Length computation of irradiated plasmid DNA molecules

2018 ◽  
Vol 13 (6) ◽  
pp. 061005 ◽  
Author(s):  
Kateřina Pachnerová Brabcová ◽  
Lembit Sihver ◽  
Egor Ukraintsev ◽  
Václav Štěpán ◽  
Marie Davídková
Keyword(s):  
Plasmid Dna ◽  
Dna Molecules

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

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.

scholarly journals Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei.

1985 ◽  
Vol 5 (1) ◽  
pp. 59-69 ◽  
Author(s):  
K R Folger ◽  
K Thomas ◽  
M R Capecchi
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Plasmid Dna ◽  
Mammalian Cells ◽  
Recombinant Dna ◽  
Dna Duplexes ◽  
Dna Molecules ◽  
Cell Nuclei ◽  
High Concentration ◽  
Length Polymorphisms

We have examined the mechanism of homologous recombination between plasmid molecules coinjected into cultured mammalian cells. Cell lines containing recombinant DNA molecules were obtained by selecting for the reconstruction of a functional Neor gene from two plasmids that bear different amber mutations in the Neor gene. In addition, these plasmids contain restriction-length polymorphisms within and near the Neor gene. These polymorphisms did not confer a selectable phenotype but were used to identify and categorize selected and nonselected recombinant DNA molecules. The striking conclusion from this analysis is that the predominant mechanism for the exchange of information between coinjected plasmid molecules over short distances (i.e., less than 1 kilobase) proceeds via nonreciprocal homologous recombination. The frequency of homologous recombination between coinjected plasmid molecules in cultured mammalian cells is extremely high, approaching unity. We demonstrate that this high frequency requires neither a high input of plasmid molecules per cell nor a localized high concentration of plasmid DNA within the nucleus. Thus, it appears that plasmid molecules, once introduced into the nucleus, have no difficulty seeking each other out and participating in homologous recombination even in the presence of a vast excess of host DNA sequences. Finally, we show that most of the homologous recombination events occur within a 1-h interval after the introduction of plasmid DNA into the cell nucleus.

scholarly journals Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules.

1996 ◽  
Vol 93 (17) ◽  
pp. 8826-8829 ◽  
Author(s):  
D. P. Allison ◽  
P. S. Kerper ◽  
M. J. Doktycz ◽  
J. A. Spain ◽  
P. Modrich ◽  
...  
Keyword(s):  
Atomic Force Microscope ◽  
Plasmid Dna ◽  
Dna Molecules ◽  
Atomic Force ◽  
Microscope Imaging

scholarly journals A candidate reference method for quantification of low concentrations of plasmid DNA by exhaustive counting of single DNA molecules in a flow stream

Metrologia ◽  
2014 ◽  
Vol 51 (5) ◽  
pp. 491-502 ◽  
Author(s):  
Hee-Bong Yoo ◽  
Donggeun Oh ◽  
Jae Yong Song ◽  
Mamoru Kawaharasaki ◽  
Jeeseong Hwang ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Reference Method ◽  
Dna Molecules ◽  
Single Dna Molecules ◽  
Low Concentrations ◽  
Flow Stream

Homologous recombination between plasmid DNA molecules in maize protoplasts

1991 ◽  
Vol 230 (1-2) ◽  
pp. 209-218 ◽  
Author(s):  
Leszek A. Lyznik ◽  
J. David McGee ◽  
Po-Yen Tung ◽  
Jeffrey L. Bennetzen ◽  
Thomas K. Hodges
Keyword(s):  
Homologous Recombination ◽  
Plasmid Dna ◽  
Dna Molecules

Recombination following transformation of Escherichia coli by heteroduplex plasmid DNA molecules

Gene ◽  
1984 ◽  
Vol 29 (3) ◽  
pp. 255-261 ◽  
Author(s):  
Chang Shing ◽  
Ho Diana ◽  
Jane R. McLaughlin ◽  
Chang Sheng-Yung
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Dna Molecules

scholarly journals Nonhomologous End-Joining Ligation Transfers DNA Regulatory Elements between Cointroduced Plasmids

2004 ◽  
Vol 24 (19) ◽  
pp. 8323-8331 ◽  
Author(s):  
Toshio Ishikawa ◽  
Eun Jig Lee ◽  
J. Larry Jameson
Keyword(s):  
Plasmid Dna ◽  
Mammalian Cells ◽  
Transfection Efficiency ◽  
Regulatory Elements ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Molecules ◽  
End Joining ◽  
Exogenous Dna ◽  
Strand Breaks

ABSTRACT Cointroduction of plasmids into mammalian cells is commonly used to investigate transcription factor regulation of reporter genes or to normalize transfection efficiency. We report here that cotransfected DNA molecules commonly transfer enhancer elements from one plasmid to another. Using separate Renilla or Firefly luciferase reporters, we found that an estrogen response element (ERE) originally linked to one of the reporters stimulated expression of the non-ERE-containing reporter. Similar enhancer transfer was seen with the cytomegalovirus enhancer. This enhancer transfer effect was not seen when cells were transfected separately with the reporters and the extracts were then combined before luciferase assays. The degree of enhancer transfer increased with transfected plasmid concentration and was greater when linearized rather than circular plasmid DNA was used. We hypothesized that double-strand breaks and heteroligation of cointroduced DNA molecules mediated the transfer of regulatory elements from one molecule to another. PCR of transfected plasmid DNA confirmed nonhomologous end-joining (NHEJ) ligation of DNA fragments originally present in separate plasmids. The NHEJ reaction was enhanced by UV light treatment to introduce double-strand breaks, and it was greater after liposome-mediated transfection than after calcium-phosphate-mediated transfection. NHEJ also occurred after adenoviral transfer of DNA into cells. We conclude that NHEJ mediates the transfer of regulatory DNA elements among cointroduced DNA molecules. These findings indicate the need for caution when interpreting results of transfection experiments containing more than one plasmid and suggest a mechanism whereby viruses or other exogenous DNA might recombine to activate unrelated genes.

scholarly journals Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency.

1991 ◽  
Vol 11 (1) ◽  
pp. 299-308 ◽  
Author(s):  
N J Marini ◽  
R M Benbow
Keyword(s):  
Xenopus Laevis ◽  
Plasmid Dna ◽  
Supramolecular Assembly ◽  
Micrococcal Nuclease ◽  
Dna Molecules ◽  
End Joining ◽  
Stage Of Development ◽  
Nuclease Digestion ◽  
Nuclease Resistance ◽  
Xenopus Laevis Embryos

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).

Sign in / Sign up

Export Citation Format

Share Document