Effect of insertions, deletions, and double-strand breaks on homologous recombination in mouse L cells.

Section: Resultsmentioning

confidence: 98%

Intermolecular homologous recombination in plants.

Baur¹,

Potrykus²,

Paszkowski³

1990

“…Based on this hypothesis, it is possible to make specific predictions about recombination between pSV2neo DL and DR. Double SmaI; E, 1-1 EcoRI; F, 1-1 KpnI; G, 1-1 Smal; H, [1][2][3][4][5][6][7][8][9] EcoRI; I, 1-9 KpnI; J, 1-9 Smal; K, 7-1 EcoRI; L, 7-1 KpnI; M, 7-1 Smal; N, pLCK EcoRI; 0, pLCK HindlIl; P, pLCK SmiaI; Q, pLCX EcoRI; R, pLCX KpnI; S, pLCX SmaI. instances two markers (deleted region and the nearest nonSmaI site) could be coconverted.…”

Section: Resultsmentioning

confidence: 99%

Effect of double-strand breaks on homologous recombination in mammalian cells and extracts.

Song¹,

Chekuri²,

Rauth³

et al. 1985

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.Purified DNA can be readily introduced into mammalian cells by microinjection (2) or by DNA transfection methods (5). Substantial evidence obtained by the use of viral and plasmid DNA molecules introduced into mammalian cells indicates that homologous recombination between input molecules occurs at a high frequency (3,4,14,15,(19)(20)(21). Based on these original observations, we developed two related systems which permit a dissection of the recombinational process in mammalian cells. One of these is an in vivo system, and the other is a cell-free system that can catalyze homologous recombination between homologous DNA molecules. The central feature of both systems is the use of a eucaryotic-procaryotic shuttle vector, pSV2neo, that carries a gene, which by virtue of its construction can be expressed equally efficiently in mammalian and bacterial cells (16). By using two nonoverlapping, nonreverting deletion mutants of this plasmid, we have shown that mammalian cells are able to mediate homologous recombination at a high frequency (7) and that purified nuclear extracts from human and rodent cells can mediate homologous recombination between these plasmids (8).Orr-Weaver et al. (13) Plasmids. pSV2neo plasmid was constructed by Southern and Berg (16). The deletion derivatives of pSV2neo were described previously (7). Briefly, pSV2neo DL (DL) was prepared by removing a 248-base-pair (bp) NarI fragment at the 5' end of the gene. pSV2neo DR (DR) was constructed by deletion of a 283-bp NaeI fragment. The unique NaeI site in DR was converted to a Sall site by linker addition (12). This plasmid is referred to as pSV2neo DR S. The deleted neo gene in this plasmid is flanked by a HindIlI site at the 5' end and by a SmaI site at the 3' end. These sites were changed to a SmaI and an XbaI site, respectively. This modified plasmid is referred to as pSV2neo DR SSalX. The chang...

“…The DNA was transferred to hybridization filters (GeneScreen Plus; New England Nuclear Corp.) using the procedure of Southern (42). After washing in 2x SSC (1 x SSC is 0.15 M sodium chloride plus 0.015 M sodium citrate), the blots were hybridized in a solution containing 50% formamide, 10% dextran sulfate, 1 M NaCl, 1% sodium dodecyl sulfate, and 200 jig of heat-denatured salmon sperm DNA (Worthington Diagnostics) per ml with 10-ng portions of single-strand-specific DNA probes at 42°C for 1 to (4). Plasmids p17G3, p17G7, and pl7G11 were rescued by using restriction endonuclease BgIII from the DNA of cell line 1-2-17 transformed with herpes simplex virus tk plus pBR322.…”

Section: Methodsmentioning

confidence: 99%

Foreign DNA introduced by calcium phosphate is integrated into repetitive DNA elements of the mouse L cell genome.

Kato¹,

Anderson²,

Camerini‐Otero³

1986

Self Cite

We investigated the sites of integration of exogenous DNA fragments introduced by DNA-mediated gene transfer. Mouse Ltk-cells were transformed with the herpes simplex virus thymidine kinase gene and pBR322 DNA by the calcium phosphate precipitation method. Some of the integrated exogenous DNA sequences were recovered from the stable tk+ transformants in the form of plasmids that were capable of propagation in bacteria. Four plasmids derived from two cloned cell lines were analyzed in detail by nucleotide sequencing and hybridization techniques. These plasmids contained a total of seven cellular-exogenous DNA junctions. In all cases, there was no sequence homology between the exogenous and cellular DNA sequences adjacent to the joining sites, and no specific exogenous or cellular sequences occurred at the junctions. Rearrangement or deletion of Ltk-DNA was always associated with the integration of exogenous DNA. All of the assignable cellular sequences at the junctions were repetitive sequences. Two of these sequences were from the MIF-1 repetitive sequence family, and a third consisted of a 40-base pair simple copolymer of alternating deoxyadenosine-deoxythymidine. Our results suggest that repetitive sequences are relatively favorable sites for the integration of exogenous DNA.Homologous recombination of foreign DNA with the host genome is sufficiently frequent in procaryotes (33) and lower eucaryotes (18) that it is possible to confidently predict the integration site of the introduced DNA. Since such efficient "targeting" of DNA into homologous sequences has not yet been demonstrated in mammalian cells (41), we asked whether the foreign DNA is integrated randomly or at some preferred sites other than the homologous sequences. Although a number of workers have reported on the DNA sequences at the integration sites of viral DNAs that have been introduced into mammalian cells (3,11,37,39), there have been no reports of a similar analysis for the integration of nonviral DNAs. Even though it has been established that at the level of chromosome analysis foreign, nonviral DNAs are integrated randomly (36), there is no information on the DNA sequences at the integration sites of exogenous nonviral DNAs.Since the site of integration may be influenced by proteins encoded by the DNA in question (as appears to be the case for the genomes of some eucaryotic viruses [34]) as opposed to DNA which relies solely on the cellular machinery for its integration, we examined integration sites resulting from the transfer of purified gene fragments and procaryotic DNA. Following transformation of mouse Ltk-cells with a herpes simplex virus thymidine kinase gene (tk) fragment and pBR322 DNA, individual colonies of stable tk+ transformants were isolated and expanded. A plasmid rescue technique was used to recover the integrated exogenous DNA and flanking cellular sequences from the genomic DNA of these cell lines (1, 2). We analyzed the sequences encompassing seven cellular-exogenous DNA junctions. MATERIALS AND METHODSDNA-mediated ge...