Novel use of synthetic oligonucleotide insertion mutants for the study of homologous recombination in mammalian cells.

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...

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confidence: 99%

“…At 4 h, the precipitate was removed, and the cells were treated with 20% dimethyl sulfoxide for 2 min. The cells were transferred to 400 ,ug of G418 per ml on the following day, and resistant colonies were scored after 14 days.…”

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confidence: 99%

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

Song¹,

Chekuri²,

Rauth³

et al. 1985

“…One of the intermediates would be the favored substrate in our original 5'-3' degradative model and the other would be the favored substrate in the alternative 3'--5' degradative model. Our results indicate that the intermediate favored by the 3'--5' model is 10 to 20 times more efficient in generating recombinant tk genes than is the other intermediate.DNA introduced into cultured mammalian cells, either as a calcium phosphate precipitate or by direct microinjection into nuclei, is capable of undergoing efficient homologous recombination before it becomes integrated into chromosomes (11,13,18,22,28,32,34,35,41,42). Frequencies of homologous recombination in the range of 10 to 20% are common in these sorts of experiments (22).…”

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confidence: 99%

Extrachromosomal recombination in mammalian cells as studied with single- and double-stranded DNA substrates.

Lin¹,

Sperle²,

Sternberg³

1987

We have previously proposed a model to account for the high levels of homologous recombination that can occur during the introduction of DNA into mammalian cells (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034). An essential feature of that model is that linear molecules with ends appropriately located between homologous DNA segments are efficient substrates for an exonuclease that acts in a 5'-)3' direction. That process generates complementary single strands that pair in homologous regions to produce an intermediate that is processed efficiently to a recombinant molecule. An alternative model, in which strand degradation occurs in the 3'-+5' direction, is also possible. In this report, we describe experiments that tested several of the essential features of the model. We first confirmed and extended our previous results with double-stranded DNA substrates containing truncated herpesvirus thymidine kinase (tk) genes (tkA5' and tkA3'). The results illustrate the importance of the location of double-strand breaks in the successful reconstruction of the tk gene by recombination. We next transformed cells with pairs of single-stranded DNAs containing truncated tk genes which should anneal in cells to generate the recombination intermediates predicted by the two alternative models. One of the intermediates would be the favored substrate in our original 5'-3' degradative model and the other would be the favored substrate in the alternative 3'--5' degradative model. Our results indicate that the intermediate favored by the 3'--5' model is 10 to 20 times more efficient in generating recombinant tk genes than is the other intermediate.DNA introduced into cultured mammalian cells, either as a calcium phosphate precipitate or by direct microinjection into nuclei, is capable of undergoing efficient homologous recombination before it becomes integrated into chromosomes (11,13,18,22,28,32,34,35,41,42). Frequencies of homologous recombination in the range of 10 to 20% are common in these sorts of experiments (22). Recent in vitro recombination studies with nuclear extracts indicate that the enzymes necessary to execute homologous recombination are indeed present in mammalian cells (9,15,17,19).Two general mechanistic models have been proposed to account for the process of recombination during the introduction of DNA into mammalian cells. One, the doublestrand (ds)-break repair model, is analogous to that proposed to explain homologous recombination in yeasts (37). The second, the single-strand (ss) annealing model (22), is analogous to a model originally proposed by Broker and Lehman (4) to explain recombination by bacteriophage T4 and one later proposed by Lai and Nathans (20) to explain the generation of deletions after the introduction of simian virus 40 DNA into mammalian cells. In the ds-break repair model, recombination is initiated by a ds break or gap in one DNA molecule which is repaired faithfully with a second unbroken copy of the homologous DNA as a template. A requirement of the model is th...

“…We have previously shown that in L cells, the joining of dissimilar DNAs follows a precise scheme (1). DNA-mediated gene transfer can also be used to study homologous recombination between cotransferred DNA fragments of selectable genes (5,14,18,21,25,29,32,35,38).The technique of altering the structure of donor molecules to study recombination has been successfully used in bacteria and fungi (6,7,9,15,17,22,23,34,40). To study homologous recombination in mammalian cells, we constructed a family of plasmids with insertions and deletions in the coding region of the herpes simplex virus type 1 thymidine kinase gene (tk).…”

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confidence: 99%

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confidence: 99%

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

Brenner¹,

Smigocki²,

Camerini‐Otero³

1985

117

We have used DNA-mediated gene transfer to study homologous recombination in cultured mammalian cells. A family of plasmids with insertion and deletion mutations in the coding region of the herpes simplex type 1 thymidine kinase (tk) gene served as substrates for DNA-mediated gene transfer into mouse Ltk-cells by the calcium phosphate technique. Intermolecular recombination events were scored by the number of colonies in hypoxanthine-aminopterin-thymidine selective medium. We used supercoiled plasmids containing tk gene fragments to demonstrate that an overlap of 62 base pairs (bp) of homologous DNA was sufficient for intermolecular recombination. Addition of 598 bp of flanking homology separated from the region of recombination by a double-strand gap, deletion, or insertion of heterologous DNA increased the frequency of recombination by 300-, 20-, or 40-fold, respectively. Linearizing one of the mutant plasmids in a pair before cotransfer by cutting in the area of homology flanking a deletion of 104 bp or an insertion of less than 24 bp increased the frequency of recombination relative to that with uncut plasmids. However, cutting an insertion mutant of .24 bp in the same manner did not increase the frequency. We show how our data are consistent with models that postulate at least two phases in the recombination process: homologous pairing and heteroduplex formation.Recombination of DNA sequences in mammalian cells occurs in germ cells and somatic cells. The analysis of meiotic and mitotic mammalian genetic recombination has been hampered by the complexity of the mammalian genome. In somatic cells, examples of recombination events include immunoglobin gene rearrangements (28, 33), chromosomal translocations in cancer (45), and sister chromatid exchange (16). Recently, general recombination between two markers on the same chromosome has been demonstrated in mitotic Chinese hamster ovarian cell hybrids in culture (41). In the initial studies on homologous recombination in mammalian cells, infectious viruses such as simian virus 40 and adenovirus were used, but the interpretation of the experiments was difficult because of the constraints placed by viral replication and packaging and the possible contribution of viral proteins. We (1, 2) and others (11,12,30,38,44) have studied the recombination of nonhomologous DNAs transferred into mammalian cells in culture. In this approach, because it is easy to locate the exact positions of the recombinant joints, it is possible to determine the sequence and structure of these joints. We have previously shown that in L cells, the joining of dissimilar DNAs follows a precise scheme (1). DNA-mediated gene transfer can also be used to study homologous recombination between cotransferred DNA fragments of selectable genes (5,14,18,21,25,29,32,35,38).The technique of altering the structure of donor molecules to study recombination has been successfully used in bacteria and fungi (6,7,9,15,17,22,23,34,40). To study homologous recombination in mammalian cells, we constructed a famil...