Recombination events during integration of transfected DNA into normal human cells

Murnane, John P.; Yezzi, Michael J.; Young, Barbara R.

doi:10.1093/nar/18.9.2733

Cited by 73 publications

(42 citation statements)

References 32 publications

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“…6 in that the Cp.3-Cp.4-containing bands of 692x3 also contain C,u1 and C,u2, arguing that the truncated C,u segment characteristic of igm692 has been deleted in this recombinant. This contrasts with the report by Murnane et al (13), in which insertions of nonhomologous DNA were associated with deletions of only -10 nucleotides.…”

Section: Resultscontrasting

confidence: 85%

Gene replacement with one-sided homologous recombination.

Berinstein¹,

Pennell²,

Ottaway³

et al. 1992

Mol. Cell. Biol.

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Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.Homologous recombination between transferred and chromosomal DNA provides a method of introducing predetermined changes into genes in their normal chromosomal location. The strategies which have been developed for replacing segments of the chromosomal genes of mammalian cells have been based on the requirements for recombination in microorganisms, and in the gene replacement vectors which have been used to date, the replacement segment has been flanked on both ends by homologous regions ranging in length from 0.1 to 5 kb (1,6,7,17,19,20). Here we describe recombination between transferred and chromosomal immunoglobulin genes in hybridoma cells when the replacement segment is bounded by homologous DNA on one side only. MATERIALS AND METHODSCell lines and plasmids. The Sp6, igm482, and igm692 cell lines, tissue culture methods, and plasmids containing the DNA fragments used for electroporation have been previously described (11). The pRCp.-E vector bears the 9.5-kb EcoRI C,u fragment from pR-Sp6 (14) ferred with a Pasteur pipette to medium in 96-well plates. These cells were expanded, and the plaque plucking was repeated. After several rounds of enrichment by this technique, the PFC were isolated and cloned by limiting dilution. In order to isolate independent recombinants, the cells were subdivided immediately after electroporation, and only one PFC was taken from each subculture.Analysis of recombinant hybridoma clones. The p. heavy chains of the recombinants were analyzed by Western immunoblots. The immu...

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Section: Resultscontrasting

confidence: 85%

Gene replacement with one-sided homologous recombination.

Berinstein¹,

Pennell²,

Ottaway³

et al. 1992

Mol. Cell. Biol.

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“…(5) The illegitimate recombination process leading to the integration of transfected plasmid DNA into the recipient genome is mediated ®rst by a number of homologous recombination events among episomal plasmid molecules and only subsequently by an illegitimate recombination resulting in the integration. The latter process is normally accompanied by the deletion of a few nucleotides from the concatenate at the integration site (Murnane et al, 1990).…”

Section: Discussionmentioning

confidence: 99%

Fine structure of translocation breakpoints in leukemic blasts with chromosomal translocation t(4;11): the DNA damage-repair model of translocation

et al. 1998

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Chromosomal translocations t(4;11) are regularly associated with a speci®c type of acute leukemias and probably initiate the development of this disease. It has been proposed by others, that these translocations are mediated by recombinases of the immune system. The breakpoints on both derivative chromosomes for three t(4;11) leukemia-derived cell lines and primary blasts from two patients have been analysed here in detail. The results revealed that: (a) multiple double-or singlestranded DNA breaks must have occured near the translocation breakpoints on both participating chromosomes; and (b) DNA fragments¯anked by these breaks must have either been deleted, inverted or duplicated during the translocation process. We found no evidence for the involvement of speci®c target sequences and recombinases of the immune system. Similar characteristic features were observed by re-interpretation of published t(6;11) and t(9;22) translocation data. Therefore we present a new model for the generation of these translocations which poses, that these translocations are reciprocal but not balanced at the ®ne structure level and that the DNA damage-repair machinery is likely involved in producing the ®nal structure of the translocation breakpoint.

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“…Small deletions in the target have been reported for hepatitis B virus (HBV) integration studied in primary hepatocellular carcinomas (e.g., Hino et al 1989) and during integration of transfected DNA in primary fibroblasts (Mumane et al 1990). Larger target rearrangements are reviewed in Roth and Wilson (1988).…”

Section: Illegitimate Recombination In Plantsmentioning

confidence: 99%

“…Larger target rearrangements are reviewed in Roth and Wilson (1988). Mumane et al (1990) suggest that the difference between minor and major target rearrangements might be explained by differences in DNA recombination and stability in the different cell types studied.…”

Section: Illegitimate Recombination In Plantsmentioning

confidence: 99%

Illegitimate recombination in plants: a model for T-DNA integration.

Gheysen¹,

Villarroel²,

Montagu³

1991

Genes Dev.

266

201

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Agrobacterium tumefaciens is a soil bacterium capable of transferring DNA (the T-DNA) to the genome of higher plants, where it is then stably integrated. Six T-DNA inserts and their corresponding preinsertion sites were cloned from Arabidopsis thaliana and analyzed. Two T-DNA integration events from Nicotiana tabacum were included in the analysis. Nucleotide sequence comparison of plant target sites before and after T-DNA integration showed that the T-DNA usually causes only a small (13-28 bp) deletion in the plant DNA, but larger target rearrangements can occur. Short homologies between the T-DNA ends and the target sites, as well as the presence of filler sequences at the junctions, indicate that T-DNA integration is mediated by illegitimate recombination and that these processes in plants are very analogous to events in mammalian cells. We propose a model for T-DNA integration on the basis of limited base-pairing for initial synapsis, followed by DNA repair at the junctions. Variations of the model can explain the formation of filler DNA at the junctions by polymerase slipping and template switching during DNA repair synthesis and the presence of larger plant target DNA rearrangements.

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Recombination events during integration of transfected DNA into normal human cells

Cited by 73 publications

References 32 publications

Gene replacement with one-sided homologous recombination.

Gene replacement with one-sided homologous recombination.

Fine structure of translocation breakpoints in leukemic blasts with chromosomal translocation t(4;11): the DNA damage-repair model of translocation

Illegitimate recombination in plants: a model for T-DNA integration.

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