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

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.

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.

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

1982 ◽  
Vol 2 (11) ◽  
pp. 1372-1387
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.

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

1982 ◽  
Vol 2 (11) ◽  
pp. 1372-1387 ◽  
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.

scholarly journals Single bacterial resolvases first exploit, then constrain intrinsic dynamics of the Holliday junction to direct recombination

2021 ◽  
Author(s):  
Sujay Ray ◽  
Nibedita Pal ◽  
Nils G Walter
Keyword(s):  
Cluster Analysis ◽  
Homologous Recombination ◽  
Single Molecule ◽  
Holliday Junction ◽  
Energetic Cost ◽  
Genomic Integrity ◽  
Dna Molecules ◽  
Single Molecule Fluorescence ◽  
And Cluster Analysis ◽  
Fluorescence Observation

Abstract Homologous recombination forms and resolves an entangled DNA Holliday Junction (HJ) crucial for achieving genetic reshuffling and genome repair. To maintain genomic integrity, specialized resolvase enzymes cleave the entangled DNA into two discrete DNA molecules. However, it is unclear how two similar stacking isomers are distinguished, and how a cognate sequence is found and recognized to achieve accurate recombination. We here use single-molecule fluorescence observation and cluster analysis to examine how prototypic bacterial resolvase RuvC singles out two of the four HJ strands and achieves sequence-specific cleavage. We find that RuvC first exploits, then constrains the dynamics of intrinsic HJ isomer exchange at a sampled branch position to direct cleavage toward the catalytically competent HJ conformation and sequence, thus controlling recombination output at minimal energetic cost. Our model of rapid DNA scanning followed by ‘snap-locking’ of a cognate sequence is strikingly consistent with the conformational proofreading of other DNA-modifying enzymes.

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.

Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laevis oocytes

1990 ◽  
Vol 10 (7) ◽  
pp. 3505-3511
Author(s):  
J B Hays ◽  
E J Ackerman ◽  
Q S Pang
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Plasmid Dna ◽  
Excision Repair ◽  
Xenopus Laevis Oocytes ◽  
Marked Contrast ◽  
Repair Synthesis ◽  
Error Frequency ◽  
Repair Activity ◽  
Good Agreement

Repair of UV-irradiated plasmid DNA microinjected into frog oocytes was measured by two techniques: transformation of repair-deficient (delta uvrB delta recA delta phr) bacteria, and removal of UV endonuclease-sensitive sites (ESS). Transformation efficiencies relative to unirradiated plasmids were used to estimate the number of lethal lesions; the latter were assumed to be Poisson distributed. These estimates were in good agreement with measurements of ESS. By both criteria, plasmid DNA was efficiently repaired, mostly during the first 2 h, when as many as 2 x 10(10) lethal lesions were removed per oocyte. This rate is about 10(6) times the average for removal of ESS from repair-proficient human cells. Repair was slower but still significant after 2 h, but some lethal lesions usually remained after overnight incubation. Most repair occurred in the absence of light, in marked contrast to differentiated frog cells, previously shown to possess photoreactivating but no excision repair activity. There was no increase in the resistance to DpnI restriction of plasmids (methylated in Escherichia coli at GATC sites) incubated in oocytes; this implies no increase in hemimethylated GATC sites, and hence no semiconservative DNA replication. Plasmid substrates capable of either intramolecular or intermolecular homologous recombination were not recombined, whether UV-irradiated or not. Repair of Lac+ plasmids was accompanied by a significant UV-dependent increase in the frequency of Lac- mutants, corresponding to a repair synthesis error frequency on the order of 10(-4) per nucleotide.

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
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