A simple method for the preparation of large quantities of pure plasmid DNA

Humphreys, G. O.; Willshaw, G. A.; Anderson, E. S.

doi:10.1016/0005-2787(75)90318-4

Cited by 575 publications

(204 citation statements)

References 20 publications

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“…All the plasmids used in this study are listed in Table 1. Plasmids were purified from overnight cultures by alkaline lysis (Birnboim & Doly, 1979) or with a Qiaprep spin Miniprep kit (Qiagen) and further purified when necessary by polyethylene glycol precipitation (Humphreys et al, 1975). A standard CaCl 2 protocol was followed to introduce plasmids into E. coli (Sambrook & Russell, 2001).…”

Section: Methodsmentioning

confidence: 99%

Identification of critical amino acid residues in the plague biofilm Hms proteins

et al. 2006

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Yersinia pestis biofilm formation causes massive adsorption of haemin or Congo red in vitro as well as colonization and eventual blockage of the flea proventriculus in vivo. This blockage allows effective transmission of plague from some fleas, like the oriental rat flea, to mammals. Four Hms proteins, HmsH, HmsF, HmsR and HmsS, are essential for biofilm formation, with HmsT and HmsP acting as positive and negative regulators, respectively. HmsH has a β-barrel structure with a large periplasmic domain while HmsF possesses polysaccharide deacetylase and COG1649 domains. HmsR is a putative glycosyltransferase while HmsS has no recognized domains. In this study, specific amino acids within conserved domains or within regions of high similarity in HmsH, HmsF, HmsR and HmsS proteins were selected for site-directed mutagenesis. Some but not all of the substitutions in HmsS and within the periplasmic domain of HmsH were critical for protein function. Substitutions within the glycosyltransferase domain of HmsR and the deacetylase domain of HmsF abolished biofilm formation in Y. pestis. Surprisingly, substitution of highly conserved residues within COG1649 did not affect HmsF function.

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Section: Methodsmentioning

confidence: 99%

Identification of critical amino acid residues in the plague biofilm Hms proteins

et al. 2006

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“…DNAs to be labeled with '*'I were further purified by banding in a cesium chloride gradient (30). Other DNAs to be used in renaturation experiments were concentrated either by polyethylene glycol precipitation (15) or by electrophoresis in a concentrator (ISCO, Lincoln, Nebr.). For labeling with 1251, the CsCI-purified DNA was dialyzed for 48 h against 0.133 M ammonium acetate-0.053 M acetic acid (pH 5) and then denatured by heating in an ethylene glycol bath at 110°C.…”

Section: Methodsmentioning

confidence: 99%

Deoxyribonucleic Acid Base Sequence Relatedness among Members of the Yeast Genus Kluyveromyces

Fuson

Presley

Phaff

1987

International Journal of Systematic Bacteriology

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Deoxyribonucleic acid (DNA) base composition and DNA base sequence relatedness comparisons were used for species delineation in the genus Kluyveromyces. Base composition values separated the members of the genus into three groups. The groups were further subdivided by comparing base sequences by using DNAIDNA renaturation experiments. Two DNA homology groups were identified. The first group included Kluyveromyces marxianus, Kluyveromyces fragilis, Kluyveromyces bulgaricus, Kluyveromyces cicerisporus, Kluyveromyces wikenii, and three anamorphs (Candida kefyr, Candida pseudotropicalis, and Torula cremoris); the members of this group exhibited 290 % DNA base sequence complementarity. The second group consisted of Kluyveromyces lactis, Kluyveromyces vanudenii, Kluyveromyces drosophilarum, and Kluyveromyces phaseolosporus; various pairs of these yeasts shared 64 to 98% of their DNA sequences. The two groups were only distantly related to each other (115% DNA base sequence complementarity). The other Kluyveromyces species appear to be unique, not being closely related to either of the two homology groups or to one another. Relationships deduced from comparisons of DNAs agreed well with those deduced by other workers from immunological comparisons of exo-P-glucanases and from isoenzyme analysis but were only in partial agreement with a taxonomic arrangement made on the basis of mating studies. We propose recognition of the following species:

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“…Radioactively labelled plasmid DNA for electron microscopy was prepared as described by Willshaw et al (1978). Large quantities of pure plasmid DNA for electrophoresis were obtained from cleared lysates of plasmid-carrying strains ; plasmid DNA was precipitated with polyethylene glycol 6000 and purified by caesium chloride-ethidium bromide dye-buoyant density gradient centrifugation (Humphreys et al, 1975).…”

Section: Methodsmentioning

confidence: 99%

Application of Agarose Gel Electrophoresis to the Characterization of Plasmid DNA in Drug-resistant Enterobacteria

Willshaw

Smith

Anderson

1979

Journal of General Microbiology

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A simple gel electrophoresis method has been described for the detection of plasmid DNA in bacteria (Meyers et al., 1976). We investigated further the problems encountered in using this method for the analysis of plasmids in wild enterobacterial strains. The migration of open circular and linear plasmid DNA was examined, since these forms sometimes caused difficulty in the interpretation of the plasmid content of uncharacterized strains. Electrophoresis at different agarose concentrations was employed to resolve clearly plasmid DNA from the chromosomal DNA fragments in the crude preparations. Dissociation of some plasmids occurs in Salmonella typhimurium, and this was detected by electrophoresis.The technique was applied to the study of drug-resistant strains of S. typhimurium phage type 208 from several Middle Eastern countries. The cultures carry a drug resistance plasmid of the F,me compatibility group, and at least two other plasmids which were detected and identified by gel electrophoresis. The studies supported and extended the genetic findings and provided information on the distribution of particular plasmids.

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A simple method for the preparation of large quantities of pure plasmid DNA

Cited by 575 publications

References 20 publications

Identification of critical amino acid residues in the plague biofilm Hms proteins

Identification of critical amino acid residues in the plague biofilm Hms proteins

Deoxyribonucleic Acid Base Sequence Relatedness among Members of the Yeast Genus Kluyveromyces

Application of Agarose Gel Electrophoresis to the Characterization of Plasmid DNA in Drug-resistant Enterobacteria

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