Rapid and simplified protocol for isolation and characterization of leptospiral chromosomal DNA for taxonomy and diagnosis

Febvre, R B Le; Foley, John W.; Thiermann, A B

doi:10.1128/jcm.22.4.606-608.1985

Cited by 25 publications

(12 citation statements)

References 13 publications

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“…Lysogens were streaked on Luria-Bertani (LB) plates containing 20 mM EGTA and then on MacConkey base plates with incubation at 308C to select for segregants that had lost the prophage. Chromosomal DNA was extracted from the segregants grown at 308C as described (Le Febvre et al, 1985) and used in PCR reactions using primers I and II (Fig. 1) to identify the mutant strain with the deletion of the P1 promoter of topA.…”

Section: Bacterial Strainsmentioning

confidence: 99%

**Effect of the deletion of the σ³²‐dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance**

Menzel

Tse‐Dinh

1996

Molecular Microbiology

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Topoisomerase I and DNA gyrase are the major topoisomerase activities responsible for the regulation of DNA supercoiling in the bacterium Escherichia coli. The P1 promoter of topA has previously been shown to be a delta 32-dependent heat-shock promoter. A mutant strain with a deletion of P1 was constructed. This mutant is > 10-fold more sensitive to heat treatment (52 degrees C) than the wild type. After brief treatment at 42 degrees C, wild-type Escherichia coli acquires an enhanced resistance to the effects of a subsequent 52 degrees C treatment. This is not the case for the P1 deletion mutant, which, and under these conditions, is about 100-fold less thermotolerant than the wild type. The presence of a plasmid expressing topoisomerase I restored the heat-survival level of the mutant to that of the wild type. During heat shock, the superhelical density of a plasmid with the heat-inducible rpoD promoter is increased in the P1 deletion mutant. We also note that the pulse-labelling pattern of proteins at 42 C (displayed on SDS-polyacrylamide gels) is different in the mutant, and, most notably, the amounts of DnaK and of GroEL protein are reduced. A model is proposed in order to unify these observations.

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Section: Bacterial Strainsmentioning

confidence: 99%

**Effect of the deletion of the σ³²‐dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance**

Menzel

Tse‐Dinh

1996

Molecular Microbiology

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“…Isolation and characterization of DNA. Whole-cell DNA was isolated by the procedure of LeFebvre et al (17). Restriction endonuclease analysis was performed as previously described (29) and according to the manufacturers' specifications.…”

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confidence: 99%

Genetic and antigenic characterization of Borrelia coriaceae, putative agent of epizootic bovine abortion

Lefebvre

Perng

1989

J Clin Microbiol

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Borrelia coriaceae was characterized genetically and antigenically by utilizing the following techniques: restriction endonuclease analysis, Southern blotting and genomic hybridization, pulsed-field electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting. The B. coriaceae genome revealed unique and characteristic banding patterns both by agarose gel electrophoresis and by hybridization when compared with several Borrelia burgdorferi isolates. Pulsed-field gel electrophoresis demonstrated several linear plasmids ranging from 65 to 30 kilobase pairs. Cross-reaction with B. burgdorferi antigens ranging from 21 to 26 kilodaltons were demonstrated by immunoblotting with rabbit anti-B. coriaceae antiserum. However, most B. coriaceae antigens were quite distinct when compared with B. burgdorferi and Leptospira interrogans antigens.

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“…suggested that we recovered a larger amount of Anaplasma DNA from the same amount of starting material with our method than we did with extraction precipitation techniques (13).…”

Section: Discussionmentioning

confidence: 85%

“…DNA cleavage patterns produced by bacterial restriction endonucleases have been used to differentiate phenotypically closely related bacteria. Such patterns have been used to study various species, strains, serovars, and isolates of Mycoplasma (19,23,24), Coxiella (21), Chlamydia (4,22), Neisseria (3,12), Streptococcus (25), Campylobacter (7), Escherichia (17), Mycobacterium (5,6), and Leptospira (13,28). Recently, a reclassification of leptospiral isolates belonging to several serogroups has been proposed (28) on the basis of DNA cleavage pattern analysis.…”

Section: Studiesmentioning

confidence: 99%

Isolation and restriction endonuclease cleavage of Anaplasma marginale DNA in situ in agarose

Krueger

Buening

1988

J Clin Microbiol

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Bacterial restriction endonucleases were used to produce DNA cleavage patterns that could be useful as tools to study the relatedness among Anaplasma marginale isolates. Bovine erythrocytes infected with A. marginale were lysed, washed, and embedded in agarose. The embedded erythrocytes and bacterial pathogens were partially digested by sequential infiltration of the agarose with acetone, lysozyme, sodium dodecyl sulfate, and proteinase K. The unfragmented genomic DNA was left supported and protected in a porous matrix. The DNA was digested in situ in agarose under the following conditions: (i) brief treatment with phenol, (ii) brief washing with distilled water, and (iii) adjustment of restriction enzyme digestion mixture to compensate for the volume of the agarose. The cleaved DNA was electrophoresed horizontally to produce a DNA cleavage pattern. Of 19 restriction enzymes screened, 12 produced distinct DNA bands from the genomes of each of the five A. marginale isolates examined. The DNA cleavage pattern produced from each isolate with a given restriction enzyme was reproducible. However, the DNA cleavage patterns produced from different isolates with a given restriction enzyme were not necessarily identical. This procedure could be modified for general bacterial DNA isolation, in situ agarose digestion, and manipulations. Anaplasma marginale is an obligately intracellular bacterial pathogen of bovine erythrocytes from the order Rickettsiales (11). A. marginale causes anaplasmosis, a significant, worldwide (20), hemolytic disease of cattle. Many isolates of A. marginale exhibit differences in morphology (9), surface antigens (18), protein structure (1), vector specificity (27), and virulence (10). Knowledge of the relatedness among the various isolates of A. marginale has importance in taxonomic, epidemiological, virulence, and vaccine development

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Rapid and simplified protocol for isolation and characterization of leptospiral chromosomal DNA for taxonomy and diagnosis

Cited by 25 publications

References 13 publications

**Effect of the deletion of the σ³²‐dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance**

**Effect of the deletion of the σ³²‐dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance**

Genetic and antigenic characterization of Borrelia coriaceae, putative agent of epizootic bovine abortion

Isolation and restriction endonuclease cleavage of Anaplasma marginale DNA in situ in agarose

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Rapid and simplified protocol for isolation and characterization of leptospiral chromosomal DNA for taxonomy and diagnosis

Cited by 25 publications

References 13 publications

Effect of the deletion of the σ32‐dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance

Effect of the deletion of the σ32‐dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance

Genetic and antigenic characterization of Borrelia coriaceae, putative agent of epizootic bovine abortion

Isolation and restriction endonuclease cleavage of Anaplasma marginale DNA in situ in agarose

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**Effect of the deletion of the σ³²‐dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance**

**Effect of the deletion of the σ³²‐dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance**