte. This rate is about 10(6) times the average for removal of ESS from repair-proficient human cells. Repair was slower but still significant after 2 h, but some lethal lesions usually remained after overnight incubation. Most repair occurred in the absence of light, in marked contrast to differentiated frog cells, previously shown to possess photoreactivating but no excision repair activity. There was no increase in the resistance to DpnI restriction of plasmids (methylated in Escherichia coli at GATC sites) incubated in oocytes; this implies no increase in hemimethylated GATC sites, and hence no semiconservative DNA replication. Plasmid substrates capable of either intramolecular or intermolecular homologous recombination were not recombined, whether UV-irradiated or not. Repair of Lac+ plasmids was accompanied by a significant UV-dependent increase in the frequency of Lac- mutants, corresponding to a repair synthesis error frequency on the order of 10(-4) per nucleotide.

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Reciprocal and non-reciprocal homologous recombination between Escherichia coli chromosomal DNA and ultraviolet light-irradiated plasmid DNA

Gene ◽

10.1016/0378-1119(86)90284-2 ◽

1986 ◽

Vol 49 (2) ◽

pp. 235-244 ◽

Cited By ~ 6

Author(s):

John S. Mudgett ◽

William D. Taylor

Keyword(s):

Escherichia Coli ◽

Homologous Recombination ◽

Ultraviolet Light ◽

Plasmid Dna ◽

Chromosomal Dna

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Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laevis oocytes.

Molecular and Cellular Biology ◽

10.1128/mcb.10.7.3505 ◽

1990 ◽

Vol 10 (7) ◽

pp. 3505-3511 ◽

Cited By ~ 13

Author(s):

J B Hays ◽

E J Ackerman ◽

Q S Pang

Keyword(s):

Escherichia Coli ◽

Homologous Recombination ◽

Plasmid Dna ◽

Excision Repair ◽

Xenopus Laevis Oocytes ◽

Marked Contrast ◽

Repair Synthesis ◽

Error Frequency ◽

Repair Activity ◽

Good Agreement

Repair of UV-irradiated plasmid DNA microinjected into frog oocytes was measured by two techniques: transformation of repair-deficient (delta uvrB delta recA delta phr) bacteria, and removal of UV endonuclease-sensitive sites (ESS). Transformation efficiencies relative to unirradiated plasmids were used to estimate the number of lethal lesions; the latter were assumed to be Poisson distributed. These estimates were in good agreement with measurements of ESS. By both criteria, plasmid DNA was efficiently repaired, mostly during the first 2 h, when as many as 2 x 10(10) lethal lesions were removed per oocyte. This rate is about 10(6) times the average for removal of ESS from repair-proficient human cells. Repair was slower but still significant after 2 h, but some lethal lesions usually remained after overnight incubation. Most repair occurred in the absence of light, in marked contrast to differentiated frog cells, previously shown to possess photoreactivating but no excision repair activity. There was no increase in the resistance to DpnI restriction of plasmids (methylated in Escherichia coli at GATC sites) incubated in oocytes; this implies no increase in hemimethylated GATC sites, and hence no semiconservative DNA replication. Plasmid substrates capable of either intramolecular or intermolecular homologous recombination were not recombined, whether UV-irradiated or not. Repair of Lac+ plasmids was accompanied by a significant UV-dependent increase in the frequency of Lac- mutants, corresponding to a repair synthesis error frequency on the order of 10(-4) per nucleotide.

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Homologous recombination catalyzed by human cell extracts.

Molecular and Cellular Biology ◽

10.1128/mcb.5.4.714 ◽

1985 ◽

Vol 5 (4) ◽

pp. 714-720 ◽

Cited By ~ 32

Author(s):

R S Kucherlapati ◽

J Spencer ◽

P D Moore

Keyword(s):

Escherichia Coli ◽

Homologous Recombination ◽

Gene Conversion ◽

Human Cell ◽

Plasmid Dna ◽

Somatic Cells ◽

Cell Extracts ◽

Escherichia Coli Cells ◽

Enzymatic Machinery

Two plasmids containing noncomplementing and nonreverting deletions in a bacterial phosphotransferase gene conferring resistance to neomycin (Neor) were incubated with human cell extracts, and the mixtures were used to transform recombination-deficient (recA-) Escherichia coli cells. We were able to obtain Neor colonies at a frequency of 2 X 10(-3). This frequency was 100 to 1,000 times higher than that obtained with no extracts. The removal of riboadenosine 5'-triphosphate, Mg2+, or deoxynucleoside triphosphates from the reaction mixture severely reduced the yield of Neor colonies. Examination of plasmid DNA from the Neor colonies revealed that they resulted from gene conversion and reciprocal recombination. On the basis of these results, we conclude that mammalian somatic cells in culture have the enzymatic machinery to catalyze homologous recombination in vitro.

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Homologous recombination catalyzed by human cell extracts

Molecular and Cellular Biology ◽

10.1128/mcb.5.4.714-720.1985 ◽

1985 ◽

Vol 5 (4) ◽

pp. 714-720

Author(s):

R S Kucherlapati ◽

J Spencer ◽

P D Moore

Keyword(s):

Escherichia Coli ◽

Homologous Recombination ◽

Gene Conversion ◽

Human Cell ◽

Plasmid Dna ◽

Somatic Cells ◽

Cell Extracts ◽

Escherichia Coli Cells ◽

Enzymatic Machinery

Two plasmids containing noncomplementing and nonreverting deletions in a bacterial phosphotransferase gene conferring resistance to neomycin (Neor) were incubated with human cell extracts, and the mixtures were used to transform recombination-deficient (recA-) Escherichia coli cells. We were able to obtain Neor colonies at a frequency of 2 X 10(-3). This frequency was 100 to 1,000 times higher than that obtained with no extracts. The removal of riboadenosine 5'-triphosphate, Mg2+, or deoxynucleoside triphosphates from the reaction mixture severely reduced the yield of Neor colonies. Examination of plasmid DNA from the Neor colonies revealed that they resulted from gene conversion and reciprocal recombination. On the basis of these results, we conclude that mammalian somatic cells in culture have the enzymatic machinery to catalyze homologous recombination in vitro.

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Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.64.12.4862-4869.1998 ◽

1998 ◽

Vol 64 (12) ◽

pp. 4862-4869 ◽

Cited By ~ 39

Author(s):

Jörg F. Rippmann ◽

Michaela Klein ◽

Christian Hoischen ◽

Bodo Brocks ◽

Wolfgang J. Rettig ◽

...

Keyword(s):

Escherichia Coli ◽

Proteus Mirabilis ◽

Binding Activity ◽

Host Cells ◽

Heterologous Proteins ◽

Single Chain Variable Fragment ◽

Single Chain ◽

E Coli ◽

Active Product ◽

Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

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An infection-induced RhoB-Beclin 1-Hsp90 complex enhances clearance of uropathogenic Escherichia coli

Nature Communications ◽

10.1038/s41467-021-22726-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chunhui Miao ◽

Mingyu Yu ◽

Geng Pei ◽

Zhenyi Ma ◽

Lisong Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Treatment Strategies ◽

Uropathogenic Escherichia Coli ◽

Beclin 1 ◽

Host Cells ◽

Infection Model ◽

Intracellular Bacteria ◽

Bladder Epithelium ◽

Binding Residue

AbstractHost cells use several anti-bacterial pathways to defend against pathogens. Here, using a uropathogenic Escherichia coli (UPEC) infection model, we demonstrate that bacterial infection upregulates RhoB, which subsequently promotes intracellular bacteria clearance by inducing LC3 lipidation and autophagosome formation. RhoB binds with Beclin 1 through its residues at 118 to 140 and the Beclin 1 CCD domain, with RhoB Arg133 being the key binding residue. Binding of RhoB to Beclin 1 enhances the Hsp90-Beclin 1 interaction, preventing Beclin 1 degradation. RhoB also directly interacts with Hsp90, maintaining RhoB levels. UPEC infections increase RhoB, Beclin 1 and LC3 levels in bladder epithelium in vivo, whereas Beclin 1 and LC3 levels as well as UPEC clearance are substantially reduced in RhoB+/− and RhoB−/− mice upon infection. We conclude that when stimulated by UPEC infections, host cells promote UPEC clearance through the RhoB-Beclin 1-HSP90 complex, indicating RhoB may be a useful target when developing UPEC treatment strategies.

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