An unusual illegitimate recombination occurs in the linear-plasmid-encoded outer-surface protein A gene of Borrelia afzelii

Wang, Jianhui; Masuzawa, Toshiyuki; Li, Muqing; Yanagihara, Yasutake

doi:10.1099/00221287-143-12-3819

Cited by 4 publications

(3 citation statements)

References 46 publications

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“…2). Short direct repeats have been reported to intervene in the generation of large deletions by a mechanism of illegitimate recombination involving strand slippage during replication that removes one of the direct repeats (40,59,60). It has been shown, in bacterial model systems, that deletion frequencies are increased by reducing the distance between the direct repeats (10), by increasing the direct repeat lengths, and by the presence of inverted repeats flanking the direct repeats (40).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Brucella Species 25-Kilobase DNA Fragment Deleted from Brucella abortus Reveals a Large Gene Cluster Related to the Synthesis of a Polysaccharide

Vizcaı́no

Cloeckaert²,

Zygmunt

et al. 2001

Infect Immun

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In the present study we completed the nucleotide sequence of a Brucella melitensis 16M DNA fragment deleted from B. abortus that accounts for 25,064 bp and show that the other Brucella spp. contain the entire 25-kb DNA fragment. Two short direct repeats of four nucleotides, detected in the B. melitensis 16M DNA flanking both sides of the fragment deleted from B. abortus, might have been involved in the deletion formation by a strand slippage mechanism during replication. In addition to omp31, coding for an immunogenic protein located in the Brucella outer membrane, 22 hypothetical genes were identified. Most of the proteins that would be encoded by these genes show significant homology with proteins involved in the biosynthesis of polysacharides from other bacteria, suggesting that they might be involved in the synthesis of a Brucella polysaccharide that would be a heteropolymer synthesized by a Wzy-dependent pathway. This polysaccharide would not be synthesized in B. abortus and would be a polysaccharide not identified until present in the genus Brucella, since all of the known polysaccharides are synthesized in all smooth Brucella species. Discovery of a novel polysaccharide not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes detected in the DNA fragment deleted in B. abortus no longer lead to the synthesis of a polysaccharide must not be excluded. They might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, with some of its members synthesizing extracellular polysaccharides and, as Brucella spp., living in association with eukaryotic cells. The genus Brucella is described as constituted by six species: Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae, each preferentially infecting an animal host. In addition, several biovars are included in the species B. melitensis, B. abortus, and B. suis. The six Brucella species and their biovars are currently differentiated by pathogenicity and host preference characteristics, serotyping, phage typing, dye sensitivities, and culture and metabolic properties (2). In spite of the high degree of DNA homology that has been found between the six species, which would justify a monospecific genus (53, 54), the classical organization of the genus Brucella has been maintained since it is in accordance with the pathogenicity and host preference characteristics of each species, and several molecular markers, allowing the differentiation between the six species and some of their biovars, have been found (55).Differences in pathogenicity and host preference found between the Brucella species and biovars could be reflected by differences at the DNA level. However, only small differences have been found between the Brucella species in the genes identified until the present (55). Small deletions in several genes have been detected in some Brucella species (11,13,...

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Section: Discussionmentioning

confidence: 99%

Characterization of a Brucella Species 25-Kilobase DNA Fragment Deleted from Brucella abortus Reveals a Large Gene Cluster Related to the Synthesis of a Polysaccharide

Vizcaı́no

Cloeckaert²,

Zygmunt

et al. 2001

Infect Immun

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“…Among the 30 strains, 4 isolates from ticks were identical to B. afzelii strain XJ23 isolated in China (19). The other 26 strains were found to be genetically variable (up to 20.6% of nucleotide difference), but the closest sequence similarity for all of them was found with different strains of B. garinii.…”

Section: Resultsmentioning

confidence: 91%

Diversity of Borrelia burgdorferi Sensu Lato in Russian Far East

Mediannikov¹,

Иванов

Zdanovskaya

et al. 2005

Microbiology and Immunology

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“…To increase homologous integration it is necessary to inhibit non-homologous recombination (Roth et al, 1985). Illegitimate recombination has two different mechanisms: (i) end-joining, mediated by enzymes which cut and join DNA, such as topoisomerases, site-specific DNases and involve sequences which include or resemble those on which such enzymes normally act, and (ii) strand slippage, where, after pausing at the replication fork, the nascent strand is able to dissociate from one template and pair with another (Wang et al, 1997).…”

Section: Mechanisms Of Transgene Integration Into the Genomementioning

confidence: 99%

437 Determining Gene Copy Number in Transfected Caprine Fibroblast Cells

Wilson¹,

Godke²,

Bondioli³

2010

Reprod. Fertil. Dev.

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Transgene expression in stably transgenic organisms is affected by many factors, including the copy number of the transgene in the genome and by interactions between the transgene and flanking DNA sequences. Very high transgene copy number has also been shown to affect genetic stability in transgenic plants and animals. Two commonly used methods for transfecting cells prior to their use in nuclear transfer (NT) are liposome-mediated transfection and electroporation. Little is known about the transgene copy number or variability of the copy number with these techniques. The objective of this study was to determine transgene copy number after liposome-mediated transfection and electroporation. The mean transgene copy number and variability between individual integration events have been determined. Q-PCR conditions were optimized for primer annealing temperature and concentration when amplifying a region of a plasmid expressing green fluorescent protein (GFP) under the control of the human elongation factor (hEF) promoter (hEFGFP) used for transfection. The quantitative nature of the Q-PCR reaction was confirmed by amplifying 10-fold dilutions of the plasmid and plotting the threshold cycle (CT) value against the log of the plasmid concentration. A correlation coefficient of 1.00 and a calculated PCR efficiency of 93.3% were obtained from this analysis. Caprine fibroblasts were transfected by electroporation with 20 μg of DNA or FuGENE® HD (Roche, Nutley, NJ, USA) reagent with 6 μg of DNA using either a circular or linearized hEFGFP plasmid. Transformed cells were plated at low density in medium containing Geneticin® (Gibco, Grand Island, NY USA). After 10 days of culture, single-cell colonies were isolated and expanded. When cultures reached 1 to 2 million cells, genomic DNA was isolated. Transgene copy number was determined by amplifying genomic DNA from individual clones representing 1 × 105 cells with Q-PCR. Transgene copy number was calculated from a standard curve of the transgene plasmid. The mean transgene copy number for electroporation circular was 2.7 ± 0.75 (n = 32 colonies) and 1.3 ± 0.65 (n = 19) when using a linear DNA construct. FuGENE HD using a circular plasmid construct generated a mean gene copy number of 0.5 ± 0.11 (n = 14) and 0.64 ± 0.13 (n = 16) for the linear plasmid construct. One-way ANOVA followed by multiple pair-wise comparisons using Tukey’s method showed significant differences when comparing electroporation circular to all other treatments. However, there were no differences when comparing electroporation linear, FuGENE HD circular, and FuGENE HD linear to each other. Because the calculated mean copy number for transfection with FuGENE HD was consistently less than 1, it is assumed that these colonies consisted predominantly of single-copy integrations. Our results indicate that the transfection method can affect gene copy number. Electroporation resulted in multiple but few copies whereas Fugene HD resulted in predominantly single-copy integrations. The probability of transgene mutation with single-copy integration suggests that electroporation is preferable forproducing transgenic animals by NT.

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An unusual illegitimate recombination occurs in the linear-plasmid-encoded outer-surface protein A gene of Borrelia afzelii

Cited by 4 publications

References 46 publications

Characterization of a Brucella Species 25-Kilobase DNA Fragment Deleted from Brucella abortus Reveals a Large Gene Cluster Related to the Synthesis of a Polysaccharide

Characterization of a Brucella Species 25-Kilobase DNA Fragment Deleted from Brucella abortus Reveals a Large Gene Cluster Related to the Synthesis of a Polysaccharide

Diversity of Borrelia burgdorferi Sensu Lato in Russian Far East

437 Determining Gene Copy Number in Transfected Caprine Fibroblast Cells

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