Double-strand cleavage at a two-base deletion mismatch in a DNA heteroduplex by nuclease S1

We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene. (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk- and RAT-2tk- cells to the TK+ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by homologous recombination.

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Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

Molecular and Cellular Biology ◽

10.1128/mcb.2.11.1372 ◽

1982 ◽

Vol 2 (11) ◽

pp. 1372-1387 ◽

Cited By ~ 232

Author(s):

K R Folger ◽

E A Wong ◽

G Wahl ◽

M R Capecchi

Keyword(s):

Homologous Recombination ◽

Plasmid Dna ◽

Mammalian Cells ◽

Recipient Cell ◽

Simian Virus 40 ◽

Linear Plasmid ◽

Dna Molecules ◽

Host Chromosome ◽

Gene Copies ◽

Physical Form

We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene. (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk- and RAT-2tk- cells to the TK+ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by homologous recombination.

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Adenovirus 2 peptide IX gene is expressed only on replicated DNA molecules

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4149-4154.1986 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4149-4154

Author(s):

T Matsui ◽

M Murayama ◽

T Mita

Keyword(s):

Quantitative Analysis ◽

Cytosine Arabinoside ◽

Plasmid Dna ◽

Adenovirus Type ◽

Plasmid Replication ◽

Gene Transcript ◽

Dna Molecules ◽

Transfected Cells ◽

Active Transcription

Transcription of the adenovirus type 2 peptide IX (pIX) gene was examined in transient expression assays. When a nonreplicating plasmid DNA containing the pIX gene was introduced into HeLa cells by the DEAE-dextran method, no pIX gene transcript was detected. In contrast, efficient transcription was observed in the cells transfected with a replicating plasmid containing the pIX gene. Adenovirus early genes did not affect the level of transcription of the pIX gene on either a nonreplicating or a replicating plasmid. Inhibition of plasmid replication with cytosine arabinoside prevented transcription of the pIX gene. By quantitative analysis of the amount of the pIX gene and its transcript in transfected cells, it was concluded that active transcription of the pIX gene occurred only on DNA molecules replicated in the cell.

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

F1000Research ◽

10.12688/f1000research.7202.1 ◽

2015 ◽

Vol 4 ◽

pp. 1145 ◽

Cited By ~ 9

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.

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Direct detection of unamplified genomic DNA based on photo-induced silver ion reduction by DNA molecules

Chemical Communications ◽

10.1039/c3cc38552c ◽

2013 ◽

Vol 49 (23) ◽

pp. 2350 ◽

Cited By ~ 12

Author(s):

Ye Lim Jung ◽

Cheulhee Jung ◽

Jung Hun Park ◽

Moon Il Kim ◽

Hyun Gyu Park

Keyword(s):

Genomic Dna ◽

Direct Detection ◽

Silver Ion ◽

Dna Molecules

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Artificial Restriction DNA Cutter Using Nuclease S1 for Site‐Selective Scission of Genomic DNA

Current Protocols in Nucleic Acid Chemistry ◽

10.1002/cpnc.72 ◽

2019 ◽

pp. e72 ◽

Cited By ~ 1

Author(s):

Arivazhagan Rajendran ◽

Narumi Shigi ◽

Jun Sumaoka ◽

Makoto Komiyama

Keyword(s):

Genomic Dna ◽

Nuclease S1 ◽

Artificial Restriction ◽

Site Selective

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Length computation of irradiated plasmid DNA molecules

Biointerphases ◽

10.1116/1.5050502 ◽

2018 ◽

Vol 13 (6) ◽

pp. 061005 ◽

Cited By ~ 1

Author(s):

Kateřina Pachnerová Brabcová ◽

Lembit Sihver ◽

Egor Ukraintsev ◽

Václav Štěpán ◽

Marie Davídková

Keyword(s):

Plasmid Dna ◽

Dna Molecules

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

Biochemical Society Transactions ◽

10.1042/bst0370023 ◽

2009 ◽

Vol 37 (1) ◽

pp. 23-28 ◽

Cited By ~ 80

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.

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