Homologous recombination in a Chinese hamster X-ray-sensitive mutant

Moore, Peter D.; Song, Kyu-Young; Chekuri, L; Wallace, Linda J.; Kucherlapati, Raju

doi:10.1016/0027-5107(86)90038-2

Cited by 24 publications

(12 citation statements)

References 17 publications

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“…Since single-stranded substrates were able to participate in homologous recombination reactions both in vivo and in vitro, it was of interest to determine whether such molecules could function in the nonhomologous process of integration Preparation of nuclear extracts and assay for recombination have been described (9,21). Substrates were incubated with the extracts and the resulting DNA was used to transform E. coli strain DH1.…”

Section: Resultsmentioning

confidence: 99%

Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts.

Rauth¹,

Song²,

Ayares³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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We have examined the ability of singlestranded DNA to participate in homologous recombination reactions in mammalian cells and nuclear extracts derived from them. We have inserted a fragment of the neo gene into the single-stranded DNA phage vector M13 mphl. The neo fragment was derived from a deletion derivative of the prokaryotic-eukaryotic shuttle vector pSV2neo. The resulting singlestranded DNA was mixed with a double-stranded deletion derivative of pSV2neo and tested for recombination in human cells, monkey cells, and nuclear extracts obtained from human cells. We were able to obtain recombinant molecules containing wild-type neo genes in all three systems. Examination of the products of recombination indicated that they resulted from correction of the deletion in the double-stranded DNA substrate. We were unable to detect any extensive conversion of single-stranded DNA into its double-stranded counterpart before it participated in the recombination reaction. We have also tested the ability of single-stranded DNA to yield transfectants. When a single-stranded DNA derivative of the herpes simplex virus thymidine kinase (TK) gene was introduced into mouse L-M(TK-) cells, we were able to obtain TKV colonies.From these results, we conclude that single-stranded DNA can participate in transfection as well as homologous recombination reactions in mammalian cells.Integration of exogenous DNA into mammalian chromosomes by homologous recombination would be the preferred strategy for gene replacement therapy and in vivo gene modification. Mammalian somatic cells have been shown to contain all of the enzymes required to mediate recombination between homologous viral or plasmid sequences cotransfected into cells or between chromosomal sequences and their homologous counterparts (1-6). The exact nature of the proteins involved and the mechanism by which homologous recombination occurs in mammalian cells is not understood.We have developed a set of three systems to study the genetic and biochemical aspects of homologous recombination in mammalian cells (7-9). All of these involve the use of two derivatives of the prokaryotic-eukaryotic shuttle vector pSV2neo, a plasmid containing the bacterial gene for aminoglycoside 3'-phosphotransferase, which can be expressed in bacterial as well as in mammalian cells, resulting in neomycin or kanamycin resistance in bacteria and G418 resistance in mammalian cells. It also has an ampicillinresistance (AmpR) gene. Each of the two recombination substrates is a nonoverlapping, nonreverting deletion derivative of pSV2neo. The two deletion derivatives are referred to as pSV2neo DL and pSV2neo DR (7). In the first system, homologous recombination is studied by cotransfecting the two deletion plasmids into mammalian cells and selecting the cells for the expression of the normal neo gene, which can be generated by homologous recombination (7). The second method, somewhat analogous to studying bacteriophage recombination, involves introducing these substrates into monkey COS cells (10) ...

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Section: Resultsmentioning

confidence: 99%

Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts.

Rauth¹,

Song²,

Ayares³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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“…The rad-52 mutants of yeast repair single-strand breaks but not doublestrand breaks (Resnick & Martin, 1976) and are extremely deficient in both mitotic and meiotic recombination (Game et al, 1980). Moore et al (1986) have studied the recombination proficiency of the most extremely deficient doublestrand break repair (xrs) mutant isolated by Kemp and Jeggo (1986). In an in vivo assay the mutants showed a 6-fold reduced homologous recombination frequency when compared to the parental cell line.…”

Section: Dna Lesions and Their Repairmentioning

confidence: 99%

Ionizing radiation-induced mutagenesis

Breimer

1988

Br J Cancer

150

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“…For pBR322-D1 the region between the BamHI and SalI restriction endonuclease sites in the tetracycline gene was deleted while in pBR322-D2 the deletion was in the region between the PVuI and PstI restriction endonuclease sites in the ampicillin gene. In addition to the plasmids and the cell extract, each 100 µl of reaction mixture contained 20 mM Tris-HCl, pH 7.5, 10 mM Mg 2 SO 4 , 85 mM NaCl, 1 mM ATP, 100 µM each dATP, dCTP, dGTP, dTTP and 0.001% gelatin [7]. The mixture was incubated for 60 min at 37°C, and the reaction was stopped by the addition of 25 mM EDTA and 100 µg/ml of proteinase K. DNA was phenol-and chloroform-extracted and precipitated overnight at -80°C.…”

Section: Methodsmentioning

confidence: 99%

“…After completion of the treatment, the cells were collected by trypsinization, washed twice with PBS, and the pellet was frozen in liquid nitrogen and stored at -80°C. Nuclear extracts were prepared as described by Moore et al [7]. Each experimental group consisted of four samples.…”

Section: Methodsmentioning

confidence: 99%

Low doses of ionizing radiation induce nuclear activity in human tumour cell lines which catalyses homologous double-strand recombination

Lehnert

Chow

1997

Radiation and Environmental Biophysics

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Activity catalysing double-strand DNA recombination has been investigated in human tumour cell lines using an in vitro assay in which nuclear extracts from tumour cells are used to catalyse homologous recombination between deletion plasmids. The cell lines investigated showed comparable constitutive levels of recombination activity. In several cell lines a two- fold to fourfold increase in the frequency of double-strand recombinational events catalysed by nuclear extracts was observed if the cells were exposed to low doses of ionizing radiation. The response was greatest for cells harvested at 6 h after radiation exposure, and the dose to produce an optimal effect was 25 cGy. Cell lines showing this response included a relatively radioresistant human colon cancer line and two cis-DDP (cis-diamminedichloroplatinum II) resistant ovarian tumour cell lines which are cross-resistant to radiation. Sub-lethal doses of cis-DDP were also effective in inducing upregulation of recombinational activity in the cis-DDP resistant cell lines. No change in recombinational activity was seen for radiation/drug-sensitive ovarian cell line following exposure to low drug or radiation doses. These findings are of particular interest since they involve a radiation-induced process with potential for direct involvement in DNA repair. Further studies will be aimed at determining if the extent of resistance to cytotoxic agents is causally related to the degree of inducible recombination activity.

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Homologous recombination in a Chinese hamster X-ray-sensitive mutant

Cited by 24 publications

References 17 publications

Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts.

Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts.

Ionizing radiation-induced mutagenesis

Low doses of ionizing radiation induce nuclear activity in human tumour cell lines which catalyses homologous double-strand recombination

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