scholarly journals Patterns of Integration of DNA Microinjected into Cultured Mammalian Cells: Evidence for Homologous Recombination Between Injected Plasmid DNA Molecules

1983 ◽  
Vol 3 (1) ◽  
pp. 147-147
Keyword(s):  
Homologous Recombination ◽  
Plasmid Dna ◽  
Mammalian Cells ◽  
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.

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 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.

Gene Targeting

1999 ◽  
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Es Cells ◽  
Practical Approach ◽  
Simple Method ◽  
Mouse Es Cells ◽  
Cell Clones ◽  
Previous Edition ◽  
Pros And Cons

Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.

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.

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