Separation of Genomic DNA from Plasmid DNA by Selective Renaturation with Immobilized Metal Affinity Capture

2005 ◽  
Vol 21 (5) ◽  
pp. 1472-1477 ◽  
Author(s):  
T. Cano ◽  
J.C. Murphy ◽  
G.E. Fox ◽  
R.C. Willson
Keyword(s):  
Plasmid Dna ◽  
Genomic Dna ◽  
Affinity Capture ◽  
Metal Affinity

scholarly journals Selective enrichment of a large size genomic DNA fragment by affinity capture: an approach for genome mapping

1990 ◽  
Vol 18 (7) ◽  
pp. 1789-1795 ◽  
Author(s):  
Rajendra P. Kandpal ◽  
David C. Ward ◽  
Sherman M. Weissman
Keyword(s):  
Genomic Dna ◽  
Genome Mapping ◽  
Affinity Capture ◽  
Selective Enrichment ◽  
Large Size ◽  
Dna Fragment

scholarly journals A paradoxical synergism between Resveratrol and copper (II) with respect to degradation of DNA and RNA

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 1145 ◽  
Author(s):  
Siddharth Subramaniam ◽  
Iqbal Vohra ◽  
Aishwarya Iyer ◽  
Naveen K Nair ◽  
Indraneel Mittra
Keyword(s):  
Synergistic Effect ◽  
Plasmid Dna ◽  
Genomic Dna ◽  
Oxygen Species ◽  
Molar Ratios ◽  
Dna Cleaving ◽  
Dna And Rna ◽  
Fixed Concentration ◽  
Anti Cancer ◽  
Low Levels

Resveratrol (R), a plant polyphenol, is known to reduce Cu (II) to Cu (I) generating reactive oxygen species that can cleave plasmid DNA. Here we report a surprising observation of a paradoxical synergistic effect between R and Cu whereby plasmid DNA cleaving / degrading activity of R-Cu increased progressively as the ratio of R to Cu was increased i.e., the concentration of Cu was successively reduced with respect to a fixed concentration R. Whereas cleavage of plasmid DNA occurred at low molar ratios of R to Cu, at higher ratios, complete degradation of DNA was achieved. By further increasing the ratio, whereby the concentration of Cu was reduced to very low levels, the DNA degrading activity of R-Cu was lost. This paradoxical synergistic effect is also seen with respect to eukaryotic genomic DNA and RNA. Since R-Cu may have anti-cancer and anti-viral activities, our findings may not only help to improve the therapeutic efficacy of R-Cu but also reduce its toxic side effects with the use of low concentration of Cu.

Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism

2009 ◽  
Vol 37 (1) ◽  
pp. 23-28 ◽  
Author(s):  
Shiraz Ali Shah ◽  
Niels R. Hansen ◽  
Roger A. Garrett
Keyword(s):  
Plasmid Dna ◽  
Genomic Dna ◽  
Inhibitory Mechanism ◽  
Archaeal Viruses ◽  
Extrachromosomal Elements ◽  
Dna Strand ◽  
High Level

Transcripts from spacer sequences within chromosomal repeat clusters [CRISPRs (clusters of regularly interspaced palindromic repeats)] from archaea have been implicated in inhibiting or regulating the propagation of archaeal viruses and plasmids. For the crenarchaeal thermoacidophiles, the chromosomal spacers show a high level of matches (∼30%) with viral or plasmid genomes. Moreover, their distribution along the virus/plasmid genomes, as well as their DNA strand specificity, appear to be random. This is consistent with the hypothesis that chromosomal spacers are taken up directly and randomly from virus and plasmid DNA and that the spacer transcripts target the genomic DNA of the extrachromosomal elements and not their transcripts.

scholarly journals Efficient co-expression of a recombinant staphopain A and its inhibitor staphostatin A in Escherichia coli

2004 ◽  
Vol 385 (1) ◽  
pp. 181-187 ◽  
Author(s):  
Benedykt WLADYKA ◽  
Katarzyna PUZIA ◽  
Adam DUBIN
Keyword(s):  
Active Site ◽  
Genomic Dna ◽  
Specific Inhibitor ◽  
Insoluble Fraction ◽  
Immobilized Metal Affinity Chromatography ◽  
Expression Vectors ◽  
Native Conformation ◽  
Natural Sources ◽  
Metal Affinity ◽  
Genes Encoding

Staphopain A is a staphylococcal cysteine protease. Genes encoding staphopain A and its specific inhibitor, staphostatin A, are localized in an operon. Staphopain A is an important staphylococcal virulence factor. It is difficult to perform studies on its interaction with other proteins due to problems in obtaining a sufficient amount of the enzyme from natural sources. Therefore efforts were made to produce a recombinant staphopain A. Sequences encoding the mature form of staphopain A and staphostatin A were PCR-amplified from Staphylococcus aureus genomic DNA and cloned into different compatible expression vectors. Production of staphopain A was observed only when the enzyme was co-expressed together with its specific inhibitor, staphostatin A. Loss of the function mutations introduced within the active site of staphopain A causes the expression of the inactive enzyme. Mutations within the reactive centre of staphostatin A result in abrogation of production of both the co-expressed proteins. These results support the thesis that the toxicity of recombinant staphopain A to the host is due to its proteolytic activity. The coexpressed proteins are located in the insoluble fraction. Ni2+-nitrilotriacetate immobilized metal-affinity chromatography allows for an efficient and easy purification of staphopain A. Our optimized refolding parameters allow restoration of the native conformation of the enzyme, with yields over 10-fold higher when compared with isolation from natural sources.

Cloning of the Bile Salt Hydrolase (bsh) Gene from Enterococcus faecium FAIR-E 345 and Chromosomal Location of bsh Genes in Food Enterococci

2004 ◽  
Vol 67 (12) ◽  
pp. 2772-2778 ◽  
Author(s):  
AGUS WIJAYA ◽  
ANETTE HERMANN ◽  
HIKMATE ABRIOUEL ◽  
INGRID SPECHT ◽  
NUHA M. K. YOUSIF ◽  
...  
Keyword(s):  
Bile Salt ◽  
Plasmid Dna ◽  
Enterococcus Faecium ◽  
Genomic Dna ◽  
High Performance ◽  
Chromosomal Location ◽  
Bile Salt Hydrolase ◽  
Gene Probe ◽  
Screening Assay ◽  
Chromatography Analysis

Enterococcus faecium strain FAIR-E 345 isolated from food was shown to possess bile salt hydrolase (Bsh) activity in a plate screening assay and by high-performance liquid chromatography analysis. The bsh gene was cloned and sequenced. DNA sequence analysis revealed that it encoded a protein of 324 amino acids, with pI 4.877. A bsh gene probe was prepared from the cloned bsh gene and was used for probing plasmid and total genomic DNA of Bsh-positive enterococci isolated from food to determine the genomic location of their bsh genes. This probe was able to detect the bsh gene among total genomic DNA preparations but not from plasmid preparations of 10 plasmid-bearing Enterococcus strains. However, the probe could detect the bsh gene from total genomic DNA preparations of 12 Enterococcus strains that did not contain detectable plasmid DNA. In no cases did the probe hybridize with plasmid DNA preparations, suggesting that the bsh gene among enterococci is probably generally chromosomally encoded. This presumptive chromosomal location of bsh genes among food enterococci suggests that transfer of this trait by conjugative plasmids is unlikely.

Transformation methods for halophilic archaebacteria

1989 ◽  
Vol 35 (1) ◽  
pp. 148-152 ◽  
Author(s):  
Steven W. Cline ◽  
Wan L. Lam ◽  
Robert L. Charlebois ◽  
Leonard C. Schalkwyk ◽  
W. Ford Doolittle
Keyword(s):  
Molecular Weight ◽  
Plasmid Dna ◽  
Genomic Dna ◽  
Halobacterium Halobium ◽  
Dna Transformation ◽  
Dna Transfection ◽  
Phage Dna ◽  
Halophilic Archaebacteria ◽  
Transformation Methods ◽  
Halobacterium Volcanii

We present a practical description of polyethylene glycol mediated spheroplast transformation of Halobacterium halobium and Halobacterium volcanii. This method has been applied to phage DNA transfection, plasmid DNA transformation, and transformation with linear fragments of high molecular weight genomic DNA. Efficient spheroplast regeneration allows uncomplicated recovery of transformed progeny. Transformations can be performed equally well using fresh or frozen cell preparations. These methods should find application in molecular cloning, genetic fine mapping, and strain construction.Key words: archaebacteria, Halobacterium, transformation methods, spheroplast.

The significance of plasmid DNA preparations contaminated with bacterial genomic DNA on inflammatory responses following delivery of lipoplexes to the murine lung

Biomaterials ◽  
2011 ◽  
Vol 32 (36) ◽  
pp. 9854-9865 ◽  
Author(s):  
Reto P. Bazzani ◽  
Ying Cai ◽  
Henry L. Hebel ◽  
Stephen C. Hyde ◽  
Deborah R. Gill
Keyword(s):  
Plasmid Dna ◽  
Genomic Dna ◽  
Inflammatory Responses ◽  
Murine Lung

scholarly journals Synapsis-mediated fusion of free DNA ends forms inverted dimer plasmids in yeast.

Genetics ◽  
1990 ◽  
Vol 124 (1) ◽  
pp. 67-80 ◽  
Author(s):  
S Kunes ◽  
D Botstein ◽  
M S Fox
Keyword(s):  
Homologous Recombination ◽  
Plasmid Dna ◽  
Genomic Dna ◽  
Chromosomal Rearrangement ◽  
Yeast Genome ◽  
Homologous Pairing ◽  
Yeast Saccharomyces Cerevisiae ◽  
Free Dna ◽  
Sister Chromatids ◽  
Dna Ends

Abstract When yeast (Saccharomyces cerevisiae) is transformed with linearized plasmid DNA and the ends of the plasmid do not share homology with the yeast genome, circular inverted (head-to-head) dimer plasmids are the principal product of repair. By measurements of the DNA concentration dependence of transformation with a linearized plasmid, and by transformation with mixtures of genetically marked plasmids, we show that two plasmid molecules are required to form an inverted dimer plasmid. Several observations suggest that homologous pairing accounts for the head-to-head joining of the two plasmid molecules. First, an enhanced frequency of homologous recombination is detected when genetically marked plasmids undergo end-to-end fusion. Second, when a plasmid is linearized within an inverted repeat, such that its ends could undergo head-to-tail homologous pairing, it is repaired by intramolecular head-to-tail joining. Last, in the joining of homologous linearized plasmids of different length, a shorter molecule can acquire a longer plasmid end by homologous recombination. The formation of inverted dimer plasmids may be related to some forms of chromosomal rearrangement. These might include the fusion of broken sister chromatids in the bridge-breakage-fusion cycle and the head-to-head duplication of genomic DNA at the sites of gene amplifications.

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